Biochemistry [1st Canadian ed.] 0176502653, 9780176502652

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Biochemistry F I RST CANAD I A N E D I T I O N

Reginald H. Garrett University of Virginia

Charles M. Grisham University of Virginia

Stavroula Andreopoulos University of Toronto

William G. Willmore

Carleton University

Imed E. Gallouzi McGill University

With molecular graphic images by Michal Sabat, University of Virginia

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

This is an electronic version of the print textbook. Due to electronic rights restrictions, some third party content may be suppressed. Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. The publisher reserves the right to remove content from this title at any time if subsequent rights restrictions require it. For valuable information on pricing, previous editions, changes to current editions, and alternate formats, please visit www.cengage.com/highered to search by ISBN#, author, title, or keyword for materials in your areas of interest.

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Biochemistry, First Canadian Edition by Reginald H. Garrett, Charles M. Grisham, Stavroula Andreopoulos, William G. Willmore, and Imed E. Gallouzi

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Library and Archives Canada Cataloguing in Publication Data

Adapted from Biochemistry, Fourth Edition, by Reginald H. Garrett and Charles M. Grisham, published by Brooks/Cole, Cengage Learning. Copyright ©2010 by Brooks/Cole, Cengage Learning. Printed and bound in the United States 1 2 3 4 15 14 13 12 For more information contact Nelson Education Ltd., 1120 Birchmount Road, Toronto, Ontario, M1K 5G4. Or you can visit our Internet site at http://www.nelson.com

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Biochemistry / Reginald H. Garrett ... [et al.].—1st Canadian ed. Includes index. ISBN 978-0-17-650265-2 1. Biochemistry—Textbooks. I. Garrett, R. (Reginald) QD415.B538 2012 C2012-905998-6

572

ISBN-13: 978-0-17-650265-2 ISBN-10: 0-17-650265-3

Every effort has been made to trace ownership of all copyrighted material and to secure permission from copyright holders. In the event of any question arising as to the use of any material, we will be pleased to make the necessary corrections in future printings.

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Dedication To Georgia —R.H.G. To Rosemary —C.M.G. To Agni —S.A. To my parents and family —W.G.W. To my wife Kerrie-Ann Trudeau and my son Jackson, with love —I.E.G.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

About the Authors

Charles M. Grisham was born and raised in Minneapolis, Minnesota, and educated at Benilde High School. He received his B.S. in chemistry from the Illinois Institute of Technology in 1969 and his Ph.D. in chemistry from the University of Minnesota in 1973. Following a postdoctoral appointment at the Institute for Cancer Research in Philadelphia, he joined the faculty of the University of Virginia, where he is Professor of Chemistry. He is the author of previous editions of Biochemistry and Principles of Biochemistry (Cengage, Brooks/Cole), and numerous papers and review articles on active transport of sodium, potassium, and calcium in mammalian systems, on protein kinase C, and on the applications of NMR and EPR spectroscopy to the study of biological systems. He has also authored Interactive Biochemistry CD-ROM and Workbook, a tutorial CD for students. His work has been supported by the National Institutes of Health, the National Science Foundation, the Muscular Dystrophy Association of America, the Research Corporation, the American Heart Association, and the American Chemical Society. He is a Research Career Development Awardee of the National Institutes of Health, and in 1983 and 1984 he was a Visiting Scientist at the Aarhus University Institute of Physiology Denmark. In 1999, he was Knapp Professor of Chemistry at the University of San Diego. He has taught biochemistry and physical chemistry at the University of Virginia for 37 years. He is a member of the American Society for Biochemistry and Molecular Biology.

Georgia Cobb Garrett

Reginald H. Garrett was educated in the Baltimore city public schools and at the Johns Hopkins University, where he received his Ph.D. in biology in 1968. Since that time, he has been at the University of Virginia, where he is currently Professor of Biology. He is the author of previous U.S. editions of Biochemistry, as well as Principles of Biochemistry (Cengage, Brooks/Cole), and numerous papers and review articles on the biochemical, genetic, and molecular biological aspects of inorganic nitrogen metabolism. His research interests focused on the pathway of nitrate assimilation in filamentous fungi. His investigations contributed substantially to our understanding of the enzymology, genetics, and regulation of this major pathway of biological nitrogen acquisition. More recently, he has collaborated in systems approaches to the metabolic basis of nutrition-related diseases. His research has been supported by the National Institutes of Health, the National Science Foundation, and private industry. He is a former Fulbright Scholar at the Universität fur Bodenkultur in Vienna, Austria, and served as Visiting Scholar at the University of Cambridge on two separate occasions. During the second, he was Thomas Jefferson Visiting Fellow in Downing College. In 2003, he was Professeur Invité at the Université Paul Sabatier/Toulouse III and the Centre National de la Recherche Scientifique, Institute for Pharmacology and Structural Biology in France. He has taught biochemistry at the University of Virginia for more than 40 years. He is a member of the American Society for Biochemistry and Molecular Biology.

Charles M. Grisham and Reginald H. Garrett

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Stavroula Andreopoulos, Ph.D., is a Senior Lecturer and Acting Undergraduate Coordinator in the department of Biochemistry at the University of Toronto. She is a passionate teacher who coordinates second- and third-year undergraduate Biochemistry courses with over 1700 students registered from a wide variety of programs. She earned her M.Sc. and Ph.D. at the University of Toronto. While she began her work in biochemical research at the University of Athens and the Centre for Addiction and Mental Health examining both the molecular mechanisms aspects of soluble guanylyl cyclase and lithium regulation of TRPC ion channels in bipolar disorder, her primary interest and love is now teaching. She is the recipient of several awards, including the NCDEU New Investigator’s Award; the Centre for Addiction and Mental Health (CAMH) Fellowship Award; and, more recently, the Excellence in Undergraduate Teaching in Life Sciences Award from the University of Toronto in 2011.

awards, including an Ontario Early Researcher Award and a Graduate Mentorship Award.

William G. Willmore

Stavroula Andreopoulos

William G. Willmore, Ph.D., is an Associate Professor and the current Chair of the Institute of Biochemistry at Carleton University. In 1997, he obtained his Ph.D. in Biochemistry at Carleton and did postdoctoral research at Brigham and Women’s Hospital, Harvard Medical School, in Boston, Massachusetts. He is the instructor for many third-year Biochemistry courses at Carleton, where he also leads a multidisciplinary team of researchers examining the role of protein posttranslational modifications in response to stress, primarily low oxygen (hypoxia) and oxidative stresses. He has established a Facility for Free Radical Research at Carleton, investigating protein structure/function modification in response to oxygen. His research attempts to discover novel therapeutics in ischemic cardiovascular diseases, such as cardiac arrest and stroke, as well as aging, have attracted international attention, including invited talks at Gordon Research Conferences. He is the recipient of numerous

Imed-Eddine Gallouzi, Ph.D., is an Associate Professor in the Department of Biochemistry at McGill University. He is also a Tier 2 Canada Research Chair in Cellular Information Systems. In 1998, he obtained his Ph.D. in Molecular and Celluar Biology from the Université Montpellier 2, Sciences et Techniques. His primary area of research is in Messenger RNA (mRNA), the stability of which influences gene expression in virtually all organisms, with a focus on mammals. His laboratory specializes in the study of RNA binding proteins and the various roles they play in physiological and pathological processes. He focuses on delineating the regulatory mechanisms that modulate mRNA expression during cell death and muscle formation. He is also testing the use of chemicals to interfere with muscle wasting induced by various diseases such as cancer, AIDS, and sepsis. His program at McGill University creates a new centre of excellence in cell biology at the institution, and his training activities have generated a strong team of cross-disciplinary researchers who bring expertise in biochemistry, cell biology, and molecular biology technologies to bear on the challenging problems related to cancer cell development and growth.

Imed-Eddine Gallouzi

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Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Contents in Brief

PART I:

Building Blocks of the Cell 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

PART II:

The Facts of Life: Chemistry Is the Logic of Biological Phenomena 1 Water: The Medium of Life 32 Thermodynamics of Biological Systems 53 Amino Acids 75 Proteins: Their Primary Structure and Biological Functions 98 Proteins: Secondary, Tertiary, and Quaternary Structure 140 Carbohydrates and the Glycoconjugates of Cell Surfaces 189 Lipids 228 Lipid Biosynthesis 252 Membranes and Membrane Transport 299 The Reception and Transmission of Extracellular Information 349

Protein Dynamics 12. 13. 14. 15.

1

403

Enzymes—Kinetics and Specificity 403 Mechanisms of Enzyme Action 442 Enzyme Regulation 477 Molecular Motors 505

PART III: Metabolism and Its Regulation 16. 17. 18. 19. 20. 21. 22. 23.

PART IV: Nucleic Acids 24. 25. 26. 27. 28.

PART V:

537

Nutrition and the Organization of Metabolism 537 Glycolysis 561 The Tricarboxylic Acid Cycle 591 Electron Transport and Oxidative Phosphorylation 621 Photosynthesis 659 Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway 693 Fatty Acid Catabolism 735 Nitrogen Acquisition and Amino Acid Metabolism 761

808

Nucleotides and Nucleic Acids 808 Structure of Nucleic Acids 833 Synthesis and Degradation of Nucleotides 871 Recombinant DNA: Cloning and Creation of Chimeric Genes 897 DNA Metabolism: Replication, Recombination, and Repair 926

Information Transfer 963 29. 30. 31. 32. 33.

Transcription and the Regulation of Gene Expression 963 Protein Synthesis 1009 Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA 1048 Completing the Protein Life Cycle: Folding, Processing, and Degradation 1086 Metabolic Integration and Organ Specialization 1109

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Detailed Contents

Archaea and Bacteria Have a Relatively Simple Structural Organization 19 The Structural Organization of Eukaryotic Cells Is More Complex Than That of Prokaryotic Cells 21

About the Authors iv Preface xxix Laboratory Techniques in Biochemistry xl

PART I

1

1.6 1.7

Building Blocks of the Cell

Prions and Viroids Can Act as Genetic Agents 26 SUMMARY 29 PROBLEMS 30

The Facts of Life: Chemistry Is the Logic of Biological Phenomena 1 1.1 1.2

What Are the Distinctive Properties of Living Systems? 2 What Kinds of Molecules Are Biomolecules? 5

FURTHER READING 31

2

Water: The Medium of Life 32 2.1

What Is the Structural Organization of Complex Biomolecules? 6 Metabolites Are Used to Form the Building Blocks of Macromolecules 6 Membranes Are Supramolecular Assemblies That Define the Boundaries of Cells 10 The Unit of Life Is the Cell 10

1.4

How Do the Properties of Biomolecules Reflect Their Fitness to the Living Condition? 10 Biological Macromolecules and Their Building Blocks Have a “Sense” or Directionality 11 Biological Macromolecules Are Informational 11 Biomolecules Have Characteristic Three-Dimensional Architecture 12 Weak Forces Maintain Biological Structure and Determine Biomolecular Interactions 12 Hydrogen Bonds Are Important in Biomolecular Interactions 13 The Defining Concept of Biochemistry Is “Molecular Recognition Through Structural Complementarity” 14 Biomolecular Recognition Is Mediated by Weak Chemical Forces 15 Weak Forces Restrict Organisms to a Narrow Range of Environmental Conditions 16 Enzymes Catalyze Metabolic Reactions 17 The Time Scale of Life 17

1.5

What Is the Organization and Structure of Cells? 17 The Evolution of Early Cells Gave Rise to Eubacteria, Archaea, and Eukaryotes 18 How Many Genes Does a Cell Need? 18

What Are the Properties of Water? 33 Water Has Unusual Properties 33 Hydrogen Bonding in Water Is Key to Its Properties 34 The Structure of Ice Is Based on H-Bond Formation 34 Molecular Interactions in Liquid Water Are Based on H Bonds 35 The Solvent Properties of Water Derive from Its Polar Nature 35 Water Can Ionize to Form H! and OH" 39

Biomolecules Are Carbon Compounds 6

1.3

What Are Viruses? 23 Do Biomolecules Ever Behave as Genetic Agents? 26

2.2

What Is pH? 40 Strong Electrolytes Dissociate Completely in Water 41 Weak Electrolytes Are Substances That Dissociate Only Slightly in Water 42 The Henderson–Hasselbalch Equation Describes the Dissociation of a Weak Acid in the Presence of Its Conjugate Base 43 Titration Curves Illustrate the Progressive Dissociation of a Weak Acid 44 Phosphoric Acid Has Three Dissociable H! 45

2.3

What Are Buffers and What Do They Do? 46 The Phosphate Buffer System Is a Major Intracellular Buffering System 46 Dissociation of the Histidine–Imidazole Group Also Serves as an Intracellular Buffering System 47 “Good” Buffers Are Buffers Useful within Physiological pH Ranges 47 HUMAN BIOCHEMISTRY: The Bicarbonate Buffer System of Blood Plasma 48 HUMAN BIOCHEMISTRY: Blood pH and Respiration 49 What Are Common Buffers Used in Biochemistry? 49

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Detailed Contents

2.4

What Properties of Water Give It a Unique Role in the Environment? 50

There Are 20 Common Amino Acids 77

SUMMARY 50

Amino Acids 21 and 22—and More? 81

PROBLEMS 51

Several Amino Acids Occur Only Rarely in Proteins 81

Are There Other Ways to Classify Amino Acids? 80

FURTHER READING 52

3

Amino Acid Derivatives 82

4.2

Thermodynamics of Biological Systems 53 3.1

What Are the Basic Concepts of Thermodynamics? 54

3.3

Side Chains of Amino Acids Undergo Characteristic Ionizations 84 Isoelectric Points of Amino Acids 85

4.3 4.4

What Can Thermodynamic Parameters Tell Us about Biochemical Events? 60 What Are the Characteristics of High-Energy Biomolecules? 61

4.5

3.6

What Are the Complex Equilibria Involved in ATP Hydrolysis? 69

4.6

A DEEPER LOOK: ATP Changes the Keq by a Factor of 108 72

SUMMARY 95 PROBLEMS 96

SUMMARY 73 FURTHER READING 74

4

Amino Acids 75 4.1

What Are the Structures and Properties of Amino Acids? 76 Typical Amino Acids Contain a Central Tetrahedral Carbon Atom 76 Amino Acids Can Join via Peptide Bonds 76

What Is the Fundamental Structural Pattern in Proteins? 91 The Peptide Bond Has Partial Double-Bond Character 92 The Polypeptide Backbone Is Relatively Polar 92 Peptides Can Be Classified According to How Many Amino Acids They Contain 94 Proteins Are Composed of One or More Polypeptide Chains 94

Why Are Coupled Processes Important to Living Things? 71 What Is the Daily Human Requirement for ATP? 71

PROBLEMS 73

What Are the Spectroscopic Properties of Amino Acids? 87 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: Discovery of Optically Active Molecules and Determination of Absolute Configuration 88 Phenylalanine, Tyrosine, and Tryptophan Absorb Ultraviolet Light 88 Amino Acids Can Be Characterized by Nuclear Magnetic Resonance 89 A DEEPER LOOK: The Murchison Meteorite— Discovery of Extraterrestrial Handedness 90 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: Rules for Description of Chiral Centres in the (R,S) System 91

The !G°’ of Hydrolysis for ATP Is pH Dependent 69 Metal Ions Affect the Free Energy of Hydrolysis of ATP 70 Concentration Affects the Free Energy of Hydrolysis of ATP 70

3.5

What Are the Post-translational Modifications of Amino Acids? 86 What Are the Optical and Stereochemical Properties of Amino Acids? 86 Amino Acids Are Chiral Molecules 86 Chiral Molecules Are Described by the d,l and R,S Naming Conventions 86 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: Green Fluorescent Protein—The “Light Fantastic” from Jellyfish to Gene Expression 87

ATP Is an Intermediate Energy-Shuttle Molecule 62 Group Transfer Potentials Quantify the Reactivity of Functional Groups 63 The Hydrolysis of Phosphoric Acid Anhydrides Is Highly Favourable 64 The Hydrolysis !G°’ of ATP and ADP Is Greater Than That of AMP 66 Acetyl Phosphate and 1,3-Bisphosphoglycerate Are Phosphoric-Carboxylic Anhydrides 67 Enol Phosphates Are Potent Phosphorylating Agents 67 Guanidino Phosphates Are Energy Reserve Compounds 68

3.4

What Are the Acid–Base Properties of Amino Acids? 82 Amino Acids Are Weak Polyprotic Acids 82

The First Law: The Total Energy of an Isolated System Is Conserved 54 Enthalpy Is a More Useful Function for Biological Systems 55 The Second Law: Systems Tend Toward Disorder and Randomness 57 A DEEPER LOOK: Entropy, Information, and the Importance of “Negentropy” 58 The Third Law: Why Is “Absolute Zero” So Important? 58 Free Energy Provides a Simple Criterion for Equilibrium 58

3.2

ix

FURTHER READING 97

5

Proteins: Their Primary Structure and Biological Functions 98 5.1

What Architectural Arrangements Characterize Protein Structure? 99 Protein Structure Is Described in Terms of Four Levels of Organization 99 Non-covalent Forces Drive Formation of the Higher Orders of Protein Structure 101

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x

Detailed Contents

Proteins Fall into Three Basic Classes According to Shape and Solubility 101 A Protein’s Conformation Can Be Described as Its Overall Three-Dimensional Structure 102

5.2

A Mutant Protein Is a Protein with a Slightly Different Amino Acid Sequence 128 A DEEPER LOOK: Bovine Spongiform Encephalopathy (BSE) and Its Effect on the Canadian Economy 130

How Are Proteins Isolated and Purified from Cells? 102

5.7

Dialysis and Ultrafiltration 103 A Number of Protein Separation Methods Exploit Differences in Size and Charge 103 A Typical Protein Purification Scheme Uses a Series of Separation Methods 104 A DEEPER LOOK: Estimation of Protein Concentrations in Solutions of Biological Origin 104

5.3

5.4

How Is the Amino Acid Analysis of Proteins Performed? 111 Acid Hydrolysis Liberates the Amino Acids of a Protein 111 Chromatographic Methods Are Used to Separate the Amino Acids 111 The Amino Acid Compositions of Different Proteins Are Different 111

5.5

How Is the Primary Structure of a Protein Determined? 111 The Sequence of Amino Acids in a Protein Is Distinctive 111 Sanger Was the First to Determine the Sequence of a Protein 112 Both Chemical and Enzymatic Methodologies Are Used in Protein Sequencing 112 Step 1. Separation of Polypeptide Chains 112 A DEEPER LOOK: The Virtually Limitless Number of Different Amino Acid Sequences 113 Step 2. Cleavage of Disulfide Bridges 113 Step 3. N- and C-Terminal Analysis 113 Steps 4 and 5. Fragmentation of the Polypeptide Chain 115 Step 6. Reconstruction of the Overall Amino Acid Sequence 118 The Amino Acid Sequence of a Protein Can Be Determined by Mass Spectrometry 118 Sequence Databases Contain the Amino Acid Sequences of Millions of Different Proteins 122

5.6

Solid-Phase Methods Are Very Useful in Peptide Synthesis 130

5.8 5.9

What Is the Nature of Amino Acid Sequences? 122 Homologous Proteins from Different Organisms Have Homologous Amino Acid Sequences 122 Computer Programs Can Align Sequences and Discover Homology between Proteins 123 Related Proteins Share a Common Evolutionary Origin 125 Apparently Different Proteins May Share a Common Ancestry 128

Do Proteins Have Chemical Groups Other Than Amino Acids? 132 What Are the Many Biological Functions of Proteins? 133 SUMMARY 135

How Are Amino Acid Mixtures Separated? 105 Amino Acids Can Be Separated by Chromatography 105 Summary of the Types of Chromatographic Techniques 105

Can Polypeptides Be Synthesized in the Laboratory? 130

PROBLEMS 136 FURTHER READING 138

6

Proteins: Secondary, Tertiary, and Quaternary Structure 140 6.1

What Non-covalent Interactions Stabilize Protein Structure? 141 Hydrogen Bonds Are Formed Whenever Possible 142 Hydrophobic Interactions Drive Protein Folding 142 Ionic Interactions Usually Occur on the Protein Surface 142 Van der Waals Interactions Are Ubiquitous 142

6.2 6.3

What Role Does the Amino Acid Sequence Play in Protein Structure? 143 What Are the Elements of Secondary Structure in Proteins, and How Are They Formed? 143 All Protein Structure Is Based on the Amide Plane 143 The Alpha-Helix Is a Key Secondary Structure 145 A DEEPER LOOK: Knowing What the Right Hand and Left Hand Are Doing 146 The b-Pleated Sheet Is a Core Structure in Proteins 149 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: In Bed with a Cold, Pauling Stumbles onto the a-Helix and a Nobel Prize 150 Helix–Sheet Composites in Spider Silk 151 b-Turns Allow the Protein Strand to Change Direction 152

6.4

How Do Polypeptides Fold into Three-Dimensional Protein Structures? 152 Fibrous Proteins Usually Play a Structural Role 153 A DEEPER LOOK: The Coiled-Coil Motif in Proteins 155 Globular Proteins Mediate Cellular Function 159 Helices and Sheets Make Up the Core of Most Globular Proteins 159 Waters on the Protein Surface Stabilize the Structure 160 Packing Considerations 160 HUMAN BIOCHEMISTRY: Collagen-Related Diseases 162 Protein Domains Are Nature’s Modular Strategy for Protein Design 162 NEL

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Detailed Contents

Classification Schemes for the Protein Universe Are Based on Domains 164 Denaturation Leads to Loss of Protein Structure and Function 166 Anfinsen’s Classic Experiment Proved That Sequence Determines Structure 168 Is There a Single Mechanism for Protein Folding? 169 What Is the Thermodynamic Driving Force for Folding of Globular Proteins? 170 Marginal Stability of the Tertiary Structure Makes Proteins Flexible 171 Motion in Globular Proteins 172 The Folding Tendencies and Patterns of Globular Proteins 172 Most Globular Proteins Belong to One of Four Structural Classes 174 Molecular Chaperones Are Proteins That Help Other Proteins to Fold 176 Some Proteins Are Intrinsically Unstructured 176 HUMAN BIOCHEMISTRY: A1-Antitrypsin: A Tale of Molecular Mousetraps and a Folding Disease 178 HUMAN BIOCHEMISTRY: Diseases of Protein Folding 179 HUMAN BIOCHEMISTRY: Structural Genomics 179

6.5

Haworth Projections Are a Convenient Device for Drawing Sugars 195 Monosaccharides Can Be Converted to Several Derivative Forms 196 A DEEPER LOOK: Honey: An Ancestral Carbohydrate Treat 199

7.3

7.4

7.5

7.6

Carbohydrates and the Glycoconjugates of Cell Surfaces 189 Synthesis of Sucrose 190

7.2

What Are Carbohydrates, and How Are They Named? 190 What Is the Structure and Chemistry of Monosaccharides? 191 Monosaccharides Are Classified as Aldoses and Ketoses 191 Stereochemistry Is a Prominent Feature of Monosaccharides 192 Monosaccharides Exist in Cyclic and Anomeric Forms 193

How Do Proteoglycans Modulate Processes in Cells and Organisms? 218 Functions of Proteoglycans Involve Binding to Other Proteins 218 Proteoglycans May Modulate Cell Growth Processes 220 Proteoglycans Make Cartilage Flexible and Resilient 221

BIOCHEMISTRY: A CANADIAN CONTEXT: The

7.1

What Are Glycoproteins, and How Do They Function in Cells? 213 Polar Fish Depend on Antifreeze Glycoproteins 215 A DEEPER LOOK: Drug Research Finds a Sweet Spot 216 N-Linked Oligosaccharides Can Affect the Physical Properties and Functions of a Protein 216 Oligosaccharide Cleavage Can Serve as a Timing Device for Protein Degradation 216 A DEEPER LOOK: N-Linked Oligosaccharides Help Proteins Fold 218

PROBLEMS 187

7

What Is the Structure and Chemistry of Polysaccharides? 203 Nomenclature for Polysaccharides Is Based on Their Composition and Structure 203 Polysaccharides Serve Energy Storage, Structure, and Protection Functions 203 Polysaccharides Provide Stores of Energy 204 Polysaccharides Provide Physical Structure and Strength to Organisms 205 A DEEPER LOOK: A Complex Polysaccharide in Red Wine: The Strange Story of Rhamnogalacturonan II 208 A DEEPER LOOK: Billiard Balls, Exploding Teeth, and Dynamite: The Colourful History of Cellulose 210 Polysaccharides Provide Strength and Rigidity to Bacterial Cell Walls 210 Peptidoglycan Is the Polysaccharide of Bacterial Cell Walls 210 Animals Display a Variety of Cell Surface Polysaccharides 212

SUMMARY 186 FURTHER READING 187

What Is the Structure and Chemistry of Oligosaccharides? 200 Disaccharides Are the Simplest Oligosaccharides 200 A DEEPER LOOK: Trehalose: A Natural Protectant for Bugs 202 A Variety of Higher Oligosaccharides Occur in Nature 202

How Do Protein Subunits Interact at the Quaternary Level of Protein Structure? 179 There Is Symmetry in Quaternary Structures 181 Quaternary Association Is Driven by Weak Forces 183 A DEEPER LOOK: Immunoglobulins: All the Features of Protein Structure Brought Together 184 Immunoglobulin-Based Techniques 184 Open Quaternary Structures Can Polymerize 184 There Are Structural and Functional Advantages to Quaternary Association 184 HUMAN BIOCHEMISTRY: Faster-Acting Insulin: Genetic Engineering Solves a Quaternary Structure Problem 185 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: Banting and Best: The Great Canadian Discovery 185

xi

7.7

Do Carbohydrates Provide a Structural Code? 222 Selectins, Rolling Leukocytes, and the Inflammatory Response 222 Galectins: Mediators of Inflammation, Immunity, and Cancer 223 C-Reactive Protein: A Lectin That Limits Inflammation Damage 224

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xii

Detailed Contents

SUMMARY 225

Acetate Units Are Committed to Fatty Acid Synthesis by Formation of Malonyl-CoA 255 Acetyl-CoA Carboxylase Is Biotin-Dependent and Displays Ping-Pong Kinetics 255 Acetyl-CoA Carboxylase in Animals Is a Multifunctional Protein 255 Phosphorylation of ACC Modulates Activation by Citrate and Inhibition by Palmitoyl-CoA 256 Acyl Carrier Proteins Carry the Intermediates in Fatty Acid Synthesis 258 In Some Organisms, Fatty Acid Synthesis Takes Place in Multi-enzyme Complexes 258 A DEEPER LOOK: Choosing the Best Organism for the Experiment 258 Decarboxylation Drives the Condensation of Acetyl-CoA and Malonyl-CoA 259 Reduction of the b-Carbonyl Group Follows a Now Familiar Route 260 Eukaryotes Build Fatty Acids on Megasynthase Complexes 261 C16 Fatty Acids May Undergo Elongation and Unsaturation 263 Unsaturation Reactions Occur in Eukaryotes in the Middle of an Aliphatic Chain 265 The Unsaturation Reaction May Be Followed by Chain Elongation 265 Mammals Cannot Synthesize Most Polyunsaturated Fatty Acids 265 Arachidonic Acid Is Synthesized from Linoleic Acid by Mammals 266 Regulatory Control of Fatty Acid Metabolism Is an Interplay of Allosteric Modifiers and Phosphorylation– Dephosphorylation Cycles 266 HUMAN BIOCHEMISTRY: v3 and v6: Essential Fatty Acids with Many Functions 267 Hormonal Signals Regulate ACC and Fatty Acid Biosynthesis 268

PROBLEMS 225 FURTHER READING 227

8

Lipids 228 8.1

What Are the Structures and Chemistry of Fatty Acids? 229 HUMAN BIOCHEMISTRY: Fast Foods in Canada 232

8.2

What Are the Structures and Chemistry of Triacylglycerols? 232 A DEEPER LOOK: Polar Bears Prefer Non-polar Food 233

8.3

What Are the Structures and Chemistry of Glycerophospholipids? 233 Glycerophospholipids Are the Most Common Phospholipids 235 Ether Glycerophospholipids Include PAF and Plasmalogens 236 HUMAN BIOCHEMISTRY: Platelet-Activating Factor: A Potent Glyceroether Mediator 237

8.4

What Are Sphingolipids, and How Are They Important for Higher Animals? 237 A DEEPER LOOK: Moby Dick and Spermaceti: A Valuable Wax from Whale Oil 239

8.5 8.6

What Are Waxes, and How Are They Used? 239 What Are Terpenes, and What Is Their Relevance to Biological Systems? 240 A DEEPER LOOK: Why Do Plants Emit Isoprene? 241 The Membranes of Archaea Are Rich in IsopreneBased Lipids 241 HUMAN BIOCHEMISTRY: Coumadin or Warfarin— Agent of Life or Death 242

8.7

What Are Steroids, and What Are Their Cellular Functions? 243 Cholesterol 243 Steroid Hormones Are Derived from Cholesterol 243

8.8

How Do Lipids and Their Metabolites Act as Biological Signals? 244

9.2

A DEEPER LOOK: Glycerophospholipid Degradation: One of the Effects of Snake Venom 245 HUMAN BIOCHEMISTRY: Plant Sterols and Stanols— Natural Cholesterol Fighters 247 HUMAN BIOCHEMISTRY: 17b-Hydroxysteroid Dehydrogenase 3 Deficiency 248 SUMMARY 248 PROBLEMS 249 FURTHER READING 250

9

Lipid Biosynthesis 252 9.1

How Are Fatty Acids Synthesized? 253 Formation of Malonyl-CoA Activates Acetate Units for Fatty Acid Synthesis 253 Fatty Acid Biosynthesis Depends on the Reductive Power of NADPH 253 Cells Must Provide Cytosolic Acetyl-CoA and Reducing Power for Fatty Acid Synthesis 254

How Are Complex Lipids Synthesized? 269 Glycerolipids Are Synthesized by Phosphorylation and Acylation of Glycerol 269 Eukaryotes Synthesize Glycerolipids from CDPDiacylglycerol or Diacylglycerol 269 Phosphatidylethanolamine Is Synthesized from Diacylglycerol and CDP-Ethanolamine 270 Exchange of Ethanolamine for Serine Converts Phosphatidylethanolamine to Phosphatidylserine 272 Eukaryotes Synthesize Other Phospholipids via CDP-Diacylglycerol 272 Dihydroxyacetone Phosphate Is a Precursor to the Plasmalogens 272 Platelet-Activating Factor Is Formed by Acetylation of 1-Alkyl-2-Lysophosphatidylcholine 275 Sphingolipid Biosynthesis Begins with Condensation of Serine and Palmitoyl-CoA 275 Ceramide Is the Precursor for Other Sphingolipids and Cerebrosides 275

9.3

How Are Eicosanoids Synthesized, and What Are Their Functions? 278 NEL

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Eicosanoids Are Local Hormones 278 Prostaglandins Are Formed from Arachidonate by Oxidation and Cyclization 278 A Variety of Stimuli Trigger Arachidonate Release and Eicosanoid Synthesis 278 A DEEPER LOOK: The Discovery of Prostaglandins 278 A DEEPER LOOK: The Molecular Basis for the Action of Non-steroidal Anti-inflammatory Drugs 280 “Take Two Aspirin and…” Inhibit Your Prostaglandin Synthesis 281

9.4

9.5

10.2

10.3

10.4

10.5 10.6

10.7

10.8

What Are the Chemical and Physical Properties of Membranes 300 The Composition of Membranes Suits Their Functions 301

How Are Certain Transport Processes Driven by Light Energy? 341 Bacteriorhodopsin Uses Light Energy to Drive Proton Transport 343

FURTHER READING 297

10.1

How Does Energy Input Drive Active Transport Processes? 335 All Active Transport Systems Are Energy-Coupling Devices 335 Many Active Transport Processes Are Driven by ATP 335 A DEEPER LOOK: Cardiac Glycosides: Potent Drugs from Ancient Times 340 ABC Transporters Use ATP to Drive Import and Export Functions and Provide Multidrug Resistance 340

10.9

299

How Does Facilitated Diffusion Occur? 329 Membrane Channel Proteins Facilitate Diffusion 329 The Bacillus cereus NaK Channel Uses a Variation on the K+ Selectivity Filter 333 CorA Is a Pentameric Mg2+ Channel 333 Chloride, Water, Glycerol, and Ammonia Flow through Single-Subunit Pores 334

PROBLEMS 296

10 Membranes and Membrane Transport

How Does Transport Occur across Biological Membranes? 327 What Is Passive Diffusion? 328 Charged Species May Cross Membranes by Passive Diffusion 329

Pregnenolone and Progesterone Are the Precursors of All Other Steroid Hormones 293 Steroid Hormones Modulate Transcription in the Nucleus 294 Cortisol and Other Corticosteroids Regulate a Variety of Body Processes 295 Anabolic Steroids Have Been Used Illegally to Enhance Athletic Performance 295 SUMMARY 295

What Are the Dynamic Processes That Modulate Membrane Function? 319 Lipids and Proteins Undergo a Variety of Movements in Membranes 319 Membrane Lipids Can Be Ordered to Different Extents 320

How Are Bile Acids Biosynthesized? 292

How Are Steroid Hormones Synthesized and Utilized? 293

How Are Biological Membranes Organized? 318 Membranes Are Asymmetric and Heterogeneous Structures 318

HUMAN BIOCHEMISTRY: Steroid 5a-Reductase: A Factor in Male Baldness, Prostatic Hyperplasia, and Prostate Cancer 293

9.7

What Are the Structure and Chemistry of Membrane Proteins? 306 Peripheral Membrane Proteins Associate Loosely with the Membrane 306 Integral Membrane Proteins Are Firmly Anchored in the Membrane 307 Lipid-Anchored Membrane Proteins Are Switching Devices 314 A DEEPER LOOK: Exterminator Proteins—Biological Pest Control at the Membrane 315 HUMAN BIOCHEMISTRY: Prenylation Reactions as Possible Chemotherapy Targets 317

How Are Lipids Transported throughout the Body? 288 Lipoprotein Complexes Transport Triacylglycerols and Cholesterol Esters 288 Lipoproteins in Circulation Are Progressively Degraded by Lipoprotein Lipase 289 The Structure of the LDL Receptor Involves Five Domains 289 The LDL Receptor !-Propeller Displaces LDL Particles in Endosomes 291 Defects in Lipoprotein Metabolism Can Lead to Elevated Serum Cholesterol 291

9.6

Lipids Form Ordered Structures Spontaneously in Water 302 The Fluid Mosaic Model Describes Membrane Dynamics 304

How Is Cholesterol Synthesized? 282 Mevalonate Is Synthesized from Acetyl-CoA via HMG-CoA Synthase 282 A Thiolase Brainteaser Asks Why Thiolase Cannot Be Used in Fatty Acid Synthesis 283 Squalene Is Synthesized from Mevalonate 283 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: The Long Search for the Route of Cholesterol Biosynthesis 285 HUMAN BIOCHEMISTRY: Statins Lower Serum Cholesterol Levels 286 Conversion of Lanosterol to Cholesterol Requires Twenty Additional Steps 286

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10.10

How Is Secondary Active Transport Driven by Ion Gradients? 343 Na+ and H+ Drive Secondary Active Transport 343 AcrB Is a Secondary Active Transport System 343

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SUMMARY 345

HUMAN BIOCHEMISTRY: Cancer, Oncogenes, and Tumour Suppressor Genes 371 Inositol Phospholipid Breakdown Yields Inositol-1,4,5-Trisphosphate and Diacylglycerol 372 Activation of Phospholipase C Is Mediated by G Proteins or by Tyrosine Kinases 372 Phosphatidylcholine, Sphingomyelin, and Glycosphingolipids Also Generate Second Messengers 373 Calcium Is a Second Messenger 373 Intracellular Calcium-Binding Proteins Mediate the Calcium Signal 374 HUMAN BIOCHEMISTRY: PI Metabolism and the Pharmacology of Li! 375 Calmodulin Target Proteins Possess a Basic Amphiphilic Helix 375

PROBLEMS 346 FURTHER READING 347

11 The Reception and Transmission of Extracellular Information 349

BIOCHEMISTRY: A CANADIAN CONTEXT: Dr. Tony

Pawson 351

11.1

What Are Hormones? 351 Steroid Hormones Act in Two Ways 351 Polypeptide Hormones Share Similarities of Synthesis and Processing 352

11.2

What Is Signal Transduction? 352 Many Signalling Pathways Involve Enzyme Cascades 353 Signalling Pathways Connect Membrane Interactions with Events in the Nucleus 353 Signalling Pathways Depend on Multiple Molecular Interactions 353

11.3

How Are Receptor Signals Transduced? 367 GPCR Signals Are Transduced by G Proteins 367 Cyclic AMP Is a Second Messenger 368 cAMP Activates Protein Kinase A 369 Ras and Other Small GTP-Binding Proteins Are Proto-oncogene Products 369 G Proteins Are Universal Signal Transducers 370 Specific Phospholipases Release Second Messengers 370

How Do Effectors Convert the Signals to Actions in the Cell? 376 Protein Kinase A Is a Paradigm of Kinases 376 A DEEPER LOOK: Mitogen-Activated Protein Kinases and Phosphorelay Systems 377 Protein Kinase C Is a Family of Isozymes 378 Protein Tyrosine Kinase pp60c-src Is Regulated by Phosphorylation/Dephosphorylation 378 Protein Tyrosine Phosphatase SHP-2 Is a Non-receptor Tyrosine Phosphatase 379

How Do Signal-Transducing Receptors Respond to the Hormonal Message? 355 The G-Protein–Coupled Receptors Are 7-TMS Integral Membrane Proteins 357 The Single TMS Receptors Are Guanylyl Cyclases or Tyrosine Kinases 357 RTKs and RGCs Are Membrane-Associated Allosteric Enzymes 358 EGF Receptor Is Activated by Ligand-Induced Dimerization 359 EGF Receptor Activation Forms an Asymmetric Tyrosine Kinase Dimer 359 The Insulin Receptor Mediates Several Signalling Pathways 361 The Insulin Receptor Adopts a Folded Dimeric Structure in the Membrane 362 Autophosphorylation of the Insulin Receptor Kinase Opens the Active Site 363 Receptor Guanylyl Cyclases Mediate Effects of Natriuretic Hormones 363 A Symmetric Dimer Binds an Asymmetric Peptide Ligand 364 Non-receptor Tyrosine Kinases Are Typified by pp60src 365 Soluble Guanylyl Cyclases Are Receptors for Nitric Oxide 366 A DEEPER LOOK: Nitric Oxide, Nitroglycerine, and Alfred Nobel 366

11.4

11.5

11.6

How Are Signalling Pathways Organized and Integrated? 379 GPCRs Can Signal Through G-Protein–Independent Pathways 380 G Protein Signalling Is Modulated by RGS/GAPs 380 GPCR Desensitization Leads to New Signalling Pathways 383 A DEEPER LOOK: Whimsical Names for Proteins and Genes 384 Receptor Responses Can Be Coordinated by Transactivation 384 Signals from Multiple Pathways Can Be Integrated 384

11.7

How Do Neurotransmission Pathways Control the Function of Sensory Systems? 386 Nerve Impulses Are Carried by Neurons 386 Ion Gradients Are the Source of Electrical Potentials in Neurons 387 Action Potentials Carry the Neural Message 387 The Action Potential Is Mediated by the Flow of Na" and K" Ions 387 Neurons Communicate at the Synapse 389 Communication at Cholinergic Synapses Depends upon Acetylcholine 390 There Are Two Classes of Acetylcholine Receptors 390 The Nicotinic Acetylcholine Receptor Is a LigandGated Ion Channel 391 Acetylcholinesterase Degrades Acetylcholine in the Synaptic Cleft 392 A DEEPER LOOK: Tetrodotoxin and Saxitoxin Are Na! Channel Toxins 393 NEL

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Detailed Contents

Muscarinic Receptor Function Is Mediated by G Proteins 394 Other Neurotransmitters Can Act Within Synaptic Junctions 395 Glutamate and Aspartate Are Excitatory Amino Acid Neurotransmitters 395 G-Aminobutyric Acid and Glycine Are Inhibitory Neurotransmitters 395 HUMAN BIOCHEMISTRY: The Biochemistry of Neurological Disorders 397 The Catecholamine Neurotransmitters Are Derived from Tyrosine 398 Various Peptides Also Act as Neurotransmitters 399

The Michaelis Constant, Km, Is Defined as (k"1!k2)/k1 413 When [S] # Km, v # Vmax/2 413 Plots of v Versus [S] Illustrate the Relationships between Vmax, Km, and Reaction Order 414 Turnover Number Defines the Activity of One Enzyme Molecule 415 The Ratio kcat/km Defines the Catalytic Efficiency of an Enzyme 415 Linear Plots Can Be Derived from the Michaelis– Menten Equation 416 Non-linear Lineweaver–Burk or Hanes–Woolf Plots Are a Property of Regulatory Enzymes 418 Enzymatic Activity Is Strongly Influenced by pH 418 A DEEPER LOOK: An Example of the Effect of Amino Acid Substitutions on Km and kcat: Wild-Type and Mutant Forms of Human Sulfite Oxidase 418 The Response of Enzymatic Activity to Temperature Is Complex 419

SUMMARY 399 PROBLEMS 400 FURTHER READING 401

PART II

Protein Dynamics

12 Enzymes—Kinetics and Specificity

12.4

What Characteristic Features Define Enzymes? 404 BIOCHEMISTRY: A CANADIAN CONTEXT: The

Discovery of Ribozymes 404 Catalytic Power Is Defined as the Ratio of the EnzymeCatalyzed Rate of a Reaction to the Uncatalyzed Rate 405 Specificity Is the Term Used to Define the Selectivity of Enzymes for Their Substrates 405 Regulation of Enzyme Activity Ensures That the Rate of Metabolic Reactions Is Appropriate to Cellular Requirements 405 Enzyme Nomenclature Provides a Systematic Way of Naming Metabolic Reactions 406 Coenzymes and Cofactors Are Non-protein Components Essential to Enzyme Activity 407

12.2

12.3

12.5

12.6

How Can Enzymes Be So Specific? 431 The “Lock and Key” Hypothesis Was the First Explanation for Specificity 431 The “Induced Fit” Hypothesis Provides a More Accurate Description of Specificity 431 “Induced Fit” Favours Formation of the Transition State 432 Specificity and Reactivity 432

What Equations Define the Kinetics of Enzyme-Catalyzed Reactions? 411 The Substrate Binds at the Active Site of an Enzyme 411 The Michaelis–Menten Equation Is the Fundamental Equation of Enzyme Kinetics 411 Assume That [ES] Remains Constant During an Enzymatic Reaction 412 Assume That Velocity Measurements Are Made Immediately After Adding S 412

What Is the Kinetic Behaviour of Enzymes Catalyzing Bimolecular Reactions? 425 HUMAN BIOCHEMISTRY: Viagra—An Unexpected Outcome in a Program of Drug Design 426 The Conversion of AEB to PEQ Is the Rate-Limiting Step in Random Single-Displacement Reactions 426 In an Ordered Single-Displacement Reaction, the Leading Substrate Must Bind First 427 Double-Displacement (Ping-Pong) Reactions Proceed via Formation of a Covalently Modified Enzyme Intermediate 428 Exchange Reactions Are One Way to Diagnose Bisubstrate Mechanisms 429 Multisubstrate Reactions Can Also Occur in Cells 430

Can the Rate of an Enzyme-Catalyzed Reaction Be Defined in a Mathematical Way? 408 Chemical Kinetics Provides a Foundation for Exploring Enzyme Kinetics 408 Bimolecular Reactions Are Reactions Involving Two Reactant Molecules 409 Catalysts Lower the Free Energy of Activation for a Reaction 409 Decreasing ∆G‡ Increases Reaction Rate 410

What Can Be Learned from the Inhibition of Enzyme Activity? 419 Enzymes May Be Inhibited Reversibly or Irreversibly 419 Reversible Inhibitors May Bind at the Active Site or at Some Other Site 419 A DEEPER LOOK: The Equations of Competitive Inhibition 421 Enzymes Can Also Be Inhibited in an Irreversible Manner 423

403

Enzymes Are the Agents of Metabolic Function 404

12.1

xv

12.7

Are All Enzymes Proteins? 432 RNA Molecules That Are Catalytic Have Been Termed “Ribozymes” 432 Antibody Molecules Can Have Catalytic Activity 434

12.8

Is It Possible to Design an Enzyme to Catalyze Any Desired Reaction? 435

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CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: Drug Discovery and Personalized Medicine 436

As Product Accumulates, the Apparent Rate of the Enzymatic Reaction Decreases 478 Genetic Regulation of Enzyme Synthesis and Decay Determines the Amount of Enzyme Present at Any Moment 478 Enzyme Activity Can Be Regulated Allosterically 478 Allosteric Regulation of Phosphofructokinase 479 Enzyme Activity Can Be Regulated Through Covalent Modification 480 Regulation of Enzyme Activity Can Also Be Accomplished in Other Ways 480 Zymogens Are Inactive Precursors of Enzymes 481 Isozymes Are Enzymes with Slightly Different Subunits 481

SUMMARY 437 PROBLEMS 438 FURTHER READING 440

13 Mechanisms of Enzyme Action 13.1 13.2 13.3 13.4

442

What Are the Magnitudes of Enzyme-Induced Rate Accelerations? 442 What Role Does Transition-State Stabilization Play in Enzyme Catalysis? 444 How Does Destabilization of ES Affect Enzyme Catalysis? 445 How Tightly Do Transition-State Analogs Bind to the Active Site? 447

14.2

A DEEPER LOOK: Transition-State Analogs Make Our World Better 448

13.5

What Are the Mechanisms of Catalysis? 450 Enzymes Facilitate Formation of Near-Attack Conformations 450 A DEEPER LOOK: How to Read and Write Mechanisms 452 Covalent Catalysis 453 General Acid–Base Catalysis 454 Low-Barrier Hydrogen Bonds 455 Metal Ion Catalysis 456 A DEEPER LOOK: How Do Active-Site Residues Interact to Support Catalysis? 457 The Hydrolytic Mechanism of Lysozyme 457

13.6

SUMMARY 472 PROBLEMS 473 FURTHER READING 475

14 Enzyme Regulation 14.1

Regulatory Enzymes Have Certain Exceptional Properties 483

14.3

477

What Factors Influence Enzymatic Activity? 478 The Availability of Substrates and Cofactors Usually Determines How Fast the Reaction Goes 478

Can Allosteric Regulation Be Explained by Conformational Changes in Proteins? 484 The Symmetry Model for Allosteric Regulation Is Based on 2 Conformational States for a Protein 484 Conformational Changes 485 Changes in the Oligomeric State of a Protein Can Also Result in Allosteric Behaviour 485

14.4

What Kinds of Covalent Modification Regulate the Activity of Enzymes? 486 Covalent Modification Through Reversible Phosphorylation 486 Protein Kinases: Target Recognition and Intrasteric Control 487 Phosphorylation Is Not the Only Form of Covalent Modification That Regulates Protein Function 488

What Can Be Learned from Typical Enzyme Mechanisms? 459 Serine Proteases 459 The Digestive Serine Proteases 459 The Chymotrypsin Mechanism in Detail: Kinetics 460 The Serine Protease Mechanism in Detail: Events at the Active Site 460 The Aspartic Proteases 462 A DEEPER LOOK: Transition-State Stabilization in the Serine Proteases 464 The Mechanism of Action of Aspartic Proteases 465 The AIDS Virus HIV-1 Protease Is an Aspartic Protease 465 Chorismate Mutase: A Model for Understanding Catalytic Power and Efficiency 466 HUMAN BIOCHEMISTRY: Protease Inhibitors Give Life to AIDS Patients 468 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: Caught in the Act! A High-Energy Intermediate in the Phosphoglucomutase Reaction 470

What Are the General Features of Allosteric Regulation? 483

14.5

Is the Activity of Some Enzymes Controlled by Both Allosteric Regulation and Covalent Modification? 489 The Pyruvate Dehydrogenase Reaction Links Glycolysis to the Citric Acid Cycle 489 Pyruvate Dehydrogenase Activity Is Regulated Both Allosterically and Covalently 489

14.6

Is There an Example in Nature of the Relationship between Quaternary Structure and the Emergence of Allosteric Properties? Hemoglobin and Myoglobin— Paradigms of Protein Structure and Function 490 The Comparative Biochemistry of Myoglobin and Hemoglobin Reveals Insights into Allostery 491 Myoglobin Is an Oxygen-Storage Protein 492 O2 Binds to the Mb Heme Group 492 O2 Binding Alters Mb Conformation 492 Cooperative Binding of Oxygen by Hemoglobin Has Important Physiological Significance 493 Hemoglobin Has an a2b2 Tetrameric Structure 493 Oxygenation Markedly Alters the Quaternary Structure of Hb 493 Movement of the Heme Iron Ion by Less Than 0.04 nm Induces the Conformational Change in Hemoglobin 493 NEL

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A DEEPER LOOK: The Oxygen-Binding Curves of Myoglobin and Hemoglobin 494 A DEEPER LOOK: The Physiological Significance of the Hb:O2 Interaction 496 The Oxy and Deoxy Forms of Hemoglobin Represent 2 Different Conformational States 496 The Allosteric Behaviour of Hemoglobin Has Both Symmetry (MWC) Model and Sequential (KNF) Model Components 496 H+ Promotes the Dissociation of Oxygen from Hemoglobin 497 CO2 Also Promotes the Dissociation of O2 from Hemoglobin 497 A DEEPER LOOK: Changes in the Heme Iron upon O2 Binding 497 2,3-Bisphosphoglycerate Is an Important Allosteric Effector for Hemoglobin 498 BPG Binding to Hb Has Important Physiological Significance 499 Fetal Hemoglobin Has a Higher Affinity for O2 Because It Has a Lower Affinity for BPG 499 HUMAN BIOCHEMISTRY: Hemoglobin and Nitric Oxide 500 Sickle-Cell Anemia Is Characterized by Abnormal Red Blood Cells 501 Sickle-Cell Anemia Is a Molecular Disease 501

HUMAN BIOCHEMISTRY: Effectors of Microtubule Polymerization as Therapeutic Agents 519 Dyneins Move Organelles in a Plus-to-Minus Direction; Kinesins, in a Minus-to-Plus Direction—Mostly 520 Cytoskeletal Motors Are Highly Processive 521 ATP Binding and Hydrolysis Drive Hand-over-Hand Movement of Kinesin 521 The Conformation Change That Leads to Movement Is Different in Myosins and Dyneins 522

15.4

How Do Molecular Motors Unwind DNA? 522 Negative Cooperativity Facilitates Hand-over-Hand Movement 525 Papillomavirus E1 Helicase Moves along DNA on a Spiral Staircase 526

15.5

How Do Bacterial Flagella Use a Proton Gradient to Drive Rotation? 527 The Flagellar Rotor Is a Complex Structure 528 Gradients of H" and Na" Drive Flagellar Rotors 528 The Flagellar Rotor Self-Assembles in a Spontaneous Process 530 Flagellar Filaments Are Composed of Protofilaments of Flagellin 531 Motor Reversal Involves Conformational Switching of Motor and Filament Proteins 531 SUMMARY 533 PROBLEMS 533

SUMMARY 502

FURTHER READING 534

PROBLEMS 503 FURTHER READING 504

15 Molecular Motors 15.1 15.2

505

What Is a Molecular Motor? 505 What Is the Molecular Mechanism of Muscle Contraction? 506 Muscle Contraction Is Triggered by Ca2" Release from Intracellular Stores 506 The Molecular Structure of Skeletal Muscle Is Based on Actin and Myosin 506 HUMAN BIOCHEMISTRY: Smooth Muscle Effectors Are Useful Drugs 507 HUMAN BIOCHEMISTRY: The Molecular Defect in Duchenne Muscular Dystrophy Involves an ActinAnchoring Protein 510 The Mechanism of Muscle Contraction Is Based on Sliding Filaments 511 A DEEPER LOOK: The P-Loop: A Common Motif in Enzymes That Hydrolyze Nucleoside Triphosphates 512 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: Molecular “Tweezers” of Light Take the Measure of a Muscle Fibre’s Force 514

15.3

What Are the Molecular Motors That Orchestrate the Mechanochemistry of Microtubules? 515 Filaments of the Cytoskeleton Are Highways That Move Cellular Cargo 515 Three Classes of Motor Proteins Move Intracellular Cargo 516

PART III

Metabolism and Its Regulation

16 Nutrition and the Organization of Metabolism 16.1

537

Is Metabolism Similar in Different Organisms? 538 Living Things Exhibit Metabolic Diversity 538 Oxygen Is Essential to Life for Aerobes 538 A DEEPER LOOK: Calcium Carbonate—A Biological Sink for CO2 539 The Flow of Energy in the Biosphere and the Carbon and Oxygen Cycles Are Intimately Related 539

16.2

What Can Be Learned from Metabolic Maps? 539 The Metabolic Map Can Be Viewed as a Set of Dots and Lines 539 Alternative Models Can Provide New Insights into Pathways 540 Multi-enzyme Systems May Take Different Forms 542

16.3

How Do Anabolic and Catabolic Processes Form the Core of Metabolic Pathways? 543 Anabolism Is Biosynthesis 543 Anabolism and Catabolism Are Not Mutually Exclusive 544 The Pathways of Catabolism Converge to a Few End Products 544 Anabolic Pathways Diverge, Synthesizing an Astounding Variety of Biomolecules from a Limited Set of Building Blocks 546

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Amphibolic Intermediates Play Dual Roles 546 Corresponding Pathways of Catabolism and Anabolism Differ in Important Ways 546 ATP Serves in a Cellular Energy Cycle 546 NAD! Collects Electrons Released in Catabolism 547 NADPH Provides the Reducing Power for Anabolic Processes 548 Coenzymes and Vitamins Provide Unique Chemistry and Essential Nutrients to Pathways 549

16.4

16.6

SUMMARY 558 PROBLEMS 559 FURTHER READING 559

17 Glycolysis 17.3

Reaction 1: Glucose Is Phosphorylated by Hexokinase or Glucokinase—The First Priming Reaction 565 Reaction 2: Phosphoglucoisomerase Catalyzes the Isomerization of Glucose-6-Phosphate 568 Reaction 3: ATP Drives a Second Phosphorylation by Phosphofructokinase—The Second Priming Reaction 569 A DEEPER LOOK: Phosphoglucoisomerase: A Moonlighting Protein 570 Reaction 4: Cleavage by Fructose Bisphosphate Aldolase Creates Two, Three-Carbon Intermediates 571 Reaction 5: Triose Phosphate Isomerase Completes the First Phase of Glycolysis 572

17.4

17.6 17.7

What Are the Chemical Principles and Features of the Second Phase of Glycolysis? 573 Reaction 6: Glyceraldehyde-3-Phosphate Dehydrogenase Creates a High-Energy Intermediate 573

How Do Cells Regulate Glycolysis? 581 Are Substrates Other Than Glucose Used in Glycolysis? 581 HUMAN BIOCHEMISTRY: Tumour Diagnosis Using Positron Emission Tomography (PET) 582 Mannose Enters Glycolysis in Two Steps 583 Galactose Enters Glycolysis via the Leloir Pathway 583 An Enzyme Deficiency Causes Lactose Intolerance 585 Glycerol Can Also Enter Glycolysis 585 HUMAN BIOCHEMISTRY: Lactose—From Mother’s Milk to Yogurt—and Lactose Intolerance 585

17.8

How Do Cells Respond to Hypoxic Stress? 586 SUMMARY 587 PROBLEMS 588 FURTHER READING 589

18 The Tricarboxylic Acid Cycle 18.1

561

What Are the Essential Features of Glycolysis? 562 Why Are Coupled Reactions Important in Glycolysis? 563 What Are the Chemical Principles and Features of the First Phase of Glycolysis? 564

What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis? 580 Anaerobic Metabolism of Pyruvate Leads to Lactate or Ethanol 580 Lactate Accumulates under Anaerobic Conditions in Animal Tissues 581

What Can the Metabolome Tell Us about a Biological System? 555 What Food Substances Form the Basis of Human Nutrition? 557 Humans Require Protein 557 Carbohydrates Provide Metabolic Energy 557 A DEEPER LOOK: A Popular Fad Diet—Low Carbohydrates, High Protein, High Fat 557 Lipids Are Essential, But in Moderation 558 Fibre May Be Soluble or Insoluble 558

17.1 17.2

17.5

What Experiments Can Be Used to Elucidate Metabolic Pathways? 550 Mutations Create Specific Metabolic Blocks 551 Isotopic Tracers Can Be Used as Metabolic Probes 551 NMR Spectroscopy Is a Non-invasive Metabolic Probe 552 Metabolic Pathways Are Compartmentalized within Cells 553

16.5

Reaction 7: Phosphoglycerate Kinase Is the BreakEven Reaction 575 Reaction 8: Phosphoglycerate Mutase Catalyzes a Phosphoryl Transfer 576 Reaction 9: Dehydration by Enolase Creates PEP 577 Reaction 10: Pyruvate Kinase Yields More ATP 577

591

What Is the Chemical Logic of the TCA Cycle? 593 The TCA Cycle Provides a Chemically Feasible Way of Cleaving a 2-Carbon Compound 593

18.2

How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA? 595 A DEEPER LOOK: The Coenzymes of the Pyruvate Dehydrogenase Complex 597

18.3

How Are 2 CO2 Molecules Produced from Acetyl-CoA? 600 The Citrate Synthase Reaction Initiates the TCA Cycle 600 Isocitrate Dehydrogenase Catalyzes the First Oxidative Decarboxylation in the Cycle 602 a-Ketoglutarate Dehydrogenase Catalyzes the Second Oxidative Decarboxylation of the TCA Cycle 603

18.4

How Is Oxaloacetate Regenerated to Complete the TCA Cycle? 604 Succinyl-CoA Synthetase Catalyzes Substrate Level Phosphorylation 604 The First 5 Steps of the TCA Cycle Produce NADH, CO2, GTP (ATP), and Succinate 605 Succinate Dehydrogenase Is FAD Dependent 605 Fumarase Catalyzes the Trans-Hydration of Fumarate to Form l-Malate 606 Malate Dehydrogenase Completes the Cycle by Oxidizing Malate to Oxaloacetate 606 NEL

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Detailed Contents

18.5

What Are the Energetic Consequences of the TCA Cycle? 607

HUMAN BIOCHEMISTRY: Solving a Medical Mystery Revolutionized Our Treatment of Parkinson’s Disease 631 Complex II Oxidizes Succinate and Reduces Coenzyme Q 631 Complex III Mediates Electron Transport from Coenzyme Q to Cytochrome c 633 Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side 635 Proton Transport across Cytochrome c Oxidase Is Coupled to Oxygen Reduction 637 The Four Electron-Transport Complexes Are Independent 638 Electron Transfer Energy Stored in a Proton Gradient: The Mitchell Hypothesis 639

The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle 607 A DEEPER LOOK: Steric Preferences in NAD!Dependent Dehydrogenases 608

18.6

Can the TCA Cycle Provide Intermediates for Biosynthesis? 610 HUMAN BIOCHEMISTRY: Mitochondrial Diseases Are Rare 611

18.7

What Are the Anaplerotic, or “Filling-Up,” Reactions? 611 A DEEPER LOOK: Fool’s Gold and the Reductive Citric Acid Cycle—The First Metabolic Pathway? 612

18.8

How Is the TCA Cycle Regulated? 613 Pyruvate Dehydrogenase Is Regulated by Phosphorylation/Dephosphorylation 613 Isocitrate Dehydrogenase Is Strongly Regulated 615

18.9

Can Any Organisms Use Acetate as Their Sole Carbon Source? 615

19.4 19.5

SUMMARY 618 PROBLEMS 619 FURTHER READING 620

19 Electron Transport and Oxidative Phosphorylation 621

Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur? 622 Mitochondrial Functions Are Localized in Specific Compartments 622 The Mitochondrial Matrix Contains the Enzymes of the TCA Cycle 623

19.2

What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? 623

19.6 19.7

Standard Reduction Potentials Are Measured in Reaction Half-Cells 624 e0’ Values Can Be Used to Predict the Direction of Redox Reactions 625 e0’ Values Can Be Used to Analyze Energy Changes in Redox Reactions 626 The Reduction Potential Depends on Concentration 626

19.3

How Is the Electron-Transport Chain Organized? 627 The Electron-Transport Chain Can Be Isolated in Four Complexes 628 Complex I Oxidizes NADH and Reduces Coenzyme Q 629

What Are the Thermodynamic Implications of Chemiosmotic Coupling? 640 How Does a Proton Gradient Drive the Synthesis of ATP? 641 ATP Synthase Is Composed of F1 and F0 641 The Catalytic Sites of ATP Synthase Adopt Three Different Conformations 643 Boyer’s 18O Exchange Experiment Identified the Energy-Requiring Step 643 Boyer’s Binding Change Mechanism Describes the Events of Rotational Catalysis 644 Proton Flow through F0 Drives Rotation of the Motor and Synthesis of ATP 644 Racker and Stoeckenius Confirmed the Mitchell Model in a Reconstitution Experiment 646 Inhibitors of Oxidative Phosphorylation Reveal Insights about the Mechanism 646 Uncouplers Disrupt the Coupling of Electron Transport and ATP Synthase 647 ATP–ADP Translocase Mediates the Movement of ATP and ADP across the Mitochondrial Membrane 648 HUMAN BIOCHEMISTRY: Endogenous Uncouplers Enable Organisms to Generate Heat 649

The Glyoxylate Cycle Operates in Specialized Organelles 616 Isocitrate Lyase Short-Circuits the TCA Cycle by Producing Glyoxylate and Succinate 616 The Glyoxylate Cycle Helps Plants Grow in the Dark 617 Glyoxysomes Must Borrow 3 Reactions from Mitochondria 617

19.1

xix

What Is the P/O Ratio for Mitochondrial Oxidative Phosphorylation? 649 How Are the Electrons of Cytosolic NADH Fed into Electron Transport? 650 The Glycerophosphate Shuttle Ensures Efficient Use of Cytosolic NADH 651 The Malate–Aspartate Shuttle Is Reversible 651 The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used 652 3.5 Billion Years of Evolution Have Resulted in a Very Efficient System 653

19.8

How Do Mitochondria Mediate Apoptosis? 653 Cytochrome c Triggers Apoptosome Assembly 654 SUMMARY 655 PROBLEMS 656 FURTHER READING 658

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xx

Detailed Contents

20 Photosynthesis 20.1

Cyclic Photophosphorylation Generates ATP but Not NADPH or O2 679

659

What Are the General Properties of Photosynthesis? 660

20.7

Photosynthesis Occurs in Membranes 660 Photosynthesis Consists of Both Light Reactions and Dark Reactions 662 Water Is the Ultimate e" Donor for Photosynthetic NADP! Reduction 663

20.2

Ribulose-1,5-Bisphosphate Is the CO2 Acceptor in CO2 Fixation 680 2-Carboxy-3-Keto-Arabinitol Is an Intermediate in the Ribulose-1,5-Bisphosphate Carboxylase Reaction 681 Ribulose-1,5-Bisphosphate Carboxylase Exists in Inactive and Active Forms 681 CO2 Fixation into Carbohydrate Proceeds via the Calvin–Benson Cycle 682 The Enzymes of the Calvin Cycle Serve 3 Metabolic Purposes 682 The Calvin Cycle Reactions Can Account for Net Hexose Synthesis 683 The Carbon Dioxide Fixation Pathway Is Indirectly Activated by Light 683 Light Induces Movement of Mg2! Ions from the Thylakoid Vesicles into the Stroma 686 Protein–Protein Interactions Mediated by an Intrinsically Unstructured Protein Also Regulate Calvin–Benson Cycle Activity 686

How Is Solar Energy Captured by Chlorophyll? 663 Chlorophylls and Accessory Light-Harvesting Pigments Absorb Light of Different Wavelengths 664 The Light Energy Absorbed by Photosynthetic Pigments Has Several Possible Fates 665 The Transduction of Light Energy into Chemical Energy Involves Oxidation–Reduction 666 Photosynthetic Units Consist of Many Chlorophyll Molecules but Only a Single Reaction Centre 667

20.3

What Kinds of Photosystems Are Used to Capture Light Energy? 667 Chlorophyll Exists in Plant Membranes in Association with Proteins 668 PSI and PSII Participate in the Overall Process of Photosynthesis 668 The Pathway of Photosynthetic Electron Transfer Is Called the Z Scheme 668 Oxygen Evolution Requires the Accumulation of 4 Oxidizing Equivalents in PSII 670 Electrons Are Taken from H2O to Replace Electrons Lost from P680 670 Electrons from PSII Are Transferred to PSI via the Cytochrome b6f Complex 670 Plastocyanin Transfers Electrons from the Cytochrome b6f Complex to PSI 671

20.4

What Is the Molecular Architecture of Photosynthetic Reaction Centres? 671 The R. viridis Photosynthetic Reaction Centre Is an Integral Membrane Protein 672 Photosynthetic Electron Transfer by the R. viridis Reaction Centre Leads to ATP Synthesis 672 The Molecular Architecture of PSII Resembles the R. viridis Reaction Centre Architecture 673 How Does PSII Generate O2 from H2O? 674 The Molecular Architecture of PSI Resembles the R. viridis Reaction Centre and PSII Architecture 675 How Do Green Plants Carry Out Photosynthesis? 675

20.5

20.8

SUMMARY 689 PROBLEMS 690 FURTHER READING 691

21 Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway 693 21.1

What Is Gluconeogenesis, and How Does It Operate? 694 BIOCHEMISTRY: A CANADIAN CONTEXT:

Determining the Kinetic Mechanism of Phosphoenolpyruvate Carboxykinase 694 The Substrates for Gluconeogenesis Include Pyruvate, Lactate, and Amino Acids 695 Nearly All Gluconeogenesis Occurs in the Liver and Kidneys in Animals 695 HUMAN BIOCHEMISTRY: The Chemistry of Glucose Monitoring Devices 695 Gluconeogenesis Is Not Merely the Reverse of Glycolysis 696 Gluconeogenesis: Something Borrowed, Something New 696 Four Reactions Are Unique to Gluconeogenesis 696 HUMAN BIOCHEMISTRY: Gluconeogenesis Inhibitors and Other Diabetes Therapy Strategies 701

What Is the Quantum Yield of Photosynthesis? 677

How Does Light Drive the Synthesis of ATP? 677 The Mechanism of Photophosphorylation Is Chemiosmotic 678 CF1CF0–ATP Synthase Is the Chloroplast Equivalent of the Mitochondrial F1F0–ATP Synthase 678 Photophosphorylation Can Occur in Either a Non-cyclic or a Cyclic Mode 678

How Does Photorespiration Limit CO2 Fixation? 686 Tropical Grasses Use the Hatch–Slack Pathway to Capture Carbon Dioxide for CO2 Fixation 686 Cacti and Other Desert Plants Capture CO2 at Night 689

Calculation of the Photosynthetic Energy Requirements for Hexose Synthesis Depends on H!/hn and ATP/H! Ratios 677

20.6

How Is Carbon Dioxide Used to Make Organic Molecules? 680

21.2

How Is Gluconeogenesis Regulated? 701 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: The Pioneering Studies of Carl and Gerty Cori 702

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Detailed Contents

Gluconeogenesis Is Regulated by Allosteric and Substrate-Level Control Mechanisms 703 Substrate Cycles Provide Metabolic Control Mechanisms 705

21.3

22 Fatty Acid Catabolism 22.1

How Is Glycogen Synthesized? 708 Glucose Units Are Activated for Transfer by Formation of Sugar Nucleotides 708 UDP–Glucose Synthesis Is Driven by Pyrophosphate Hydrolysis 708 Glycogen Synthase Catalyzes Formation of a(1→4) Glycosidic Bonds in Glycogen 709 HUMAN BIOCHEMISTRY: Advanced Glycation End Products: A Serious Complication of Diabetes 710 Glycogen Branching Occurs by Transfer of Terminal Chain Segments 710

21.5

21.6

22.2

Coenzyme A Activates Fatty Acids for Degradation 740 Carnitine Carries Fatty Acyl Groups Across the Inner Mitochondrial Membrane 741 b-Oxidation Involves a Repeated Sequence of 4 Reactions 741 HUMAN BIOCHEMISTRY: Carnitine Deficiency 742 Repetition of the b-Oxidation Cycle Yields a Succession of Acetate Units 746 Complete b-Oxidation of 1 Palmitic Acid Yields 106 Molecules of ATP 746 Migratory Birds Travel Long Distances on Energy from Fatty Acid Oxidation 747 HUMAN BIOCHEMISTRY: Exercise Can Reverse the Consequences of Metabolic Syndrome 748 Fatty Acid Oxidation Is an Important Source of Metabolic Water for Some Animals 748

22.3

22.4

FURTHER READING 733

How Are Unsaturated Fatty Acids Oxidized? 752 An Isomerase and a Reductase Facilitate the b-Oxidation of Unsaturated Fatty Acids 752 A DEEPER LOOK: Can Natural Antioxidants in Certain Foods Improve Fat Metabolism? 752 Degradation of Polyunsaturated Fatty Acids Requires 2,4-Dienoyl-CoA Reductase 753

22.5

Are There Other Ways to Oxidize Fatty Acids? 753 Peroxisomal b-Oxidation Requires FAD-Dependent Acyl-CoA Oxidase 753 Branched-Chain Fatty Acids Are Degraded via a-Oxidation 753 v-Oxidation of Fatty Acids Yields Small Amounts of Dicarboxylic Acids 753 HUMAN BIOCHEMISTRY: Refsum’s Disease Is a Result of Defects in a-Oxidation 755

SUMMARY 731 PROBLEMS 731

How Are Odd-Carbon Fatty Acids Oxidized? 749 b-Oxidation of Odd-Carbon Fatty Acids Yields Propionyl-CoA 749 A B12-Catalyzed Rearrangement Yields Succinyl-CoA from l-Methylmalonyl-CoA 750 Net Oxidation of Succinyl-CoA Requires Conversion to Acetyl-CoA 750 A DEEPER LOOK: The Activation of Vitamin B12 751

Can Glucose Provide Electrons for Biosynthesis? 722 The Pentose Phosphate Pathway Operates Mainly in Liver and Adipose Cells 722 The Pentose Phosphate Pathway Begins with Two Oxidative Steps 722 There Are Four Non-oxidative Reactions in the Pentose Phosphate Pathway 722 HUMAN BIOCHEMISTRY: Aldose Reductase and Diabetic Cataract Formation 725 Utilization of Glucose-6-P Depends on the Cell’s Need for ATP, NADPH, and Ribose-5-P 728 Xylulose-5-Phosphate Is a Metabolic Regulator 730

How Are Fatty Acids Broken Down? 739 Knoop Elucidated the Essential Feature of b-Oxidation 739

How Is Glycogen Metabolism Controlled? 711 Glycogen Metabolism Is Highly Regulated 711 Glycogen Synthase Is Regulated by Covalent Modification 711 The Glycogen Phosphorylase Reaction Converts Glycogen into Readily Usable Fuel in the Form of Glucose-1-Phosphate 712 Glycogen Phosphorylase Is a Homodimer 713 Glycogen Phosphorylase Activity Is Regulated Allosterically 713 Covalent Modification of Glycogen Phosphorylase Trumps Allosteric Regulation 716 Enzyme Cascades Regulate Glycogen Phosphorylase Covalent Modification 716 Hormones Regulate Glycogen Synthesis and Degradation 717 A DEEPER LOOK: Carbohydrate Utilization in Exercise 718 HUMAN BIOCHEMISTRY: Von Gierke’s Disease: A Glycogen-Storage Disease 719

735

How Are Fats Mobilized from Dietary Intake and Adipose Tissue? 736 Modern Diets Are Often High in Fat 736 Triacylglycerols Are a Major Form of Stored Energy in Animals 736 Hormones Trigger the Release of Fatty Acids from Adipose Tissue 736 Degradation of Dietary Fatty Acids Occurs Primarily in the Duodenum 737

How Are Glycogen and Starch Catabolized in Animals? 705 Dietary Starch Breakdown Provides Metabolic Energy 705 Metabolism of Tissue Glycogen Is Regulated 706

21.4

xxi

22.6

What Are Ketone Bodies, and What Role Do They Play in Metabolism? 756

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Detailed Contents

A DEEPER LOOK: Amino Acid Biosynthesis Inhibitors as Herbicides 795 A DEEPER LOOK: Intramolecular Tunnels Connect Distant Active Sites in Some Enzymes 796 Histidine Biosynthesis and Purine Biosynthesis Are Connected by Common Intermediates 796

Ketone Bodies Are a Significant Source of Fuel and Energy for Certain Tissues 756 HUMAN BIOCHEMISTRY: Large Amounts of Ketone Bodies Are Produced in Diabetes Mellitus 757 SUMMARY 757 PROBLEMS 758 FURTHER READING 759

23.5

23 Nitrogen Acquisition and Amino Acid

The Twenty Common Amino Acids Are Degraded by Twenty Different Pathways That Converge to Just Seven Metabolic Intermediates 798 A DEEPER LOOK: Histidine—A Clue to Understanding Early Evolution? 800 A DEEPER LOOK: The Serine Dehydratase Reaction: A b-Elimination 801 HUMAN BIOCHEMISTRY: Hereditary Defects in Phe Catabolism Underlie Alkaptonuria and Phenylketonuria 804 Animals Differ in the Form of Nitrogen That They Excrete 804

Metabolism 761 23.1

Which Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen? 762 Nitrogen Is Cycled between Organisms and the Inanimate Environment 762 Nitrate Assimilation Is the Principal Pathway for Ammonium Biosynthesis 763 Nitrate Reductase Contains Cytochrome b557 and Molybdenum Cofactor 763 Organisms Gain Access to Atmospheric N2 via the Pathway of Nitrogen Fixation 764

23.2

SUMMARY 804

What Is the Metabolic Fate of Ammonium? 768

PROBLEMS 805

The Major Pathways of Ammonium Assimilation Lead to Glutamine Synthesis 769

23.3

FURTHER READING 806

What Regulatory Mechanisms Act on Escherichia coli Glutamine Synthetase? 770 Glutamine Synthetase Is Allosterically Regulated 771 Glutamine Synthetase Is Regulated by Covalent Modification 771 Glutamine Synthetase Is Regulated through Gene Expression 773

23.4

How Does Amino Acid Catabolism Lead into Pathways of Energy Production? 798

PART IV

24 Nucleotides and Nucleic Acids 24.1

How Do Organisms Synthesize Amino Acids? 773 HUMAN BIOCHEMISTRY: Human Dietary Requirements for Amino Acids 775 Amino Acids Are Formed from a-Keto Acids by Transamination 775 A DEEPER LOOK: The Mechanism of the Aminotransferase (Transamination) Reaction 776 The Pathways of Amino Acid Biosynthesis Can Be Organized into Families 776 The a-Ketoglutarate Family of Amino Acids Includes Glu, Gln, Pro, Arg, and Lys 777 The Urea Cycle Acts to Excrete Excess N Through Arg Breakdown 778 A DEEPER LOOK: The Urea Cycle as Both an Ammonium and a Bicarbonate Disposal Mechanism 782 The Aspartate Family of Amino Acids Includes Asp, Asn, Lys, Met, Thr, and Ile 782 HUMAN BIOCHEMISTRY: Asparagine and Leukemia 783 The Pyruvate Family of Amino Acids Includes Ala, Val, and Leu 785 The 3-Phosphoglycerate Family of Amino Acids Includes Ser, Gly, and Cys 788 The Aromatic Amino Acids Are Synthesized from Chorismate 790 HUMAN BIOCHEMISTRY: Phenylketonuria and Aspartame 793

Nucleic Acids 808

What Are the Structure and Chemistry of Nitrogenous Bases? 809 Three Pyrimidines and 2 Purines are Commonly Found in Cells 810 The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature 810

24.2

What Are Nucleosides? 811 HUMAN BIOCHEMISTRY: Adenosine: A Nucleoside with Physiological Activity 812

24.3

What Are the Structure and Chemistry of Nucleotides? 813 Cyclic Nucleotides Are Cyclic Phosphodiesters 813 Nucleoside Diphosphates and Triphosphates Are Nucleotides with 2 or 3 Phosphate Groups 813 NDPs and NTPs Are Polyprotic Acids 814 Nucleoside 5$-Triphosphates Are Carriers of Chemical Energy 814

24.4

What Are Nucleic Acids? 815 The Base Sequence of a Nucleic Acid Is Its Distinctive Characteristic 815

24.5

What Are the Different Classes of Nucleic Acids? 816 The Fundamental Structure of DNA Is a Double Helix 817 Various Forms of RNA Serve Different Roles in Cells 819 A DEEPER LOOK: Do the Properties of DNA Invite Practical Applications? 820 NEL

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Detailed Contents

A DEEPER LOOK: The RNA World and Early Evolution 823 The Chemical Differences between DNA and RNA Have Biological Significance 824

24.6

Single-Stranded DNA Can Renature to Form DNA Duplexes 849 The Rate of DNA Renaturation Is an Index of DNA Sequence Complexity 849 A DEEPER LOOK: The Buoyant Density of DNA 849 Nucleic Acid Hybridization: Different DNA Strands of Similar Sequence Can Form Hybrid Duplexes 850

Are Nucleic Acids Susceptible to Hydrolysis? 825 RNA Is Susceptible to Hydrolysis by Base, but DNA Is Not 826 The Enzymes That Hydrolyze Nucleic Acids Are Phosphodiesterases 826 Nucleases Differ in Their Specificity for Different Forms of Nucleic Acid 827 Restriction Enzymes Are Nucleases That Cleave Double-Stranded DNA Molecules 827 Type II Restriction Endonucleases Are Useful for Manipulating DNA in the Lab 827 Restriction Endonucleases Can Be Used to Map the Structure of a DNA Fragment 828

25.4

25.5

25 Structure of Nucleic Acids 25.1

25.2

833

25.3

25.7

Can the Secondary Structure of DNA Be Denatured and Renatured? 847 Thermal Denaturation of DNA Can Be Observed by Changes in UV Absorbance 847 pH Extremes or Strong H-Bonding Solutes Also Denature DNA Duplexes 847

What Are the Secondary and Tertiary Structures of RNA? 858 Transfer RNA Adopts Higher-Order Structure through Intrastrand Base Pairing 861 Ribosomal RNA Also Adopts Higher-Order Structure through Intrastrand Base Pairing 863 Aptamers Are Oligonucleotides Specifically Selected for Their Ligand-Binding Ability 865

What Sorts of Secondary Structures Can DoubleStranded DNA Molecules Adopt? 837 Conformational Variation in Polynucleotide Strands 837 DNA Usually Occurs in the Form of Double-Stranded Molecules 838 Watson–Crick Base Pairs Have Virtually Identical Dimensions 838 The DNA Double Helix Is a Stable Structure 839 Double-Helical Structures Can Adopt a Number of Stable Conformations 839 A-Form DNA Is an Alternative Form of Right-Handed DNA 841 Z-DNA Is a Conformational Variation in the Form of a Left-Handed Double Helix 841 The Double Helix Is a Very Dynamic Structure 843 Alternative Hydrogen-Bonding Interactions Give Rise to Novel DNA Structures: Cruciforms, Triplexes, and Quadruplexes 845

Can Nucleic Acids Be Synthesized Chemically? 856 HUMAN BIOCHEMISTRY: Telomeres and Tumours 857 Phosphoramidite Chemistry Is Used to Form Oligonucleotides from Nucleotides 857 Genes Can Be Synthesized Chemically 858

How Do Scientists Determine the Primary Structure of Nucleic Acids? 834 The Nucleotide Sequence of DNA Can Be Determined from the Electrophoretic Migration of a Defined Set of Polynucleotide Fragments 834 Sanger’s Chain Termination, or Dideoxy, Method Uses DNA Replication to Generate a Defined Set of Polynucleotide Fragments 834 High-Throughput DNA Sequencing by the Light of Fireflies 836

What Is the Structure of Eukaryotic Chromosomes? 853 Nucleosomes Are the Fundamental Structural Unit in Chromatin 853 Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes 854 SMC Proteins Establish Chromosome Organization and Mediate Chromosome Dynamics 855

25.6

FURTHER READING 832

Can DNA Adopt Structures of Higher Complexity? 850 Supercoils Are One Kind of Structural Complexity in DNA 851

SUMMARY 831 PROBLEMS 831

xxiii

SUMMARY 867 PROBLEMS 868 FURTHER READING 869

26 Synthesis and Degradation of Nucleotides 26.1 26.2

871

Can Cells Synthesize Nucleotides? 872 How Do Cells Synthesize Purines? 872 IMP Is the Immediate Precursor to GMP and AMP 872 A DEEPER LOOK: Tetrahydrofolate and 1-Carbon Units 875 AMP and GMP Are Synthesized from IMP 876 HUMAN BIOCHEMISTRY: Folate Analogs as Antimicrobial and Anticancer Agents 877 The Purine Biosynthetic Pathway Is Regulated at Several Steps 877 ATP-Dependent Kinases Form Nucleoside Diphosphates and Triphosphates from the Nucleoside Monophosphates 878

26.3 26.4

Can Cells Salvage Purines? 879 How Are Purines Degraded? 880 The Major Pathways of Purine Catabolism Lead to Uric Acid 880

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Detailed Contents

HUMAN BIOCHEMISTRY: Lesch–Nyhan Syndrome— HGPRT Deficiency Leads to a Severe Clinical Disorder 880 The Purine Nucleoside Cycle in Skeletal Muscle Serves as an Anaplerotic Pathway 881 Xanthine Oxidase 882 Gout Is a Disease Caused by an Excess of Uric Acid 882 HUMAN BIOCHEMISTRY: Severe Combined Immunodeficiency Syndrome—A Lack of Adenosine Deaminase Is One Cause of this Inherited Disease 883 Different Animals Oxidize Uric Acid to Form Excretory Products 883

26.5

How Do Cells Synthesize Pyrimidines? 884 “Metabolic Channelling” by Multifunctional Enzymes of Mammalian Pyrimidine Biosynthesis 886 UMP Synthesis Leads to Formation of the 2 Most Prominent Ribonucleotides—UTP and CTP 887 Pyrimidine Biosynthesis Is Regulated at ATCase in Bacteria and at CPS-II in Animals 887 HUMAN BIOCHEMISTRY: Mammalian CPS-II Is Activated In Vitro by MAP Kinase and In Vivo by Epidermal Growth Factor 887

26.6 26.7

27.3

Reporter Gene Constructs Are Chimeric DNA Molecules Composed of Gene Regulatory Sequences Positioned Next to an Easily Expressible Gene Product 915 Specific Protein–Protein Interactions Can Be Identified Using the Yeast Two-Hybrid System 916

27.4

In Vitro Mutagenesis 919

27.5

FURTHER READING 896

SUMMARY 922 PROBLEMS 923 FURTHER READING 924

28 DNA Metabolism: Replication, Recombination, and Repair 926

BIOCHEMISTRY: A CANADIAN CONTEXT:

27 Recombinant DNA: Cloning and Creation of Chimeric Genes 897

BIOCHEMISTRY: A CANADIAN CONTEXT: Michael

Dr. Jack W. Szostak 927

28.1

What Does It Mean “to Clone”? 898 Plasmids Are Very Useful in Cloning Genes 898 Shuttle Vectors Are Plasmids That Can Propagate in Two Different Organisms 904 Artificial Chromosomes Can Be Created from Recombinant DNA 904

27.2

What Is a DNA Library? 904

How Is DNA Replicated? 927 DNA Replication Is Bidirectional 927 Replication Requires Unwinding of the DNA Helix 928 DNA Replication Is Semidiscontinuous 928 The Lagging Strand Is Formed from Okazaki Fragments 929

Smith 898

27.1

Is It Possible to Make Directed Changes in the Heredity of an Organism? 920 Human Gene Therapy Can Repair Genetic Deficiencies 920 HUMAN BIOCHEMISTRY: The Biochemical Defects in Cystic Fibrosis and ADA"SCID 922

A DEEPER LOOK: Fluoro-Substituted Analogs as Therapeutic Agents 892 HUMAN BIOCHEMISTRY: Fluoro-Substituted Pyrimidines in Cancer Chemotherapy, Fungal Infections, and Malaria 894 PROBLEMS 895

What Is the Polymerase Chain Reaction (PCR)? 917 Real-Time PCR 917

How Are Thymine Nucleotides Synthesized? 891

SUMMARY 894

Can the Cloned Genes in Libraries Be Expressed? 912 Expression Vectors Are Engineered So That the RNA or Protein Products of Cloned Genes Can Be Expressed 912

How Are Pyrimidines Degraded? 888 How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis? 888 E. Coli Ribonucleotide Reductase Has 3 Different Nucleotide-Binding Sites 889 Thioredoxin Provides the Reducing Power for Ribonucleotide Reductase 889 Both the Specificity and the Catalytic Activity of Ribonucleotide Reductase Are Regulated by Nucleotide Binding 890

26.8

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: Combinatorial Libraries 905 Genomic Libraries Are Prepared from the Total DNA in an Organism 905 Libraries Can Be Screened for the Presence of Specific Genes 906 Probes for Southern Hybridization Can Be Prepared in a Variety of Ways 906 cDNA Libraries Are DNA Libraries Prepared from mRNA 907 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: Identifying Specific DNA Sequences by Southern Blotting (Southern Hybridization) 908 DNA Microarrays (Gene Chips) Are Arrays of Different Oligonucleotides Immobilized on a Chip 910 HUMAN BIOCHEMISTRY: The Human Genome Project 911

28.2

What Are the Properties of DNA Polymerases? 930 E. coli Cells Have Several Different DNA Polymerases 930 The First DNA Polymerase Discovered Was E. coli DNA Polymerase I 930 NEL

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Detailed Contents

E. coli DNA Polymerase I Has 3 Active Sites on Its Single Polypeptide Chain 931 E. coli DNA Polymerase I Is Its Own Proofreader and Editor 931 E. coli DNA Polymerase III Holoenzyme Replicates the E. coli Chromosome 932 A DNA Polymerase III Holoenzyme Sits at Each Replication Fork 933 DNA Ligase Seals the Nicks between Okazaki Fragments 934 DNA Replication Terminates at the Ter Region 934 A DEEPER LOOK: A Mechanism for All Polymerases 935 DNA Polymerases Are Immobilized in Replication Factories 935

28.3

Transposons Are DNA Sequences That Can Move from Place to Place in the Genome 949 HUMAN BIOCHEMISTRY: The Breast Cancer Susceptibility Genes BRCA1 and BRCA2 Are Involved in DNA Damage Control and DNA Repair 950

28.8

28.9

28.5 28.6

SUMMARY 959

How Is DNA Replicated in Eukaryotic Cells? 936

How Are the Ends of Chromosomes Replicated? 939 How Are RNA Genomes Replicated? 940

PROBLEMS 960 FURTHER READING 960

PART V

Expression 963 29.1

How Are Genes Transcribed in Prokaryotes? 963 Prokaryotic RNA Polymerases Use Their Sigma Subunits to Identify Sites Where Transcription Begins 964 The Process of Transcription Has Four Stages 965 A DEEPER LOOK: Conventions Used in Expressing the Sequences of Nucleic Acids and Proteins 965 A DEEPER LOOK: DNA Footprinting: Identifying the Nucleotide Sequence in DNA Where a Protein Binds 968

How Is the Genetic Information Shuffled by Genetic Recombination? 941 A DEEPER LOOK: RNA as Genetic Material 941 General Recombination Requires Breakage and Reunion of DNA Strands 942 Homologous Recombination Proceeds According to the Holliday Model 942 The Enzymes of General Recombination Include RecA, RecBCD, RuvA, RuvB, and RuvC 943 The RecBCD Enzyme Complex Unwinds dsDNA and Cleaves Its Single Strands 943 The RecA Protein Can Bind ssDNA and Then Interact with Duplex DNA 946 RuvA, RuvB, and RuvC Proteins Resolve the Holliday Junction to Form the Recombination Products 947 A DEEPER LOOK: The 3 R’s of Genomic Manipulation: Replication, Recombination, and Repair 948 A DEEPER LOOK: “Knockout” Mice: A Method to Investigate the Essentiality of a Gene 949 Recombination-Dependent Replication Restarts DNA Replication at Stalled Replication Forks 949

Information Transfer

29 Transcription and the Regulation of Gene

HUMAN BIOCHEMISTRY: Telomeres—A Timely End to Chromosomes? 940 The Enzymatic Activities of Reverse Transcriptases 941

28.7

What Is the Molecular Basis of Mutation? 955 Point Mutations Arise by Inappropriate Base Pairing 956 Mutations Can Be Induced by Base Analogs 957 Chemical Mutagens React with the Bases in DNA 957 Insertions and Deletions 957

Why Are There So Many DNA Polymerases? 935

The Cell Cycle Controls the Timing of DNA Replication 936 Proteins of the Prereplication Complex Are AAA! ATPase Family Members 938 Geminin Provides Another Control over Replication Initiation 938 Eukaryotic Cells Contain a Number of Different DNA Polymerases 938

Can DNA Be Repaired? 950 A DEEPER LOOK: Transgenic Animals Are Animals Carrying Foreign Genes 953 Mismatch Repair Corrects Errors Introduced During DNA Replication 954 Damage to DNA by UV Light or Chemical Modification Can Also Be Repaired 954

Cells Have Different Versions of DNA Polymerase, Each for a Particular Purpose 935 The Common Architecture of DNA Polymerases 935

28.4

xxv

29.2

How Is Transcription Regulated in Prokaryotes? 970 Transcription of Operons Is Controlled by Induction and Repression 971 The lac Operon Serves as a Paradigm of Operons 972 lac Repressor Is a Negative Regulator of the lac Operon 972 CAP Is a Positive Regulator of the lac Operon 974 A DEEPER LOOK: Quantitative Evaluation of lac Repressor:DNA Interactions 974 Negative and Positive Control Systems Are Fundamentally Different 975 The araBAD Operon Is Both Positively and Negatively Controlled by AraC 975 The trp Operon Is Regulated Through a Co-repressor– Mediated Negative Control Circuit 977 Attenuation Is a Prokaryotic Mechanism for Posttranscriptional Regulation of Gene Expression 978

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DNA:Protein Interactions and Protein:Protein Interactions Are Essential to Transcription Regulation 980 Proteins That Activate Transcription Work through Protein:Protein Contacts with RNA Polymerase 980 DNA Looping Allows Multiple DNA-Binding Proteins to Interact with One Another 981

29.3

How Are Genes Transcribed in Eukaryotes? 981 Eukaryotes Have 3 Classes of RNA Polymerases 982 RNA Polymerase II Transcribes Protein-Coding Genes 983 The Regulation of Gene Expression Is More Complex in Eukaryotes 984 Gene Regulatory Sequences in Eukaryotes Include Promoters, Enhancers, and Response Elements 985 Transcription Initiation by RNA Polymerase II Requires TBP and the GTFs 987 The Role of Mediator in Transcription Activation and Repression 988 Mediator as a Repressor of Transcription 990 Chromatin-Remodelling Complexes and HistoneModifying Enzymes Alleviate the Repression Due to Nucleosomes 990 Chromatin-Remodelling Complexes Are Nucleic Acid–Stimulated Multi-subunit ATPases 990 Covalent Modification of Histones 991 Covalent Modification of Histones Forms the Basis of the Histone Code 992 Methylation and Phosphorylation Act as a Binary Switch in the Histone Code 992 Chromatin Deacetylation Leads to Transcription Repression 992 Nucleosome Alteration and Interaction of RNA Polymerase II with the Promoter Are Essential Features in Eukaryotic Gene Activation 992 A SINE of the Times 993

29.4

SUMMARY 1005 PROBLEMS 1006 FURTHER READING 1006

30 Protein Synthesis

How Are Eukaryotic Transcripts Processed and Delivered to Ribosomes for Translation? 997 Eukaryotic Genes Are Split Genes 997 The Organization of Exons and Introns in Split Genes Is Both Diverse and Conserved 998

1009

BIOCHEMISTRY: A CANADIAN CONTEXT: Dr. Nahum

Sonenberg 1010

30.1

What Is the Genetic Code? 1010 The Genetic Code Is a Triplet Code 1010 Codons Specify Amino Acids 1011

30.2

How Is an Amino Acid Matched with Its Proper tRNA? 1012 Aminoacyl-tRNA Synthetases Interpret the Second Genetic Code 1012 A DEEPER LOOK: Natural and Unnatural Variations in the Standard Genetic Code 1012 Evolution Has Provided 2 Distinct Classes of Aminoacyl-tRNA Synthetases 1014 Aminoacyl-tRNA Synthetases Can Discriminate between the Various tRNAs 1014 Escherichia coli Glutaminyl-tRNAGln Synthetase Recognizes Specific Sites on tRNAGln 1016 The Identity Elements Recognized by Some AminoacyltRNA Synthetases Reside in the Anticodon 1016 A Single G:U Base Pair Defines tRNAAlas 1016

How Do Gene Regulatory Proteins Recognize Specific DNA Sequences? 994 a-Helices Fit Snugly into the Major Groove of B-DNA 994 HUMAN BIOCHEMISTRY: Storage of Long-Term Memory Depends on Gene Expression Activated by CREB-Type Transcription Factors 994 Proteins with the Helix-Turn-Helix Motif Use One Helix to Recognize DNA 995 Some Proteins Bind to DNA via Zn-Finger Motifs 995 Some DNA-Binding Proteins Use a Basic RegionLeucine Zipper (bZIP) Motif 996 The Zipper Motif of bZIP Proteins Operates through Intersubunit Interaction of Leucine Side Chains 997 The Basic Region of bZIP Proteins Provides the DNA-Binding Motif 997

29.5

Post-transcriptional Processing of Messenger RNA Precursors Involves Capping, Methylation, Polyadenylylation, and Splicing 999 Nuclear Pre-mRNA Splicing 1000 The Splicing Reaction Proceeds via Formation of a Lariat Intermediate 1001 Splicing Depends on snRNPs 1002 snRNPs Form the Spliceosome 1002 Alternative RNA Splicing Creates Protein Isoforms 1003 Fast Skeletal Muscle Troponin T Isoforms Are an Example of Alternative Splicing 1003 RNA Editing: Another Mechanism That Increases the Diversity of Genomic Information 1004 Enzymatic RNA: Self-splicing Introns and the Discovery of Ribozymes 1004

30.3

What Are the Rules in Codon–Anticodon Pairing? 1017 Francis Crick Proposed the “Wobble” Hypothesis for Codon–Anticodon Pairing 1017 Some Codons Are Used More Than Others 1017 Nonsense Suppression Occurs When Suppressor tRNAs Read Nonsense Codons 1019

30.4

What Is the Structure of Ribosomes, and How Are They Assembled? 1019 Prokaryotic Ribosomes Are Composed of 30S and 50S Subunits 1019 Prokaryotic Ribosomes Are Made from 50 Different Proteins and 3 Different RNAs 1020 Ribosomes Spontaneously Self-Assemble in Vitro 1021 NEL

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Detailed Contents

Ribosomes Have a Characteristic Anatomy 1021 The Cytosolic Ribosomes of Eukaryotes Are Larger Than Prokaryotic Ribosomes 1022

30.5

What Are the Mechanics of mRNA Translation? 1023 Peptide Chain Initiation in Prokaryotes Requires a G-Protein Family Member 1024 Peptide Chain Elongation Requires 2 G-Protein Family Members 1026 The Elongation Cycle 1026 Aminoacyl-tRNA Binding 1027 GTP Hydrolysis Fuels the Conformational Changes That Drive Ribosomal Functions 1031 A DEEPER LOOK: Molecular Mimicry—The Structures of EF-Tu:Aminoacyl-tRNA, EF-G, and RF-3 1031 Peptide Chain Termination Requires a G-Protein Family Member 1032 The Ribosomal Subunits Cycle between 70S Complexes and a Pool of Free Subunits 1032 Polyribosomes Are the Active Structures of Protein Synthesis 1034

30.6

A DEEPER LOOK: Journey into the Exosome 1058 trans-Factors and Effectors in mRNA Turnover 1059

31.3

31.4

What Cellular Processes Serve to Maintain RNA Integrity? 1066 mRNA Surveillance Systems 1066 The Players in NMD 1066 General Mechanism of NMD 1066 How Does NMD Distinguish between Normal Stop Codons and Premature Stop (Nonsense) Codons? 1067 How Do the UPFs and the EJC Work Together to Trigger NMD? 1069 Translation Dependence of NMD 1069 How Are NMD Substrates Degraded? 1069 Non-stop Decay 1070

31.5

What Is miRNA-Mediated Gene Regulation? 1071 miRNA Biogenesis 1072 Mechanisms of miRNA-Mediated Repression 1076 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: siRNA and shRNA 1076 miRNA-Mediated Translation Repression 1077 miRNA-Mediated mRNA Decay 1078 miRNAs and AUBPs: A Potential Link? 1079

SUMMARY 1044 PROBLEMS 1045

What Is AU-Rich–Mediated Decay? 1059 The AU-Rich Element 1060 Classification of ARE-Containing mRNAs 1060 ARE-Binding Proteins 1061 A DEEPER LOOK: Physiological Significance of AMD 1061 The Effects of AUBPs on AMD 1062 Identifying RBP-mRNA Interactions through RNA-EMSA 1062 CRITICAL DEVELOPMENTS IN BIOCHEMISTRY: RIP-Chip and PAR-CLIP: Advanced Techniques to Study RBP-mRNA Interactions 1064 GU-Rich Elements 1066

How Are Proteins Synthesized in Eukaryotic Cells? 1034 Peptide Chain Initiation in Eukaryotes 1034 Control of Eukaryotic Peptide Chain Initiation by Phosphorylation 1038 Control of Eukaryotic Translation by Cis-Elements in the 5’ and 3’ UTRs 1039 Peptide Chain Elongation in Eukaryotes Resembles the Prokaryotic Process 1040 Eukaryotic Peptide Chain Termination Requires Just 1 Release Factor 1040 Inhibitors of Protein Synthesis 1040 HUMAN BIOCHEMISTRY: Diphtheria Toxin ADPRibosylates eEF2 1041

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FURTHER READING 1046

Can We Propose a Unified Theory of Gene Expression? 1079

Expression: mRNA Turnover and Micro-RNA 1048

RNA Regulation Is Coordinated: RNA Operons and Regulons 1080 Can mRNA Manipulation Serve as a Therapeutic Approach? 1083

31.1

What Is mRNA Turnover? 1049

SUMMARY 1083

Importance of mRNA Turnover 1049 How Is the Half-Life of an mRNA Determined? 1049 The Life Cycle of an mRNA 1051 Location of mRNA Turnover: P-Bodies 1051 A DEEPER LOOK: Measuring the Effect of an RBP on mRNA Half-Life Using a Pulse-Chase Experiment 1052

PROBLEMS 1084

31 Post-transcriptional Regulation of Gene

31.2

How Is mRNA Decay Carried Out? 1055 Deadenylation 1055 5’→3’ Decay 1056 3’→5’ Decay 1057 Deadenylation-Independent mRNA Decay 1057 cis-Elements in mRNA Turnover 1057

FURTHER READING 1084

32 Completing the Protein Life Cycle: Folding, Processing, and Degradation 1086 32.1

How Do Newly Synthesized Proteins Fold? 1087 HUMAN BIOCHEMISTRY: Alzheimer’s, Parkinson’s, and Huntington Disease Are Late-Onset Neurodegenerative Disorders Caused by the Accumulation of Protein Deposits 1088 Chaperones Help Some Proteins Fold 1088 Hsp70 Chaperones Bind to Hydrophobic Regions of Extended Polypeptides 1089

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The GroES–GroEL Complex of E. coli Is an Hsp60 Chaperonin 1089 The Eukaryotic Hsp90 Chaperone System Acts on Proteins of Signal Transduction Pathways 1091

32.2

33.3

Is There a Good Index of Cellular Energy Status? 1114 Adenylate Kinase Interconverts ATP, ADP, and AMP 1114 Energy Charge Relates the ATP Levels to the Total Adenine Nucleotide Pool 1114 Key Enzymes Are Regulated by Energy Charge 1115 Phosphorylation Potential Is a Measure of Relative ATP Levels 1115

33.4

How Is Overall Energy Balance Regulated in Cells? 1115 AMPK Targets Key Enzymes in Energy Production and Consumption 1116 AMPK Controls Whole-Body Energy Homeostasis 1117

33.5

How Is Metabolism Integrated in a Multicellular Organism? 1118 The Major Organ Systems Have Specialized Metabolic Roles 1119 HUMAN BIOCHEMISTRY: Athletic Performance Enhancement with Creatine Supplements? 1121 HUMAN BIOCHEMISTRY: Fat-Free Mice: A Snack Food for Pampered Pets? No, a Model for One Form of Diabetes 1122

How Does Protein Degradation Regulate Cellular Levels of Specific Proteins? 1098 Eukaryotic Proteins Are Targeted for Proteasome Destruction by the Ubiquitin Pathway 1098 Proteins Targeted for Destruction Are Degraded by Proteasomes 1100 ATPase Modules Mediate the Unfolding of Proteins in the Proteasome 1101 Ubiquitination Is a General Regulatory Protein Modification 1101 Small Ubiquitin-Like Protein Modifiers Are Posttranscriptional Regulators 1101 HtrA Proteases Also Function in Protein Quality Control 1103 HUMAN BIOCHEMISTRY: Proteasome Inhibitors in Cancer Chemotherapy 1103 A DEEPER LOOK: Protein Triage—A Model for Quality Control 1104 A DEEPER LOOK: Methods for Examining Protein Folding 1105

What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism? 1112 ATP Coupling Stoichiometry Determines the Keq for Metabolic Sequences 1113 ATP Has Two Metabolic Roles 1114

How Do Proteins Find Their Proper Place in the Cell? 1093 Proteins Are Delivered to the Proper Cellular Compartment by Translocation 1094 Prokaryotic Proteins Destined for Translocation Are Synthesized as Preproteins 1094 Eukaryotic Proteins Are Routed to Their Proper Destinations by Protein Sorting and Translocation 1095 The Synthesis of Secretory Proteins and Many Membrane Proteins Is Coupled to Translocation Across the ER Membrane 1095 Interaction between the RNC-SRP and the SR Delivers the RNC to the Membrane 1095

32.4

33.2

How Are Proteins Processed Following Translation? 1091 A DEEPER LOOK: How Does ATP Drive ChaperoneMediated Protein Folding? 1092 Proteolytic Cleavage Is the Most Common Form of Post-translational Processing 1092

32.3

Phototrophs Have an Additional Metabolic System: The Photochemical Apparatus 1112

33.6

What Regulates Our Eating Behaviour? 1124 The Hormones That Control Eating Behaviour Come from Many Different Tissues 1124 Ghrelin and Cholecystokinin Are Short-Term Regulators of Eating Behaviour 1124 HUMAN BIOCHEMISTRY: The Metabolic Effects of Alcohol Consumption 1125 Insulin and Leptin Are Long-Term Regulators of Eating Behaviour 1126 AMPK Mediates Many of the Hypothalamic Responses to These Hormones 1126

33.7

Can You Really Live Longer by Eating Less? 1126 Caloric Restriction Leads to Longevity 1126 Mutations in the SIR2 Gene Decrease Life Span 1127 SIRT1 Is a Key Regulator in Caloric Restriction 1128 Resveratrol, a Compound Found in Red Wine, Is a Potent Activator of Sirtuin Activity 1128

SUMMARY 1105 PROBLEMS 1106 FURTHER READING 1107

33 Metabolic Integration and Organ

SUMMARY 1128

Specialization 1109

PROBLEMS 1130

33.1

FURTHER READING 1131

Can Systems Analysis Simplify the Complexity of Metabolism? 1109 BIOCHEMISTRY: A CANADIAN CONTEXT:

Dr. Frederick Banting 1110 Only a Few Intermediates Interconnect the Major Metabolic Systems 1110 ATP and NADPH Couple Anabolism and Catabolism 1111

Abbreviated Answers to Problems A-1 Index I-1

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Preface FMT

Why Write the First Canadian Edition? When we were approached to prepare a Canadian edition of Biochemistry, we were excited to build on Dr. Garrett and Dr. Grisham’s wonderful book. In the process, we learned so much about the diversity of work in the field of biochemistry within Canada, and we were reminded of the richness of Canadian experience. We hope that we have captured that richness for both the students and the faculty who will use this book. We embarked on a first Canadian edition for two reasons. First, it was our intention to offer university students a highly prestigious and up-to-date biochemistry text with inclusion of documented Canadian research data when appropriate. The Canadian market lacked an appropriate resource that included current biochemical findings, as well as topics and trends ideal for the Canadian educational system (for example, the findings of Canadian researchers Banting and Best that eventually lead to the discovery of insulin). Second, the lack of a textbook containing an in-depth look at post-transcriptional regulation of gene expression, an emerging and exciting research field, prompted us to introduce a new chapter to Biochemistry in this first Canadian edition. We hope that while highlighting these new and important findings and bringing them to the forefront in the Canadian edition, we are also addressing this shortcoming in other major biochemistry texts by being the first to include such a chapter. Ultimately, we visualized the Canadian adaptation of Biochemistry as a more comprehensive text and resource for Canadian students with the ultimate goal of enabling them to master the exciting topic of biochemistry with ease.

Biochemistry’s Expanding Boundaries Scientific understanding of the molecular nature of life is growing at an astounding rate. Significantly, society is the prime beneficiary of this increased understanding. Cures for diseases, better public health, remedies for environmental pollution, and the development of cheaper and safer natural products are just a few practical benefits of this knowledge. In addition, this expansion of information fuels, in the words of Thomas Jefferson, “the illimitable freedom of the human mind.” Scientists can use the tools of biochemistry and molecular biology to explore all aspects of an organism—from basic questions about its chemical composition, through inquiries into the complexities of its metabolism, its differentiation and development, to analysis of its evolution and even its behaviour. New procedures based on the results of these explorations lie at the heart of the many modern medical miracles. Biochemistry is a science whose boundaries now encompass all aspects of biology, from molecules to cells, to organisms, to ecology, and to all aspects of health care. This first Canadian edition of Biochemistry embodies and reflects the expanse of this knowledge. We hope that this text will encourage students to ask questions of their own and to push the boundaries of their curiosity about science.

Making Connections As the explication of natural phenomena rests more and more on biochemistry, its inclusion in undergraduate and graduate curricula in biology, chemistry, and the health sciences becomes imperative. The challenge to authors and instructors is a formidable one: how to NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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familiarize students with the essential features of modern biochemistry in an introductory course or textbook. Fortunately, the increased scope of knowledge allows scientists to make generalizations connecting the biochemical properties of living systems with the character of their constituent molecules. As a consequence, these generalizations, validated by repetitive examples, emerge in time as principles of biochemistry, principles that are useful in discerning and describing new relationships between diverse biomolecular functions and in predicting the mechanisms underlying newly discovered biomolecular processes. Nevertheless, it is increasingly apparent that students must develop skills in inquiry-based learning, so that, beyond this first encounter with biochemical principles and concepts, students are equipped to explore science on their own. Much of the design of this new edition is meant to foster the development of such skills. We are biochemists, working in different departments. Undoubtedly, we each view biochemistry through the lens of our respective disciplines. We believe, however, that our collaboration on this textbook represents a melding of our perspectives that will provide new dimensions of appreciation and understanding for all students.

Our Audience Biochemistry, First Canadian Edition, is designed to communicate the fundamental principles governing the structure, function, and interactions of biological molecules to students encountering biochemistry for the first time. We aim to bring an appreciation of biochemistry to a broad audience that includes undergraduates majoring in the life sciences, physical sciences, or premedical programs, as well as medical students and graduate students in the various health sciences for whom biochemistry is an important route to understanding human physiology. To make this subject matter more relevant and interesting to all readers, we emphasize, where appropriate, the biochemistry of humans.

Objectives Biochemistry, First Canadian Edition, carries forward the clarity of purpose found in previous editions; namely, to illuminate for students the principles governing the structure, function, and interactions of biological molecules. At the same time, this edition has been revised to reflect tremendous developments in biochemistry. Significantly, emphasis is placed on the interrelationships of ideas so that students can begin to appreciate the overarching questions of biochemistry. Overall, the textbook chapters were reorganized to represent a more logical flow of biochemical information, beginning with the fundamental components or building blocks of the cell (such as amino acids, proteins, carbohydrates, and lipids) and followed by protein dynamics, metabolism and its regulation, nucleic acids, and with information transfer and the processes associated with it. At the chapter level, we capitalize on information already present by supplementing it with relevant trends and recent developments in Canadian research. Finally, we introduce a new and distinct chapter (Chapter 31) entitled “Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA.”

Features • Why It Matters Every chapter of the first Canadian edition begins with an examination of the conceptual relevance of each topic that explains to students how it applies to “everyday life” situations. These ideas are intended to promote critical thinking of the subject material and highlight the importance of biochemical processes in the development and well-being of living organisms. • Biochemistry: A Canadian Context Unique to the first Canadian edition, these boxes highlight the discoveries of Canadian scientists who are international leaders in their field. These boxes showcase the works of Canadian researchers who are biochemists and molecular biologists, of whom many are winners of the Nobel Prize in either Physiology or Medicine or Chemistry. Other Canadian Context boxes focus on NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Preface

•

•

•

•

• • • • •

•

•

Canadian groups or organizations that work on key topics related to the material presented in each chapter of this book. The aim of the Canadian Context boxes is to bring Canadian scientific achievements to the attention of our students and emphasize Canada’s leading role in global biochemical, molecular, and biomedical research. Clarity of Instruction This edition continues to be written with clarity and readability. Many of the lengthier figure legends have been shortened and more information is included directly within illustrations. These changes will help the more visual reader. Visual Instruction The richness of the Protein Data Bank (www.pdb.org) and availability of molecular graphics software have been exploited to enliven this text. Over 330 images of prominent proteins and nucleic acids involved with essential biological functions illustrate and inform the subject matter and were prepared especially for this book. Revised End-of-Chapter Problems These problems serve as meaningful exercises that help students develop problem-solving skills useful in achieving their learning goals. Some problems require students to employ calculations to find mathematical answers to relevant structural or functional questions. Other questions address conceptual problems whose answers require application and integration of ideas and concepts introduced in the chapter. Human Biochemistry The central role of basic biochemistry in medicine and the health sciences is emphasized in these essays. They often present clinically important issues such as diet, diabetes, and cardiovascular health. A Deeper Look Highlighting selected topics or experimental observations, these essays expand on the text. Critical Developments in Biochemistry Recent and historical advances in the field are emphasized in these essays. Up-to-Date References End-of-chapter references make it easy for students to find additional information about each topic. Laboratory Techniques The experimental nature of biochemistry is highlighted, and a list of laboratory techniques found in this book is provided on page xl. Essential Questions Each chapter in this book is framed around an Essential Question that invites students to become actively engaged in their learning and encourages curiosity and imagination about the subject matter. For example, the Essential Question of Chapter 3 asks, “What are the laws and principles of thermodynamics that allow us to describe the flows and interchange of heat, energy, and matter in systems of interest?” The section heads then pose key questions such as, “What Is the Daily Human Requirement for ATP?” The end-of-chapter summary then brings the question and a synopsis of the answer together for the student. In addition, the OWLv2 site at www.cengage.com/login expands on this Essential Question theme by asking students to explore their knowledge of key concepts. Key Questions The section headings within chapters are phrased as important questions that serve as organizing principles for a lecture. The subheadings are designed to be concept statements that respond to the section headings. Linking Key Questions to Chapter Summaries The end-of-chapter summaries recite the key questions posed as section heads and then briefly summarize the important concepts and facts to aid students in organizing and understanding the material.

New to the First Canadian Edition Chapter 1 • New Section 1.7, “Do Biomolecules Ever Behave as Genetic Agents?” • Expanded synopsis for Section 1.6 in Chapter Summary • Three new figures: Figure 1.20, the phylogenetic tree of life; Figure 1.26, secondary structures of representative viroids from the two viroid families, Avsunviroidae: ASBVd and PLMVd, and Pospiviroidae: PSTVd; and Figure 1.27, distinct steps of systemic infection of ASBVd and PSTVd, type members of the two viroid families • New primary references added to “Papers on Prions and Virons” in the Further Reading section NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Chapter 2 • New Section 2.4, “What Are Common Buffers Used in Biochemistry?” • New Figure 2.18, common buffers used in biochemistry and their pKa values at 20°C • Condensed summary of essential features of water that make it amenable to support life

Chapter 3 • New subsection “Guanidino Phosphates are Energy Reserve Compounds” in Section 3.3 • New Figure 3.13, the phosphocreatine shuttle

Chapter 4 • Expanded content on amino acid derivatives and pKa values of common amino acids • New discussion of posttranslational modifications of amino acids (expanded on in Section 5.5) • New discussion of pI along with two new problems at the end of the chapter.

Chapter 5 • New discussion of separation methods • Expanded content in Section 5.1, “What Architectural Arrangements Characterize Protein Structure?” • Expanded discussion and reorganization of protein techniques • New “A Deeper Look” essay on bovine spongiform encephalopathy (BSE) and its effects on the Canadian economy, along with supporting references in Further Reading • Two new problems at the end of the chapter on peptide structure and sequencing

Chapter 6 • New discussion of immunoglobulin-based techniques • New “Critical Developments in Biochemistry” essay on Frederick Banting and Charles Best • New “Critical Developments in Biochemistry” essay on Linus Pauling’s discovery of the a-Helix, which led to his Nobel Prize in Chemistry • New problem at the end of the chapter regarding Anfinsen’s experiment

Chapter 7 • New “Biochemistry: A Canadian Context” box on Dr. Raymond Urgel Lemieux and the synthesis of sucrose • Expanded content on carbohydrates and how they are named • New content outlining the enzymes that add sugar moieties to amino acid side chains and the enzymes that remove sugar moieties from substrates

Chapter 8 • New “Human Biochemistry” essay on the consumption of fast foods in Canada

Chapter 9 • Reorganized topic progression so that Lipid Biosynthesis now follows Chapter 8, Lipids • New discussion of Tay-Sach’s Disease (TSD) • New discussion of Canadian sprinter Ben Johnson and the use of anabolic steroids by athletes

Chapter 10 • Revised Figure 9.4 with additional relevant detergents that are used in biochemistry, as well as their molecular weights, critical micelle concentrations, and micelle molecular weights

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Preface

Chapter 11 • Additional coverage regarding posttranslational modifications via cleavage being non-reversible • New “Biochemistry: A Canadian Context” box on Dr. Antony Pawson, who discovered the SH2 domain that mediates protein-protein interactions of key players of cell metabolism and development

Chapter 12 • New “Biochemistry: A Canadian Context” box on Sidney Altman and the discovery of ribozymes and the kinetic mechanism of RNase P • Expanded content discussing “directed enzyme evolution” in Section 12.8 (Section 13.8 in the fourth edition of Biochemistry) and in the Summary synopsis to emphasize the phrase used in the field to custom design enzymes • New “Critical Developments in Biochemistry” essay on drug discovery and personalized medicine

Chapter 13 • New discussion of kinetic mechanism of lysozyme, including new Figure 13.5

Chapter 14 • New Section 14.5, “Is the Activity of Some Enzymes Controlled by Both Allosteric Regulation and Covalent Modification?” • New discussion of allosteric regulation of phosphofructokinase • New Figure 14.1, conformational changes in the phosphofructokinase tetramer • New Figure 14.12, allosteric and covalent modification of the activity of the pyruvate dehydrogenase complex

Chapter 15 • New Canadian references included in the Further Reading section

Chapter 17 • New Table 17.3, “Regulation of the Glycolytic Enzymes”, in Section 17.4

Chapter 18 • Discussion of Sir Hans Krebs, Nobel Prize Winner in Physiology/Medicine in “Why It Matters” • New “Human Biochemistry” essay on mitochondrial diseases. • Two new problems at the end of the chapter • Updated references in Further Reading, General section

Chapter 19 • Updated references to Further Reading, Bioenergetics

Chapter 20 • Updated references in Further Reading, General section

Chapter 21 • New discussion on glycogen phosphorylase reaction and regulation • New “Biochemistry, A Canadian Context Box” on Louis Delbaere and the discovery of the kinetic mechanism of phosphoenolpyrvate carboxykinase • New Figure 21.14, glycogenesis and glycogenolysis are reciprocal pathways • New Figure 21.22, mechanism of covalent modification and allosteric regulation of glycogen phosphorylase

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Chapter 22 • Updated discussion of hormones that trigger the release of fatty acids from adipose tissue, reflecting current knowledge of the initial breakdown of triacylglycerols to free fatty acids and glycerol • New discussion on the percentage of fatty acids of total caloric intake along with Canadian references • New “Human Biochemistry” essay on carnitine deficiency • New Figure 22.2, liberation of fatty acids from triacylglycerols in adipose tissue is hormone-dependent (changed to include perilipin [PLIN], coactivator, adipose triglyceride lipase [ATGL] and hormone-sensitive lipase)

Chapter 23 • Updated figure legends include the names of the enzymes that catalyze the individual steps indicated • New “Human Biochemistry” essay on phenylketonuria and aspartame, including figure depicting aspartame

Chapter 24 • New references in the Further Reading section, in The History of Discovery of the DNA Double Helix.

Chapter 25 • Expanded discussion of superhelix density • Revised and expanded discussion of ball-and-stick content

Chapter 26 • Revised and updated online directions for viewing proteins in the Protein Data Bank

Chapter 27 • New “Biochemistry: A Canadian Context” box on Canadian Nobel Prize winner Michael Smith and his achievements • Expanded and revised of Table 27.2 , “Gene Fusion Systems for Isolation of Cloned Fusion Proteins” • New discussion of real-time PCR, including new references in Further Reading section • New Figure 27.19, fluorophore-conjugated oligonucleotides specific for the target sequence are added to the reaction mixture (red and green) to support the new content on real-time PCR

Chapter 28 • New “Biochemistry: A Canadian Context” box on Jack W. Szostak and his achievements • Expanded discussion of senescent cells • New Canadian references included in the Further Reading section

Chapter 29 • Expanded discussion of the effects of a-amanitin on the three classes of eukaryotic RNA polymerases • New discussion of self-splicing introns • New Figure 29.47, the mechanisms of self-splicing introns

Chapter 30 • Expanded discussion of peptide chain initiation in eukaryotes with accompanying new Figure 30.28, the six stages in the initiation of translation in eukaryotic cells • New discussion of control of eukaryotic peptide chain initiation by phosphorylation, with accompanying new Figure 30.30, eIF4E binding protein (4EBP) as a key modulator of translation initiation NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Preface

• New discussion of control of eukaryotic translation by cis-elements in the 5’ and 3’ UTRs, with accompanying new Figure 30.32, regarding iron-responsive proteins (IRPs) • New “Biochemistry: A Canadian Content” box on Nahum Sonenberg, who discovered the eukaryotic translation initiation initiation factor 4E (eIF4E)

Chapter 31 • Brand new chapter, Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA

Chapter 32 • New discussion of the Prion Centre at the University of Alberta in the Why It Matters section • New reference to the role of carbohydrates in glycoproteins for protein processing, targeting, and function • New “A Deeper Look” essay on ITC and DSC references in protein folding techniques

Chapter 33 • New “Biochemistry: A Canadian Context” box on Fredrick Banting and his accomplishments • New “Human Biochemistry” essay on Donovan Bailey and the use of phosphocreatine as an energy source for improving athletic performance • New discussion of the cytological differences between brown and white fat cells, as well as accompanying new Figure 33.11 • New discussion of the obesity statistics for Canadians

Instructor Ancillaries The Nelson Education Teaching Advantage (NETA) program delivers research-based instructor resources that promote student engagement and higher-order thinking to enable the success of Canadian students and educators. Instructors today face many challenges. Resources are limited, time is scarce, and a new kind of student has emerged: one who is juggling school with work, has gaps in his or her basic knowledge, and is immersed in technology in a way that has led to a completely new style of learning. In response, Nelson Education has gathered a group of dedicated instructors to advise us on the creation of richer and more flexible ancillaries and online learning platforms that respond to the needs of today’s teaching environments. Whether your course is offered in-class, online, or both, Nelson is pleased to provide pedagogicallydriven, research-based resources to support you. The members of our editorial advisory board have experience across a variety of disciplines and are recognized for their commitment to teaching. They include: Norman Althouse, Haskayne School of Business, University of Calgary Brenda Chant-Smith, Department of Psychology, Trent University Scott Follows, Manning School of Business Administration, Acadia University Jon Houseman, Department of Biology, University of Ottawa Glen Loppnow, Department of Chemistry, University of Alberta Tanya Noel, Department of Biology, York University Gary Poole, Senior Scholar, Centre for Health Education Scholarship, and Associate Director, School of Population and Public Health, University of British Columbia Dan Pratt, Department of Educational Studies, University of British Columbia Mercedes Rowinsky-Geurts, Department of Languages and Literatures, Wilfrid Laurier University David DiBattista, Department of Psychology, Brock University Roger Fisher, PhD In consultation with the editorial advisory board, Nelson Education has completely rethought the structure, approaches, and formats of our key textbook ancillaries and online learning platforms. We’ve also increased our investment in editorial support for our NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Preface

ancillary and digital authors. The result is the Nelson Education Teaching Advantage and its key components: NETA Assessment, NETA Presentation, and NETA Digital. Each component includes one or more ancillaries prepared according to our best practices, and may also be accompanied by documentation explaining the theory behind the practices. NETA Assessment relates to testing materials. Under NETA Assessment, Nelson’s authors create multiple-choice questions that reflect research-based best practices for constructing effective questions and testing not just recall but also higher-order thinking. Our guidelines were developed by David DiBattista, a 3M National Teaching Fellow whose recent research as a professor of psychology at Brock University has focused on multiple-choice testing. All Test Bank authors receive training at workshops conducted by Prof. DiBattista, as do the copyeditors assigned to each Test Bank. A copy of Multiple Choice Tests: Getting Beyond Remembering, Prof. DiBattista’s guide to writing effective tests, is included with every Nelson Test Bank/ Computerized Test Bank package. (Information about the NETA Test Bank prepared for Biochemistry, First Canadian Edition, is included in the description of the IRCD below.) NETA Presentation has been developed to help instructors make the best use of PowerPoint® in their classrooms. With a clean and uncluttered design developed by Maureen Stone of StoneSoup Consulting, NETA Presentation features slides with improved readability, more multi-media and graphic materials, activities to use in class, and tips for instructors on the Notes page. A copy of NETA Guidelines for Classroom Presentations by Maureen Stone is included with each set of PowerPoint slides. (Information about the NETA PowerPoint® prepared for Biochemistry, First Canadian Edition, is included in the description of the IRCD below.) NETA Digital is a framework based on Arthur Chickering and Zelda Gamson’s seminal work “Seven Principles of Good Practice In Undergraduate Education” (AAHE Bulletin, 1987) and the follow-up work by Chickering and Stephen C. Ehrmann, “Implementing the Seven Principles: Technology as Lever”(AAHE Bulletin, 1996). This aspect of the NETA program guides the writing and development of our digital products to ensure that they appropriately reflect the core goals of contact, collaboration, multimodal learning, time on task, prompt feedback, active learning, and high expectations. The resulting focus on pedagogical utility, rather than technological wizardry, ensures that all of our technology supports better outcomes for students.

Instructor’s Resource CD-ROM (ISBN: 0176502653) • NETA Assessment: The Test Bank was adapted for the first Canadian edition by Stavroula Andreopoulous, co-author of the textbook. It includes almost 1000 multiplechoice questions vetted according to NETA guidelines for effective construction and development of higher-order questions. Test Bank files are provided in Word format for easy editing and in PDF format for convenient printing whatever your system. The Computerized Test Bank by ExamView® includes all the questions from the Test Bank. The easy-to-use ExamView software is compatible with Microsoft Windows and Mac OS. Create tests by selecting questions from the question bank, modifying these questions as desired, and adding new questions you write yourself. You can administer quizzes online and export tests to WebCT, Blackboard, and other formats. • Instructor Solutions Manual: written by David K. Jemiolo (Vassar College) and Steven M. Theg (University of California, Davis) and revised by the Canadian author team, this comprehensive combination resource contains chapter summaries, important definitions, illustrations of major metabolic pathways, self-tests, and detailed solutions to all end-of-chapter problems with answers. • NETA Presentation: Microsoft® PowerPoint® lecture slides for every chapter have been adapted by textbook co-author Stavroula Andreopulos. There is an average of 40 slides per chapter, many featuring key figures, tables, and photographs from Biochemisry, First Canadian Edition. NETA principles of clear design and engaging content have been incorporated throughout. • Image Library: This resource consists of digital copies of figures, short tables, and photographs used in the book. Instructors may use these jpegs to create their own PowerPoint presentations. • DayOne: Day One — Prof InClass is a PowerPoint presentation that you can customize to orient your students to the class and their text at the beginning of the course. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Preface

Student Ancillaries • Student Solutions Manual: written by David K. Jemiolo (Vassar College) and Steven M. Theg (University of California, Davis) and revised and updated by the Canadian author team, this comprehensive combination resource contains all odd chapter summaries, important definitions, illustrations of major metabolic pathways, self-tests, and detailed solutions to all odd numbered end-of-chapter problems with answers. • OWLv2 with MindTap Reader: Conceived and developed by Chemistry instructors for Chemistry courses at the University of Massachusetts, Amherst, OWLv2 is the most widely used online chemistry solution in the world. OWLv2 not only saves instructors time and helps students learn, but also solves the very real teaching and learning problems that instructors and students face in the classroom. OWLv2 meets students “where they are” using a mastery learning approach and is based on principles of active learning, time on task, and prompt feedback. Students work through each concept-based problem until they master it. Students who need intensive help continue to work problems using a wealth of learning resources until they can show they have mastered the concepts. OWLv2 helps all students based on their individual needs. Adding to the challenge, each time a student tries a problem, OWLv2 randomizes the chemical, numerical and contextual aspects of the question. Rather than just memorizing a formula and plugging in new numbers, students must understand the underlying chemical concepts before they gain mastery and move on.

Acknowledgments to the First Canadian Edition We thank the many experts who have contributed in the reviewing process throughout the development of this book. We are grateful for your suggestions in helping create a streamlined, concise, current, Canadian text that would not have been possible without you. These reviewers include: Stavroula Andreopoulos, University of Toronto Gerald Audette, York University Amanda Cockshutt, Mount Allison University Scott Covey, University of British Columbia Oleg Dmitriev, University of Saskatchewan James W. Gauld, University of Windsor Eric Gauthier, Laurentian University Dr. Dara Gilbert, University of Waterloo Michelle MacDonald, McMaster University William Willmore, Carleton University Adrienne Wright, University of Alberta We are also indebted to the amazing and hardworking editorial professionals at Nelson Education, in particular Paul Fam, Publisher, and Candace Morrison, Developmental Editor, who took the idea of a Canadian edition of Biochemistry and made the project a reality. We would also like to thank Sean Chamberland, Executive Marketing Manager; Leanne Newell, Marketing Manager; Jennifer Hare, Content Production Manager; Frances Robinson and Julia Cochrane, copy editors; Lynn McLeod, photo researcher and permissions editor; and Suwathiga Velayutham, project manager, for their tremendous dedication to this project – they helped drive the success of this text. Roula Andreopoulos would like to acknowledge her mom, Agni, “for sacrificing so much in her life so that I could be where I am today. Thank you for everything mom. Also, to Roy, I am extremely grateful for having you as a wonderful colleague throughout my academic career.” William Willmore would like to acknowledge his family, “my wife Azine and my daughter Kianna, for keeping my life sane. To my parents and brothers, I would like to thank for providing the best environment that one could grow up in. I would also like to thank my previous mentors for providing the opportunity to stand on the shoulders of giants.” NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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xxxviii Preface

Imed Gallouzi would like to thank his wife Kerrie-Ann Trudeau, “for her continuous support despite my crazy hours and for the unconditional love she is giving me. To my son Jackson and his gorgeous smile that kept me going even when progress seemed impossible. To my parents, for their infinitive love and support, without whom nothing would have been possible. I would like give a special thanks to my students Angelo Macri and Derek Hall who helped me at every step of the editing and the writing processes. I would also like to thank all members of my laboratory for their help in making this work possible.” Finally, to all of our colleagues and students, who contributed in many different ways, thanks so much. Stavroula Andreopoulos Toronto, ON William G. Willmore Ottawa, ON Imed Gallouzi Montreal, QC September 2012

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Acknowledgments to the U.S. Edition

We are indebted to the many experts in biochemistry and molecular biology who carefully reviewed this book at several stages for their outstanding and invaluable advice on how to construct an effective textbook. Guillaume Chanfreau, University of California, Los Angeles Jeffrey Cohlberg, California State University, Long Beach Bansidhar Datta, Kent State University Clyde Denis ,University of New Hampshire Gregg B. Fields, Florida Atlantic University Eric Fisher, University of Illinois, Springfield Nancy Gerber, San Francisco State University Donavan Haines, University of Texas, Dallas Nicole Horenstein, University of Florida Gary Kunkel, Texas A&M University Scott Lefler, Arizona State University Susanne Nonekowski, University of Toledo Wendy Pogozelski, State University of New York, Geneseo Michael Reddy, University of Wisconsin Mary Rigler, California Polytechnic State University Huiping Zhou,Virginia Commonwealth University Brent Znosko, St. Louis University We also wish to warmly and gratefully acknowledge many other people who assisted and encouraged us in this endeavor. A special thank you to Scott Lefler, Arizona State University, who read page proofs with an eye for accuracy. This book remains a legacy of Publisher John Vondeling, who originally recruited us to its authorship. We sense his presence still nurturing our book and we are grateful for it. Lisa Lockwood, our new publisher, has brought enthusiasm and an unwavering emphasis on student learning as the fundamental purpose of our collective endeavor. Sandi Kiselica, Senior Developmental Editor, is a biochemist in her own right. Her fascination with our shared discipline has given her a particular interest in our book and a singular purpose: to keep us focused on the matters at hand, the urgencies of the schedule, and limits of scale in a textbook’s dimensions. The dint of her efforts has been a major factor in the fruition of our writing projects. She is truly a colleague in these endeavors. We also applaud the unsung but absolutely indispensable contributions by those whose efforts transformed a rough manuscript into this final

product: Teresa Trego, project manager; Carol O’Connell, production editor; Lisa Weber, media editor; and Ashley Summers, assistant editor. If this book has visual appeal and editorial grace, it is due to them. The beautiful illustrations that not only decorate this text, but explain its contents are a testament to the creative and tasteful work of Cindy Geiss, Director of Graphic World Illustration Studio, and to the legacy of John Woolsey and Patrick Lane at J/B Woolsey Associates. We are thankful to our many colleagues who provided original art and graphic images for this work, particularly Professor Jane Richardson of Duke University. We are eager to acknowledge the scientific and artistic contributions of Michal Sabat, Senior Scientist in the Department of Chemistry at the University of Virginia. Michal was the creator of most of the PyMOL-based molecular graphics in this book. Much of the visual appeal that you will find in these pages gives testimony to his fine craftsmanship and his unflagging dedication to our purpose. We owe a very special thank-you to Rosemary Jurbala Grisham, devoted spouse of Charles and wonderfully tolerant friend of Reg. Also to be acknowledged with love and pride are Georgia Grant, to whom this book is also dedicated, and our children, Jeffrey, Randal, and Robert Garrett, and David, Emily, and Andrew Grisham. Also to be appreciated are Jatszi, Jazmine, and Jasper, three Hungarian Pulis whose unseen eyes view life with an energetic curiosity we all should emulate. Memories of Clancy, a Golden Retriever of epic patience and perspicuity, are companions to our best thoughts. We hope this fourth edition of our textbook has captured the growing sense of wonder and imagination that researchers, teachers, and students share as they explore the everchanging world of biochemistry. “Imagination is more important than knowledge. For while knowledge defines all we currently know and understand, imagination points to all we might yet discover and create.” —Albert Einstein Reginald H. Garrett Charles M. Grisham Charlottesville, VA Ivy, VA December 2008

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Laboratory Techniques in Biochemistry All of our knowledge of biochemistry is the outcome of experiments. For the most part, this text presents biochemical knowledge as established fact, but students should never lose sight of the obligatory connection between scientific knowledge and its validation by observation and analysis. The path of discovery by experimental research is often indirect, tortuous, and confounding before the truth is realized. Laboratory techniques lie at the heart of scientific inquiry, and many techniques of biochemistry are presented within these pages to foster a deeper understanding of the biochemical principles and concepts that they reveal.

Recombinant DNA Techniques Restriction endonuclease digestion of DNA 827-828 Restriction mapping 828-830 Nucleotide sequencing 834-837 Nucleic acid hybridization 850 Chemical synthesis of oligonucleotides 856-858 Cloning; recombinant DNA constructions 898-904 Construction of genomic DNA libraries 904-906 Combinatorial libraries of synthetic oligomers 905 Screening DNA libraries by colony hybridization 906 mRNA isolation 907 Construction of cDNA libraries 907 Expressed sequence tags 907 Southern blotting 908-909 Gene chips (DNA microarrays) 910-911 Protein expression from cDNA inserts 913-914 Screening protein expression libraries with antibodies 914 Two-hybrid systems to identify protein:protein interactions 916-917 Reporter gene constructs 916 Polymerase chain reaction (PCR) 917 In vitro mutagenesis 919-920

Probing the Function of Biomolecules Green fluorescent protein 87 Plotting enzyme kinetic data 416-418 Enzyme inhibition 419-424 Optical trapping to measure molecular forces 514 Isotopic tracers as molecular probes 551-552 NMR spectroscopy 552-553 Transgenic animals 953 DNA footprinting 968

Techniques Relevant to Clinical Biochemistry Metabolomic analysis 555 Tumor diagnosis with positron emission tomography (PET) 582

Glucose monitoring devices 695 Fluoro-substituted analogs as therapeutic agents 892 Gene therapy 920-922 “Knockout” mice 949

Isolation/Purification of Macromolecules Protein purification protocols 102-104 Dialysis and ultrafiltration 103 Size exclusion chromatography 107-108 Ion exchange chromatography 105-107 SDS-polyacrylamide gel electrophoresis 108-109 Isoelectric focusing 109 Two-dimensional gel electrophoresis 109-110 Hydrophobic interaction chromatography 110 High-performance liquid chromatography 110 Affinity chromatography 110 Ultracentrifugation 110

Analyzing the Physical and Chemical Properties of Biomolecules Titration of weak acids 42-45 Preparation of buffers 46-49 Estimation of protein concentration 104 Amino acid analysis of proteins 111 Amino acid sequence determination 111-122 Mass spectrometry of proteins 118-122 Peptide mass fingerprinting 121-122 Solid-phase peptide synthesis 130-132 Membrane lipid phase transitions 320-322 Measurement of standard reduction potentials 623-626 DNA nanotechnology 820 Nucleic acid hydrolysis 825-827 DNA sequencing 834-837 Density gradient (isopycnic) centrifugation 849

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

1

PART I BUILDING BLOCKS OF THE CELL

The Facts of Life: Chemistry Is the Logic of Biological Phenomena ! Sperm approaching an egg.

“…everything that living things do can be understood in terms of the jigglings and wigglings of atoms.” Richard P. Feynman Lectures on Physics, Addison-Wesley, 1963

Dennis Wilson/CORBIS

KEY QUESTIONS

ESSENTIAL QUESTION Molecules are lifeless. Yet, the properties of living things derive from the properties of molecules. Despite the spectacular diversity of life, the elaborate structure of biological molecules, and the complexity of vital mechanisms, can life functions be ultimately interpretable in chemical terms?

1.1

What Are the Distinctive Properties of Living Systems?

1.2

What Kinds of Molecules Are Biomolecules?

1.3

What Is the Structural Organization of Complex Biomolecules?

1.4

How Do the Properties of Biomolecules Reflect Their Fitness to the Living Condition?

1.5

What Is the Organization and Structure of Cells?

1.6

What Are Viruses?

1.7

Do Biomolecules Ever Behave as Genetic Agents?

Molecules are lifeless. Yet, in appropriate complexity and number, molecules compose living things. These living systems are distinct from the inanimate world because they have certain extraordinary properties. They can grow, move, perform the incredible chemistry of metabolism, respond to stimuli from the environment, and, most significantly, replicate themselves with exceptional fidelity. The complex structure and behaviour of living organisms conceal the basic truth that their molecular constitution can be described and understood. The chemistry of the living cell resembles the chemistry of organic reactions. Indeed, cellular constituents or biomolecules must conform to the chemical and physical principles that govern all matter. Despite the spectacular diversity of life, the intricacy of biological structures, and the complexity of vital mechanisms, life functions are ultimately interpretable in chemical terms. Chemistry is the logic of biological phenomena.

WHY IT MATTERS Before we begin our discussion of the chemistry of life, we pose this question: “Why does the chemistry of biomacromolecules matter?” As will be demonstrated in this and other chapters of this book, the chemistry of biomacromolecules is essential for the processes that maintain life, including the flow of energy, the maintenance of structure, and the passage of genetic information from generation to generation. Without the constant maintenance of proper energy, structure, and organization, macromolecular structure would fall to entropy and life could not be supported. Such cellular entropy is the basis for disease

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

2

Part 1 Building Blocks of the Cell

" “The Tree of Life and Death” by Canadian

Rick Leong

artist Rick Leong—Proper functioning of macromolecules is dependent upon their chemistry and could mean the difference between life and death of the cell.

and aging. Energy, stored in the form of chemical bonds, can be released from these bonds under controlled conditions and utilized for the purpose of building macromolecules. Throughout this book, we will also emphasize the importance of the structure of biomacromolecules to their function. The shape and folding of biomacromolecules will determine whether they function within the cell or not. If non-functioning macromolecules cannot be identified and broken down, they generally lead to disease states or death.

1.1

What Are the Distinctive Properties of Living Systems?

© Herbert Kehrer/zefa/CORBIS

Thomas C. Boydon/Marie Selby Botanical Gardens

First, the most obvious quality of living organisms is that they are complicated and highly organized (Figure 1.1). For example, organisms large enough to be seen with the naked eye are composed of many cells, typically of many types. In turn, these cells possess subcellular structures, called organelles, which are complex assemblies of very large polymeric molecules, called macromolecules. These macromolecules themselves show an exquisite degree of organization in their intricate 3-dimensional architecture, even though they are composed of simple sets of chemical building blocks, such as sugars and amino acids. Indeed, the complex 3-dimensional structure of a macromolecule, known as its conformation, is a consequence of interactions between the monomeric units, according to their individual chemical properties.

(a)

(b)

FIGURE 1.1 (a) Gelada (Theropithecus gelada), a baboon native to the Ethiopian highlands. (b) Tropical orchid (Bulbophyllum blumei), New Guinea. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 1

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

FIGURE 1.2 The food pyramid. Photosynthetic organisms at the base capture light energy. Herbivores and carnivores derive their energy ultimately from these primary producers.

hν

Carnivore product (0.4 g)

Carnivores 2° Consumers

Herbivore product (6 g)

Herbivores 1° Consumers

Primary productivity (270 g)

Photosynthesis 1° Producers

Productivity per square metre of a Saskatchewan field

Second, biological structures serve functional purposes. That is, biological structures play a role in the organism’s existence. From parts of organisms, such as limbs and organs, down to the chemical agents of metabolism, such as enzymes and metabolic intermediates, a biological purpose can be given for each component. Indeed, it is this functional characteristic of biological structures that separates the science of biology from studies of the inanimate world such as chemistry, physics, and geology. In biology, it is always meaningful to seek the purpose of observed structures, organizations, or patterns, that is, to ask what functional role they serve within the organism. Third, living systems are actively engaged in energy transformations. Maintenance of the highly organized structure and activity of living systems depends on their ability to extract energy from the environment. The ultimate source of energy is the sun. Solar energy flows from photosynthetic organisms (organisms able to capture light energy by the process of photosynthesis) through food chains to herbivores and ultimately to carnivorous predators at the apex of the food pyramid (Figure 1.2 above). The biosphere is thus a system through which energy flows. Organisms capture some of this energy, be it from photosynthesis or the metabolism of food, by forming special energized biomolecules, of which ATP and NADPH are the 2 most prominent examples (Figure 1.3).

NH2 O–

O– –O

P O

O

P O

N

O– O

P

N

OCH2

H

H

N CH2

O –O

H

H

H

P

O

NH2

FIGURE 1.3 ATP and NADPH, 2 biochemically important energy-rich compounds.

N O

O

OH OH ATP

O C

N

O

O

H

–O

OH OH P O

O

NH2 N

CH2

O

N

N N

OH O –O

P

O–

O NADPH NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

3

Part 1 Building Blocks of the Cell

Thinkstock

Kristin Garrett

4

(b)

Thomas Cooke

(a)

FIGURE 1.4 Organisms resemble their parents. (a) The Garrett guys. Left to right: (seated) Reg Garrett, flanked by grandsons Reggie and Ricky; (standing) grandson Jackson, and sons Jeffrey, Randal, and Robert. (b) Orangutan with infant. (c) The Grisham family. Left to right: David, Emily, Charles, Rosemary, and Andrew.

(c)

Entropy: A thermodynamic term used to designate that amount of energy in a system that is unavailable to do work.

(Commonly used abbreviations such as ATP and NADPH are defined on the inside back cover of this book.) ATP and NADPH are energized biomolecules because they represent chemically useful forms of stored energy. We explore the chemical basis of this stored energy in subsequent chapters. For now, suffice it to say that when these molecules react with other molecules in the cell, the energy released can be used to drive favourable processes. That is, ATP, NADPH, and related compounds are the power sources that drive the energy-requiring activities of the cell, including biosynthesis, movement, osmotic work against concentration gradients, and, in special instances, light emission (bioluminescence). Only upon death does an organism reach equilibrium with its inanimate environment. The living state is characterized by the flow of energy through the organism. At the expense of this energy flow, the organism can maintain its intricate order and activity far removed from equilibrium with its surroundings and yet exist in a state of apparent constancy over time. This state of apparent constancy, or so-called steady state, is actually a very dynamic condition: Energy and material are consumed by the organism and used to maintain its stability and order. In contrast, inanimate matter, as exemplified by the universe in totality, is moving to a condition of increasing disorder or, in thermodynamic terms, maximum entropy. Fourth, living systems have a remarkable capacity for self-replication. Generation after generation, organisms reproduce virtually identical copies of themselves. This selfreplication can proceed by a variety of mechanisms, ranging from simple division in bacteria to sexual reproduction in plants and animals; but in every case, it is characterized by an astounding degree of fidelity (Figure 1.4). Indeed, if the accuracy of selfreplication were significantly greater, the evolution of organisms would be hampered. NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 1

A G

5'

T

A

T

T

C

G C

C G

A 3'

A T

G

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

C

C

G

G

A T

C

T

5'

A

3'

FIGURE 1.5 The DNA double helix. Two complementary polynucleotide chains running in opposite directions can pair through hydrogen bonding between their nitrogenous bases. Their complementary nucleotide sequences give rise to structural complementarity.

This is so because evolution depends upon natural selection operating on individual organisms that vary slightly in their fitness for the environment. The fidelity of selfreplication resides ultimately in the chemical nature of the genetic material. This substance consists of polymeric chains of deoxyribonucleic acid, or DNA, which are structurally complementary to one another (Figure 1.5 above). These molecules can generate new copies of themselves in a rigorously executed polymerization process that ensures a faithful reproduction of the original DNA strands. In contrast, the molecules of the inanimate world lack this capacity to replicate. A crude mechanism of replication must have existed at life’s origin.

1.2

What Kinds of Molecules Are Biomolecules?

The elemental composition of living matter differs markedly from the relative abundance of elements in Earth’s crust (Table 1.1). Hydrogen, oxygen, carbon, and nitrogen constitute more than 99% of the atoms in the human body, with most of the H and O occurring as H2O. Oxygen, silicon, aluminum, and iron are the most abundant atoms in Earth’s crust, with hydrogen, carbon, and nitrogen being relatively rare (less than 0.2% each). Nitrogen as dinitrogen (N2) is the predominant gas in the atmosphere, and carbon dioxide (CO2) is present at a level of 0.04%, a small but critical amount. Oxygen is also abundant in the atmosphere and in the oceans. What property unites H, O, C, and N and renders these atoms so suitable to the chemistry of life? It is their ability to form covalent bonds by electron-pair sharing. Furthermore, H, C, N, and O are among the lightest elements of the

TABLE 1.1 Composition of the Earth’s Crust, Seawater, and the Human Body* Earth’s Crust Element

Human Body†

Seawater %

Compound

mM

Element

%

O

47

Cl!

548

H

63

Si

28

Na"

470

O

25.5

Al

7.9

Mg2"

54

C

9.5

Fe

4.5

SO42!

28

N

1.4

Ca

3.5

Ca2"

10

Ca

0.31

Na

2.5

K"

10

P

0.22

K

2.5

Mg

2.2

Ti

0.46

HCO! 3 NO! 3 HPO42!

H C

2.3

Cl

0.08

0.01

K

0.06

#0.001

S

0.05

0.22

Na

0.03

0.19

Mg

0.01

*Figures for Earth’s crust and the human body are presented as percentages of the total number of atoms; seawater data are in millimoles per litre. Figures for Earth’s crust do not include water, whereas figures for the human body do. † Trace elements found in the human body serving essential biological functions include Mn, Fe, Co, Cu, Zn, Mo, I, Ni, and Se. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

5

6

Part 1 Building Blocks of the Cell

Atoms

Covalent e– pairing bond

Bond energy (kJ/mol)

H

+

H

H H

H

H

436

C

+

H

C H

C

H

414

C

+

C

C C

C

C

343

C

+

N

C N

C

N

292

C

+

O

C O

C

O

351

C

+

C

C C

C

C

615

C

+

N

C N

C

N

615

C

+

O

C O

C

O

686

O

+

O

O O

O

O

142

O

+

O

O O

O

O

402

N

+

N

N N

N

N

946

N

+

H

N H

N

H

393

O

+

H

O H

O

H

460

FIGURE 1.6 Covalent bond formation by e– pair sharing.

periodic table capable of forming such bonds (Figure 1.6). Because the strength of covalent bonds is inversely proportional to the atomic weights of the atoms involved, H, C, N, and O form the strongest covalent bonds. Two other covalent bond–forming elements, phosphorus [as phosphate (—OPO32–) derivatives] and sulfur, also play important roles in biomolecules.

Biomolecules Are Carbon Compounds All biomolecules contain carbon. The prevalence of C is due to its unparalleled versatility in forming stable covalent bonds through electron-pair sharing. Carbon can form as many as 4 such bonds by sharing each of the 4 electrons in its outer shell with electrons contributed by other atoms. Atoms commonly found in covalent linkage to C are C itself, H, O, and N. Hydrogen can form 1 such bond by contributing its single electron to the formation of an electron pair. Oxygen, with 2 unpaired electrons in its outer shell, can participate in 2 covalent bonds, and nitrogen, which has 3 unshared electrons, can form 3 such covalent bonds. Furthermore, C, N, and O can share 2 electron pairs to form double bonds with one another within biomolecules, a property that enhances their chemical versatility. Carbon and nitrogen can even share 3 electron pairs to form triple bonds. Two properties of carbon covalent bonds merit particular attention: one is the ability of carbon to form covalent bonds with itself, and the other is the tetrahedral nature of the 4 covalent bonds when carbon atoms form only single bonds. Together these properties hold the potential for an incredible variety of linear, branched, and cyclic compounds of C. This diversity is multiplied further by the possibilities for including N, O, and H atoms in these compounds (Figure 1.7). We can therefore envision the ability of C to generate complex structures in 3 dimensions. These structures, by virtue of appropriately included N, O, and H atoms, can display unique chemistries suitable to the living state. Thus, we may ask, is there any pattern or underlying organization that brings order to this astounding potentiality?

1.3

What Is the Structural Organization of Complex Biomolecules?

Examination of the chemical composition of cells reveals a dazzling variety of organic compounds covering a wide range of molecular dimensions (Table 1.2 on page 8). As this complexity is sorted out and biomolecules are classified according to the similarities of their sizes and chemical properties, an organizational pattern emerges. The biomolecules are built according to a structural hierarchy: Simple molecules are the units for building complex structures. The molecular constituents of living matter do not randomly reflect the infinite possibilities for combining C, H, O, and N atoms. Instead, only a limited set of the many possibilities is found, and these collections share certain properties essential to the establishment and maintenance of the living state. The most prominent aspect of biomolecular organization is that macromolecular structures are constructed from simple molecules according to a hierarchy of increasing structural complexity. What properties do these biomolecules possess that make them so appropriate for the condition of life?

Metabolites Are Used to Form the Building Blocks of Macromolecules The major precursors for the formation of biomolecules are water, carbon dioxide, and 3 inorganic nitrogen compounds: ammonium (NH4+), nitrate (NO3–), and dinitrogen (N2). Metabolic processes assimilate and transform these inorganic precursors through ever more complex levels of biomolecular order (Figure 1.8 on page 9). In the first step, precursors are converted to metabolites, simple organic compounds that are intermediates in cellular energy transformation and in the biosynthesis of various sets of building blocks: amino NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 1

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

LINEAR ALIPHATIC: Stearic acid HOOC

(CH2)16

CH3

CH2

O

CH2 CH2

C

CH2 CH2

CH2 CH2

CH2 CH2

CH2 CH2

CH2 CH2

CH2 CH2

CH3 CH2

OH

CH3

CYCLIC: H

Cholesterol

C

H CH2

CH2

CH2

C

H3C

CH3

CH3

H3C

HO

BRANCHED: !-Carotene H3C

CH3

CH3

H3C

CH3

CH3

CH3

H3C

CH3

CH3

PLANAR: Chlorophyll a H3C

H2C

HC

CH2CH3

CH3

N N

2+

Mg

N

N

H3C

H3C

O

CH2 CH2

C

OCH3

O CH3

C O

O

CH2

CH

C

CH2

CH3 CH2

CH2

CH

CH3 CH2

CH2

CH2

CH

CH3 CH2

CH2

CH2

CH

CH3

FIGURE 1.7 Examples of the versatility of C—C bonds in building complex structures: linear, cyclic, branched, and planar. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

7

8

Part 1 Building Blocks of the Cell

TABLE 1.2 Biomolecular Dimensions The dimensions of mass* and length for biomolecules are given typically in daltons and nanometers,† respectively. One dalton (D) is the mass of 1 hydrogen atom, 1.67 $ 10!24 g. One nanometer (nm) is 10!9 m, or 10 Å (angstroms). Mass Length (long dimension, nm)

Biomolecule

Daltons

Water

0.3

Alanine

0.5

89

Glucose

0.7

180

18

Phospholipid

3.5

Ribonuclease (a small protein)

4

12 600

14

150 000

Immunoglobulin G (IgG) Myosin (a large muscle protein)

750

160

470 000

Ribosome (bacteria)

18

2 520 000

Bacteriophage !X174 (a very small bacterial virus)

25

4 700 000

Pyruvate dehydrogenase complex (a multienzyme complex) Tobacco mosaic virus (a plant virus) Mitochondrion (liver)

60

7 000 000

300

40 000 000

1500

Picograms

6.68 $ 10!5 1.5

Escherichia coli cell

2000

2

Chloroplast (spinach leaf)

8000

60

Liver cell

20 000

8000

*Molecular mass is expressed in units of daltons (D) or kilodaltons (kD) in this book; alternatively, the dimensionless term molecular weight, symbolized by Mr, and defined as the ratio of the mass of a molecule to 1 D of mass, is used. †Prefixes used for powers of 10 are mega M 10!6 micro " 106 kilo k 10!9 nano n 103 !1 !12 deci d 10 pico p 10 !2 !15 centi c 10 femto f 10 m 10!3 milli

acids, sugars, nucleotides, fatty acids, and glycerol. Through covalent linkage of these building blocks, the macromolecules are constructed: proteins, polysaccharides, polynucleotides (DNA and RNA), and lipids. (Strictly speaking, lipids contain relatively few building blocks and are therefore not really polymeric like other macromolecules; however, lipids are important contributors to higher levels of complexity.) Interactions among macromolecules lead to the next level of structural organization, supramolecular complexes. Here, various members of 1 or more of the classes of macromolecules come together to form specific assemblies that serve important subcellular functions. Examples of these supramolecular assemblies are multifunctional enzyme complexes, ribosomes, chromosomes, and cytoskeletal elements. For example, a eukaryotic ribosome contains 4 different RNA molecules and at least 70 unique proteins. These supramolecular assemblies are an interesting contrast to their components because their structural integrity is maintained by non-covalent forces, not by covalent bonds. These non-covalent forces include hydrogen bonds, ionic attractions, van der Waals forces, and hydrophobic interactions between macromolecules. Such forces maintain these supramolecular assemblies in a highly ordered functional state. Although non-covalent forces are weak (less than 40 kJ/mol), they are numerous in these assemblies and thus can collectively maintain the essential architecture of the supramolecular complex under conditions of temperature, pH, and ionic strength that are consistent with cell life. Organelles Represent a Higher Order in Biomolecular Organization The next higher rung in the hierarchical ladder is occupied by the organelles, entities of considerable dimensions compared with the cell itself. Organelles are found only in eukaryotic cells, that is, the cells of “higher” organisms (eukaryotic cells are described in Section 1.5). Several kinds, such as mitochondria and chloroplasts, evolved from bacteria that gained entry to NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 1

O

C

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

The inorganic precursors: (18–64 daltons) Carbon dioxide, Water, Ammonia, Nitrogen(N2), Nitrate(NO3–)

O

Carbon dioxide Metabolites: (50–250 daltons) Pyruvate, Citrate, Succinate, Glyceraldehyde-3-phosphate, Fructose-1,6-bisphosphate, 3-Phosphoglyceric acid

O H

C

C H

H

C

O O

–

Pyruvate H H H

N

H

H C

+

O

H C

C

–

O

Building blocks: (100–350 daltons) Amino acids, Nucleotides, Monosaccharides, Fatty acids, Glycerol

H Alanine (an amino acid)

Macromolecules: (103–109 daltons) Proteins, Nucleic acids, Polysaccharides, Lipids

–OOC

NH3+ Protein Supramolecular complexes: (106–109 daltons) Ribosomes, Cytoskeleton, Multienzyme complexes

Organelles: Nucleus, Mitochondria, Chloroplasts, Endoplasmic reticulum, Golgi apparatus, Vacuole

The cell

FIGURE 1.8 Molecular organization in the cell is a hierarchy.

the cytoplasm of early eukaryotic cells. Organelles share 2 attributes: They are cellular inclusions, usually membrane bounded, and they are dedicated to important cellular tasks. Organelles include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, and vacuoles, as well as other relatively small cellular inclusions, such as peroxisomes, lysosomes, and chromoplasts. The nucleus is the repository of genetic information NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

9

10

Part 1 Building Blocks of the Cell

as contained within the linear sequences of nucleotides in the DNA of chromosomes. Mitochondria are the “power plants” of cells by virtue of their ability to carry out the energy-releasing aerobic metabolism of carbohydrates and fatty acids, capturing the energy in metabolically useful forms such as ATP. Chloroplasts endow cells with the ability to carry out photosynthesis. They are the biological agents for harvesting light energy and transforming it into metabolically useful chemical forms.

Membranes Are Supramolecular Assemblies That Define the Boundaries of Cells Membranes define the boundaries of cells and organelles. As such, they are not easily classified as supramolecular assemblies or organelles, although they share the properties of both. Membranes resemble supramolecular complexes in their construction because they are complexes of proteins and lipids maintained by non-covalent forces. Hydrophobic interactions are particularly important in maintaining membrane structure. Hydrophobic interactions arise because water molecules prefer to interact with each other rather than with non-polar substances. The presence of non-polar molecules lessens the range of opportunities for water–water interaction by forcing the water molecules into ordered arrays around the nonpolar groups. Such ordering can be minimized if the individual non-polar molecules redistribute from a dispersed state in the water into an aggregated organic phase surrounded by water. The spontaneous assembly of membranes in the aqueous environment where life arose and exists is the natural result of the hydrophobic (“water fearing”) character of their lipids and proteins. Hydrophobic interactions are the creative means of membrane formation and the driving force that presumably established the boundary of the first cell. The membranes of organelles, such as nuclei, mitochondria, and chloroplasts, differ from one another, with each having a characteristic protein and lipid composition tailored to the organelle’s function. Furthermore, the creation of discrete volumes or compartments within cells is not only an inevitable consequence of the presence of membranes but usually an essential condition for proper organellar function.

The Unit of Life Is the Cell The cell is characterized as the unit of life, the smallest entity capable of displaying the attributes associated uniquely with the living state: growth, metabolism, stimulus response, and replication. In the previous discussions, we explicitly narrowed the infinity of chemical complexity potentially available to organic life and we previewed an organizational arrangement, moving from simple to complex, that provides interesting insights into the functional and structural plan of the cell. Nevertheless, we find no obvious explanation within these features for the living characteristics of cells. Can we find other themes represented within biomolecules that are explicitly chemical yet anticipate or illuminate the living condition?

1.4

How Do the Properties of Biomolecules Reflect Their Fitness to the Living Condition?

If we consider what attributes of biomolecules render them so fit as components of growing, replicating systems, several biologically relevant themes of structure and organization emerge. Furthermore, as we study biochemistry, we will see that these themes serve as principles of biochemistry. Prominent among them is the necessity for information and energy in the maintenance of the living state. Some biomolecules must have the capacity to contain the information, or “recipe,” of life. Other biomolecules must have the capacity to translate this information so that the organized structures essential to life are synthesized. Interactions between these structures are the processes of life. An orderly mechanism for abstracting energy from the environment must also exist in order to obtain the energy needed to drive these processes. What properties of biomolecules endow them with the potential for such remarkable qualities? NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 1

11

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

Biological Macromolecules and Their Building Blocks Have a “Sense” or Directionality The macromolecules of cells are built of units—amino acids in proteins, nucleotides in nucleic acids, and carbohydrates in polysaccharides—that have structural polarity. That is, these molecules are not symmetrical, and so they can be thought of as having a “head” and a “tail.” Polymerization of these units to form macromolecules occurs by head-to-tail linear connections. Because of this, the polymer also has a head and a tail, and hence, the macromolecule has a “sense” or direction to its structure (Figure 1.9).

Biological Macromolecules Are Informational Because biological macromolecules have a sense to their structure, the sequential order of their component building blocks, when read along the length of the molecule, has the capacity to specify information in the same manner that the letters of the alphabet can form

(a) Amino acid R1

+

C H+3N

...

R2

C H2O

C

Sugar 4

HO

CH2OH

+

O 3

HO

2 1

4

6

5

Polysaccharide HO

CH2OH

HO

3

HO

2 1

1

Sense

OH

HO

P

N

5'

OCH2

O

2'

O

N

1' 3'

OH OH

N

N HO

P

NH2 3'

OH OH

Sense

OH

FIGURE 1.9 (a) Amino acids build proteins. (b) Polysaccharides are built by joining sugars together. (c) Nucleic acids are polymers of nucleotides. All these polymerization processes involve bond formations accompanied by the elimination of water (dehydration synthesis reactions).

O

H2O

2'

3'

O

N

5'

OCH2

O–

4'

OH

NH2

O

....

...........

+

O– 1'

3'

CH2OH

Nucleic acid

N

O

4'

4

NH2

O

N

5'

O

HO

O

O–

PO4

1

OH

N

5'

HO

H2O

Nucleotide

O OCH2

O

HO

Nucleotide

P

CH2OH

HO

NH2

HO

R2

O

OH

4

(c)

H

O

.....

HO

C

Sugar

6

5

................

HO

COO–

N C

H+3N

COO–

Sense

(b)

R1 H

H

C H+3N

COO–

N

HO

H

...

H

Polypeptide

Amino acid

O

N

2'

O

OH

P

OCH2

O–

N O

3'

OH OH

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

N N

12

Part 1 Building Blocks of the Cell

FIGURE 1.10 The sequence of monomeric units in a biological polymer has the potential to contain information if the diversity and order of the units are not overly simple or repetitive. Nucleic acids and proteins are information-rich molecules; polysaccharides are not.

A strand of DNA 5'

T T C

A G C A A T A A G G G T C C T A C G G A G

3'

A polypeptide segment Phe

Ser

Asn

Lys

Gly

Pro

Thr

Glu

A polysaccharide chain Glc

Glc

Glc

Glc

Glc

Glc

Glc

Glc

Glc

words when arranged in a linear sequence (Figure 1.10 above). Not all biological macromolecules are rich in information. Polysaccharides are often composed of the same sugar unit repeated over and over, as in cellulose or starch, which are homopolymers of many glucose units. On the other hand, proteins and polynucleotides are typically composed of building blocks arranged in no obvious repetitive way; that is, their sequences are unique, akin to the letters and punctuation that form this descriptive sentence. In these unique sequences lies meaning. Discerning the meaning, however, requires some mechanism for recognition.

Biomolecules Have Characteristic Three-Dimensional Architecture The structure of any molecule is a unique and specific aspect of its identity. Molecular structure reaches its pinnacle in the intricate complexity of biological macromolecules, particularly the proteins. Although proteins are linear sequences of covalently linked amino acids, the course of the protein chain can turn, fold, and coil in the 3 dimensions of space to establish a specific, highly ordered architecture that is an identifying characteristic of the given protein molecule (Figure 1.11).

Weak Forces Maintain Biological Structure and Determine Biomolecular Interactions Covalent bonds hold atoms together so that molecules are formed. In contrast, weak chemical forces or non-covalent bonds (hydrogen bonds, van der Waals forces, ionic interactions, and hydrophobic interactions) are intramolecular or intermolecular attractions between atoms. None of these forces, which typically range from 4 to 30 kJ/mol, are strong enough to bind free atoms together (Table 1.3). The average kinetic energy of molecules at 25°C is 2.5 kJ/mol, so the energy of weak forces is only several times greater than the dissociating tendency due to thermal motion of molecules. Thus, these weak forces create interactions that are constantly forming and breaking at physiological temperature, unless by cumulative number they impart stability to the structures generated by their collective action. These weak forces merit further discussion because their attributes profoundly influence the nature of the biological structures they build.

FIGURE 1.11 Antigen-binding domain of immunoglobulin G (IgG).

Van der Waals Attractive Forces Play an Important Role in Biomolecular Interactions van der Waals forces are the result of induced electrical interactions between closely approaching atoms or molecules as their negatively charged electron clouds fluctuate instantaneously in time. These fluctuations allow attractions to occur between the positively charged nuclei and the electrons of nearby atoms. Van der Waals attractions operate only over a very limited interatomic distance (0.3–0.6 nm) and are an effective bonding interaction at physiological temperatures only when a number of atoms in a molecule can interact with several atoms in a neighbouring molecule. For this to occur, the atoms on interacting molecules must pack together neatly. That is, their molecular surfaces must possess a degree of structural complementarity (Figure 1.12). NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 1

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

13

TABLE 1.3 Weak Chemical Forces and Their Relative Strengths and Distances Strength (kJ/mol)

Force

Distance (nm)

Description

Van der Waals interactions

0.4–4.0

0.3–0.6

Strength depends on the relative size of the atoms or molecules and the distance between them. The size factor determines the area of contact between 2 molecules: The greater the area, the stronger the interaction.

Hydrogen bonds

12–30

0.3

Relative strength is proportional to the polarity of the H bond donor and H bond acceptor. More polar atoms form stronger H bonds.

Ionic interactions

20

0.25

Strength also depends on the relative polarity of the interacting charged . . . !OCO h species. Some ionic interactions are also H bonds: h NH" 3

Hydrophobic interactions

#40

—

Force is a complex phenomenon determined by the degree to which the structure of water is disordered as discrete hydrophobic molecules or molecular regions coalesce.

At best, van der Waals interactions are weak and individually contribute 0.4–4.0 kJ/ mol of stabilization energy. However, the sum of many such interactions within a macromolecule or between macromolecules can be substantial. Calculations indicate that the attractive van der Waals energy between the enzyme lysozyme and a sugar substrate that it binds is about 60 kJ/mol. When 2 atoms approach each other so closely that their electron clouds interpenetrate, strong repulsive van der Waals forces occur, as shown in Figure 1.13. Between the repulsive and attractive domains lies a low point in the potential curve. This low point defines the distance known as the van der Waals contact distance, which is the interatomic distance that results if only van der Waals forces hold 2 atoms together. The limit of approach of 2 atoms is determined by the sum of their van der Waals radii (Table 1.4).

Hydrogen Bonds Are Important in Biomolecular Interactions Hydrogen bonds form between a hydrogen atom covalently bonded to an electronegative atom (such as oxygen or nitrogen) and a second electronegative atom that serves as the hydrogen bond acceptor. Several important biological examples are given in Figure 1.14. Hydrogen bonds, at a strength of 12–30 kJ/mol, are stronger than van der Waals forces and have an additional property: H bonds are cylindrically symmetrical and tend to be highly directional, forming straight bonds between donor, hydrogen, and acceptor atoms. Hydrogen bonds are also more specific than van der Waals interactions because they require the presence of complementary hydrogen donor and acceptor groups.

(a)

(b) Phe 91 Tyr 32

...

Tyr 101

Trp 92

Gln 121

FIGURE 1.12 Van der Waals packing is enhanced in molecules that are structurally complementary. Gln121, a surface protuberance on lysozyme, is recognized by the antigen-binding site of an antibody against lysozyme. Gln121 (pink) fits nicely in a pocket formed by Tyr32 (orange), Phe91 (light green), Trp92 (dark green), and Tyr101 (blue) components of the antibody. (See also Figure 1.16.) (a) Ball-and-stick model. (b) Space-filling representation. (From Amit, A.G., et al., 1986. Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. Science 233: 747–753, figure 5.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

14

Part 1 Building Blocks of the Cell Sum of van der Waals radii

Energy (kJ/mol)

2.0

TABLE 1.4 Radii of the Common Atoms of Biomolecules Van der Waals Radius (nm)

Covalent Radius (nm)

H

0.1

0.037

C

0.17

0.077

N

0.15

0.070

O

0.14

0.066

P

0.19

0.096

S

0.185

0.104

Half-thickness of an aromatic

0.17

—

Atom 1.0

0 van der Waals contact distance

–1.0 0

0.2

0.4 r (nm)

0.6

0.8

FIGURE 1.13 The van der Waals interaction energy profile as a function of the distance, r, between the centres of 2 atoms.

Approximate bond length*

H bonds Bonded atoms O O O N +N N

H H H H H H

0.27 nm 0.26 nm 0.29 nm 0.30 nm 0.29 nm 0.31 nm

O O– N O O N

* Lengths given are distances from the atom covalently linked to the H to the atom H bonded to the hydrogen: O

H O 0.27 nm

Functional groups that are important H-bond donors and acceptors: Donors Acceptors O C

C

O

OH R C

OH

R O

H N H R N

H O

Atom Represented to Scale

Ionic Interactions Ionic interactions are the result of attractive forces between oppositely charged structures, such as negative carboxyl groups and positive amino groups (Figure 1.15). These electrostatic forces average about 20 kJ/mol in aqueous solutions. Typically, the electrical charge is radially distributed, so these interactions may lack the directionality of hydrogen bonds or the precise fit of van der Waals interactions. Nevertheless, because the opposite charges are restricted to sterically defined positions, ionic interactions can impart a high degree of structural specificity. The strength of electrostatic interactions is highly dependent on the nature of the interacting species and the distance, r, between them. Electrostatic interactions may involve ions (species possessing discrete charges), permanent dipoles (having a permanent separation of positive and negative charge), or induced dipoles (having a temporary separation of positive and negative charge induced by the environment). Hydrophobic Interactions Hydrophobic interactions result from the strong tendency of water to exclude non-polar groups or molecules (see Chapter 2). Hydrophobic interactions arise not so much because of any intrinsic affinity of non-polar substances for one another (although van der Waals forces do promote the weak bonding of non-polar substances), but because water molecules prefer the stronger interactions that they share with one another, compared to their interaction with non-polar molecules. Hydrogen-bonding interactions between polar water molecules can be more varied and numerous if non-polar molecules come together to form a distinct organic phase. This phase separation raises the entropy of water because fewer water molecules are arranged in orderly arrays around individual non-polar molecules. It is these preferential interactions between water molecules that “exclude” hydrophobic substances from aqueous solution and drive the tendency of non-polar molecules to cluster together. Thus, non-polar regions of biological macromolecules are often buried in the molecule’s interior to exclude them from the aqueous milieu. The formation of oil droplets as hydrophobic non-polar lipid molecules coalesce in the presence of water is an approximation of this phenomenon. These tendencies have important consequences in the creation and maintenance of the macromolecular structures and supramolecular assemblies of living cells.

N

H P

O

FIGURE 1.14 Some biologically important H bonds.

The Defining Concept of Biochemistry Is “Molecular Recognition Through Structural Complementarity” Structural complementarity is the means of recognition in biomolecular interactions. The complicated and highly organized patterns of life depend on the ability of biomolecules to recognize and interact with one another in very specific ways. Such interactions are NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 1

Ligand: A molecule (or atom) that binds specifically to another molecule (from Latin ligare, to bind).

Mg2+. .

Weak chemical forces underlie the interactions that are the basis of biomolecular recognition. It is important to realize that because these interactions are sufficiently weak, they are readily reversible. Consequently, biomolecular interactions tend to be transient; rigid, static lattices of biomolecules that might paralyze cellular activities are not formed. Instead, a dynamic interplay occurs between metabolites and macromolecules, hormones and receptors, and all the other participants instrumental to life processes. This interplay is initiated upon specific recognition between complementary molecules and ultimately culminates in unique physiological activities. Biological function is achieved through mechanisms based on structural complementarity and weak chemical interactions. This principle of structural complementarity extends to higher interactions essential to the establishment of the living condition. For example, the formation of supramolecular complexes occurs because of recognition and interaction between their various macromolecular components, as governed by the weak forces formed between them. If a sufficient number of weak bonds can be formed, as in macromolecules complementary in structure to one another, larger structures assemble spontaneously. The tendency for non-polar molecules and parts of molecules to come together through hydrophobic interactions also promotes the formation of supramolecular assemblies. Very complex subcellular structures are actually spontaneously formed in an assembly process that is driven by weak forces accumulated through structural complementarity.

O–

.....

...

Biomolecular Recognition Is Mediated by Weak Chemical Forces

NH2

Magnesium ATP

...

fundamental to metabolism, growth, replication, and other vital processes. The interaction of one molecule with another, a protein with a metabolite, for example, can be most precise if the structure of one is complementary to the structure of the other, as in 2 connecting pieces of a puzzle or, in the more popular analogy for macromolecules and their ligands, a lock and its key (Figure 1.16). This principle of structural complementarity is the very essence of biomolecular recognition. Structural complementarity is the significant clue to understanding the functional properties of biological systems. Biological systems from the macromolecular level to the cellular level operate via specific molecular recognition mechanisms based on structural complementarity: A protein recognizes its specific metabolite, a strand of DNA recognizes its complementary strand, sperm recognize an egg. All these interactions involve structural complementarity between molecules.

Puzzle

15

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

O–

N

. O–

N

N N

–O P O P O P O CH 2 O

O

O

O HO OH

Intramolecular ionic bonds between oppositely charged groups on amino acidresidues in a protein – O O C O

...

+ NH3 –O

C

H2C

...

H2C C

+ O– H3N (CH2)4

O

Protein strand

FIGURE 1.15 Ionic bonds in biological molecules.

Lock and key

Ligand Ligand

FIGURE 1.16 Structural complementarity: the pieces of a puzzle, the lock and its key, a biological macromolecule and its ligand—an antigen–antibody complex. The antigen on the right (gold) is a small protein, lysozyme, from hen egg white. The antibody molecule (IgG) (left) has a pocket that is structurally complementary to a surface feature (red) on the antigen. (See also Figure 1.12.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

16

Part 1 Building Blocks of the Cell

FIGURE 1.17 Denaturation and renaturation of the intricate structure of a protein.

Native protein

Denatured protein

Weak Forces Restrict Organisms to a Narrow Range of Environmental Conditions Because biomolecular interactions are governed by weak forces, living systems are restricted to a narrow range of physical conditions. Biological macromolecules are functionally active only within a narrow range of environmental conditions, such as temperature, ionic strength, and relative acidity. Extremes of these conditions disrupt the weak forces essential to maintaining the intricate structure of macromolecules. The loss of structural order in these complex macromolecules, so-called denaturation, is accompanied by loss of function (Figure 1.17 above). As a consequence, cells cannot tolerate reactions in which large amounts of energy are released, nor can they generate a large energy burst to drive energyrequiring processes. Instead, such transformations take place via sequential series of chemical reactions whose overall effect achieves dramatic energy changes, even though any given reaction in the series proceeds with only modest input or release of energy (Figure 1.18). These sequences of reactions are organized to provide for the release of useful energy to The combustion of glucose: C6H12O6 + 6 O2

6 CO2 + 6 H2O + 2870 kJ energy (b) In a bomb calorimeter

(a) In an aerobic cell Glucose

Glucose

Glycolysis

ATP ATP

ATP ATP

ATP 2 Pyruvate ATP

ATP ATP

ATP ATP

ATP ATP Citric acid cycle and oxidative phosphorylation 6 CO2 + 6 H2O

ATP

2870 kJ energy as heat

ATP

ATP

ATP

ATP

ATP

ATP ATP

30–38 ATP

6 CO2 + 6 H2O

FIGURE 1.18 Metabolism is the organized release or capture of small amounts of energy in processes whose overall change in energy is large. (a) Cells can release the energy of glucose in a stepwise fashion and the small “packets” of energy appear in ATP. (b) Combustion of glucose in a bomb calorimeter results in an uncontrolled, explosive release of energy in its least useful form, heat. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 1

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

17

the cell from the breakdown of food or to take such energy and use it to drive the synthesis of biomolecules essential to the living state. Collectively, these reaction sequences constitute cellular metabolism—the ordered reaction pathways by which cellular chemistry proceeds and biological energy transformations are accomplished.

Enzymes Catalyze Metabolic Reactions The sensitivity of cellular constituents to environmental extremes places another constraint on the reactions of metabolism. The rate at which cellular reactions proceed is a very important factor in maintenance of the living state. However, the common ways chemists accelerate reactions are not available to cells; the temperature cannot be raised, acid or base cannot be added, the pressure cannot be elevated, and concentrations cannot be dramatically increased. Instead, biomolecular catalysts mediate cellular reactions. These catalysts, called enzymes, accelerate the reaction rates many orders of magnitude and, by selecting the substances undergoing reaction, determine the specific reaction that takes place. Virtually every metabolic reaction is catalyzed by an enzyme (Figure 1.19). Metabolic Regulation Is Achieved by Controlling the Activity of Enzymes Thousands of reactions mediated by an equal number of enzymes are occurring at any given instant within the cell. Collectively, these reactions constitute cellular metabolism. Metabolism has many branch points, cycles, and interconnections, as subsequent chapters reveal. All these reactions, many of which are at apparent cross-purposes in the cell, must be fine-tuned and integrated so that metabolism and life proceed harmoniously. The need for metabolic regulation is obvious. This metabolic regulation is achieved through controls on enzyme activity so that the rates of cellular reactions are appropriate to cellular requirements. Despite the organized pattern of metabolism and the thousands of enzymes required, cellular reactions nevertheless conform to the same thermodynamic principles that govern any chemical reaction. Enzymes have no influence over energy changes (the thermodynamic component) in their reactions. Enzymes only influence reaction rates. Thus, cells are systems that take in food, release waste, and carry out complex degradative and biosynthetic reactions essential to their survival while operating under conditions of essentially constant temperature and pressure and maintaining a constant internal environment (homeostasis) with no outwardly apparent changes. Cells are open thermodynamic systems exchanging matter and energy with their environment and functioning as highly regulated isothermal chemical engines.

FIGURE 1.19 Carbonic anhydrase, a representative enzyme.

The Time Scale of Life Individual organisms have lifespans ranging from a day or less to a century or more, but the phenomena that characterize and define living systems have durations ranging over 33 orders of magnitude, from 10–15 sec (electron transfer reactions, photo-excitation in photosynthesis) to 1018 sec (the period of evolution, spanning from the first appearance of organisms on Earth more than 3 billion years ago to today) (Table 1.5). Because proteins are the agents of biological function, phenomena involving weak interactions and proteins dominate the shorter times. As time increases, more stable interactions (covalent bonds) and phenomena involving the agents of genetic information (the nucleic acids) come into play.

1.5

What Is the Organization and Structure of Cells?

All living cells fall into 1 of 3 broad categories: archaea, bacteria, and eukarya (Figure 1.20 on page 19). Archaea and bacteria are referred to collectively as prokaryotes. As a group, prokaryotes are single-celled organisms that lack nuclei and other organelles; the word is derived from pro meaning “prior to” and karyot meaning “nucleus.” In conventional biological classification schemes, prokaryotes are grouped together as members of the kingdom Monera. The other 4 living kingdoms are all Eukaryota: the single-celled protists, such as amoebas, and all multicellular life forms, including the fungi, plant, and animal kingdoms. Eukaryotic cells have true nuclei and other organelles such as mitochondria, with the prefix eu meaning “true.” NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

18

Part 1 Building Blocks of the Cell

TABLE 1.5 Life Times Time (sec)

Process

Example

10!15

Electron transfer

The light reactions in photosynthesis

10!13

Transition states

Transition states in chemical reactions have lifetimes of 10!11 to 10!15 sec (the reciprocal of the frequency of bond vibrations)

10!11

H-bond lifetimes

H bonds are exchanged between H2O molecules due to the rotation of the water molecules themselves

10!12 – 103

Motion in proteins

Fast: tyrosine ring flips, methyl group rotations Slow: bending motions between protein domains

10!6 – 100

Enzyme catalysis

10!6 sec: fast enzyme reactions 10!3 sec: typical enzyme reactions 100 sec: slow enzyme reactions

100

Diffusion in membranes

A typical membrane lipid molecule can diffuse from one end of a bacterial cell to the other in 1 sec; a small protein would go half as far

101 – 102

Protein synthesis

Some ribosomes synthesize proteins at a rate of 20 amino acids added per second

104 – 105

Cell division

Prokaryotic cells can divide as rapidly as every hour or so; eukaryotic cell division varies greatly (from hours to years)

107 – 108

Embryonic development

Human embryonic development takes 9 months (2.4 $ 108 sec)

105 – 109

Life span

Human life expectancy is 77.6 years (about 2.5 $ 109 sec)

1018

Evolution

The first organisms appeared 3.5. $ 109 years ago and evolution has continued since then

The Evolution of Early Cells Gave Rise to Eubacteria, Archaea, and Eukaryotes For a long time, most biologists believed that eukaryotes evolved from the simpler prokaryotes in some linear progression from simple to complex over the course of geological time. However, contemporary evidence favours the view that present-day organisms are better grouped into the 3 classes mentioned: eukarya, bacteria, and archaea. All are believed to have evolved approximately 3.5 billion years ago from an ancestral communal gene pool shared among primitive cellular entities. Furthermore, contemporary eukaryotic cells are, in reality, composite cells that harbour various bacterial contributions. Despite great diversity in form and function, cells and organisms share much biochemistry in common. This commonality and diversity has been substantiated by the results of whole genome sequencing, the determination of the complete nucleotide sequence within the DNA of an organism. For example, the genome of the metabolically divergent archaea Methanococcus jannaschii shows 44% similarity to known genes in eubacteria and eukaryotes, yet 56% of its genes are new to science.

How Many Genes Does a Cell Need? Gene: A unit of hereditary information, physically defined by a specific sequence of nucleotides in DNA; in molecular terms, a gene is a nucleotide sequence that encodes a protein or RNA product.

The genome of the Mycoplasma genitalium consists of 523 genes, encoding 484 proteins, in just 580 074 base pairs (Table 1.6 on page 20). This information sparks an interesting question: How many genes are needed for cellular life? Any minimum gene set must encode all the information necessary for cellular metabolism, including the vital functions essential to reproduction. The simplest cell must show at least (1) some degree of metabolism and energy production; (2) genetic replication based on a template molecule that encodes information (DNA or RNA?); and (3) formation and maintenance of a cell boundary (membrane). Top-down studies aim to discover from existing cells what a minimum gene set might be. These studies have focused on simple parasitic bacteria, because parasites often obtain many substances from their hosts and do not have to synthesize them from scratch; thus, they require fewer genes. One study concluded that 206 genes are sufficient to form NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Animals Vertebrates

Mosses, liverworts

Echinoderms

Ferns Brown algae Red algae

Urochordates

Arthropods

Neurosporas

Nematodes Mollusks

Green algae

Coelenterates Diatoms

Yeasts

Sponges

Slime molds

Amoeba Heliozoans

oto

a zo

Pr

Ciliates Dinoflagellates

Microsporidiae Cyanobacteria

Chloroplasts Purple bacteria

Myxobacteria Flavobacteria

Methanogens

Mitochondria Gram positive bacteria Bacteria

Ancestral prokaryote

Thermoacidophiles

Archaea

Halophiles

Modified from Voet, D. and Voet, J.G., 2011. Biochemistry, 4th ed. John Wiley and Sons, Hoboken, NJ; Figure 1-11.

Fungi Ascobolis

Prokaryotes

Angiosperms

Multicellular Eukaryotes

Plants Gymnosperms

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

Single-Cell Eukaryotes

Chapter 1

FIGURE 1.20 The phylogenetic “tree of life.” All life arose from an ancestral prokaryote which gave rise to the archaea, the bacteria, and the eukarya.

a minimum gene set. The set included genes for DNA replication and repair, transcription, translation, protein processing, cell division, membrane structure, nutrient transport, metabolic pathways for ATP synthesis, and enzymes to make a small number of metabolites that might not be available, such as pentoses for nucleotides. Yet another study based on computer modelling decided that a minimum gene set might have only 105 protein-coding genes. Bottom-up studies aim to create a minimal cell by reconstruction based on known cellular components. At this time, no such bottom-up creation of an artificial cell has been reported. The simplest functional artificial cell capable of replication would contain an informational macromolecule (presumably a nucleic acid) and enough metabolic apparatus to maintain a basic set of cellular components within a membrane-like boundary.

Archaea and Bacteria Have a Relatively Simple Structural Organization The bacteria form a widely spread group. Certain of them are pathogenic to humans. The archaea, about which we know less, are remarkable because they can be found in unusual environments where other cells cannot survive. Archaea include the thermoacidophiles (heat- and acid-loving bacteria) of hot springs, the halophiles (salt-loving bacteria) of salt lakes and ponds, and the methanogens (bacteria that generate methane from CO2 and H2). Prokaryotes are typically very small, on the order of several microns in length, and are usually surrounded by a rigid cell wall that protects the cell and gives it its shape. The characteristic structural organization of 1 of these cells is depicted in Figure 1.21. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

19

Part 1 Building Blocks of the Cell

TABLE 1.6 How Many Genes Does It Take To Make An Organism? Number of Cells in Adult*

Organism Mycobacterium genitalium

Number of Genes

1

523

1

1800

1

4400

1

6000

959

19 000

104

13 500

107

27 000

a pathogenic bacterium Methanococcus jannaschii an archaeal methanogen Escherichia coli K12 an intestinal bacterium Saccharomyces cereviseae baker’s yeast (eukaryote) Caenorhabditis elegans a nematode worm Drosophila melanogaster a fruit fly Arabidopsis thaliana a flowering plant Fugu rubripes

1012

38 000 (est.)

1014

20 500 (est.)

a pufferfish Homo sapiens a human

David M. Phillips/The Population Council/Science Source/Photo Researchers, Inc.

The first 4 of the 9 organisms in the table are single-celled microbes; the last 6 are eukaryotes; the last 5 are multicellular, 4 of which are animals; the final 2 are vertebrates. Although pufferfish and humans have roughly the same number of genes, the pufferfish genome, at 0.365 billion nucleotide pairs, is only one-eighth the size of the human genome. *Numbers for Arabidopsis thaliana, the pufferfish, and human are “order-of-magnitude” rough estimates.

E. coli bacteria

A BACTERIAL CELL

Martin Rotker/ Phototake

20

Ribosomes Nucleoid (DNA) Capsule Flagella

FIGURE 1.21 This bacterium is Escherichia coli, a member of the coliform group of bacteria that colonize the intestinal tract of humans. (See Table 1.7.)

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The Facts of Life: Chemistry Is the Logic of Biological Phenomena

21

Prokaryotic cells have only a single membrane, the plasma membrane or cell membrane. Because they have no other membranes, prokaryotic cells contain no nucleus or organelles. Nevertheless, they possess a distinct nuclear area where a single circular chromosome is localized, and some have an internal membranous structure called a mesosome that is derived from and continuous with the cell membrane. Reactions of cellular respiration are localized on these membranes. In cyanobacteria, flat, sheetlike membranous structures called lamellae are formed from cell membrane infoldings. These lamellae are the sites of photosynthetic activity, but they are not contained within plastids, the organelles of photosynthesis found in higher plant cells. Prokaryotic cells also lack a cytoskeleton; the cell wall maintains their structure. Some bacteria have flagella, single, long filaments used for motility. Prokaryotes largely reproduce by asexual division, although sexual exchanges can occur. Table 1.7 lists the major features of bacterial cells.

The Structural Organization of Eukaryotic Cells Is More Complex Than That of Prokaryotic Cells Compared with prokaryotic cells, eukaryotic cells are much greater in size, typically having cell volumes 103–104 times larger. They are also much more complex. These 2 features require that eukaryotic cells partition their diverse metabolic processes into organized compartments, with each compartment dedicated to a particular function. A system of internal membranes accomplishes this partitioning. A typical animal cell is shown in Figure 1.22 and a typical plant cell in Figure 1.23 (on page 23). Tables 1.8 (on page 24) and 1.9 (on page 25) list the major features of a typical animal cell and a higher plant cell respectively.

TABLE 1.7 Major Features of Prokaryotic Cells Structure

Molecular Composition

Function

Cell wall

Peptidoglycan: a rigid framework of polysaccharide cross-linked by short peptide chains. Some bacteria possess a lipopolysaccharide- and protein-rich outer membrane.

Mechanical support, shape, and protection against swelling in hypotonic media. The cell wall is a porous nonselective barrier that allows most small molecules to pass.

Cell membrane

The cell membrane is composed of about 45% lipid and 55% protein. The lipids form a bilayer that is a continuous non-polar hydrophobic phase in which the proteins are embedded.

The cell membrane is a highly selective permeability barrier that controls the entry of most substances into the cell. Important enzymes in the generation of cellular energy are located in the membrane.

Nuclear area or nucleoid

The genetic material is a single, tightly coiled DNA molecule 2 nm in diameter but more than 1 mm in length (molecular mass of E. coli DNA is 3 $ 109 D; 4.64 $ 106 nucleotide pairs).

DNA provides the operating instructions for the cell; it is the repository of the cell’s genetic information. During cell division, each strand of the double-stranded DNA molecule is replicated to yield 2 double-helical daughter molecules. Messenger RNA (mRNA) is transcribed from DNA to direct the synthesis of cellular proteins.

Ribosomes

Bacterial cells contain about 15 000 ribosomes. Each is composed of a small (30S) subunit and a large (50S) subunit. The mass of a single ribosome is 2.3 $ 106 D. It consists of 65% RNA and 35% protein.

Ribosomes are the sites of protein synthesis. The mRNA binds to ribosomes, and the mRNA nucleotide sequence specifies the protein that is synthesized.

Storage granules

Bacteria contain granules that represent storage forms of polymerized metabolites such as sugars or #-hydroxybutyric acid.

When needed as metabolic fuel, the monomeric units of the polymer are liberated and degraded by energy-yielding pathways in the cell.

Cytosol

Despite its amorphous appearance, the cytosol is an organized, gelatinous compartment that is 20% protein by weight and rich in the organic molecules that are the intermediates in metabolism.

The cytosol is the site of intermediary metabolism, the interconnecting sets of chemical reactions by which cells generate energy and form the precursors necessary for biosynthesis of macromolecules essential to cell growth and function.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Part 1 Building Blocks of the Cell AN ANIMAL CELL

Rough endoplasmic reticulum (plant and animal)

Smooth endoplasmic reticulum Nuclear membrane

Dwight R. Kuhn

Rough endoplasmic reticulum Nucleolus Lysosome

Nucleus

D. W. Fawcett/Visuals Unlimited

Smooth endoplasmic reticulum (plant and animal)

Plasma membrane

Mitochondrion

Golgi body Mitochondrion (plant and animal)

Cytoplasm Filamentous cytoskeleton (microtubules) Keith Porter/Photo Researchers, Inc.

22

FIGURE 1.22 This figure diagrams a rat liver cell, a typical higher animal cell.

Eukaryotic cells possess a discrete, membrane-bounded nucleus, the repository of the cell’s genetic material, which is distributed among a few or many chromosomes. During cell division, equivalent copies of this genetic material must be passed to both daughter cells through duplication and orderly partitioning of the chromosomes by the process known as mitosis. Like prokaryotic cells, eukaryotic cells are surrounded by a plasma membrane. Unlike prokaryotic cells, eukaryotic cells are rich in internal membranes that are differentiated into specialized structures such as the endoplasmic reticulum (ER) and the Golgi apparatus. Membranes also surround certain organelles (e.g., mitochondria and chloroplasts) and various vesicles, including vacuoles, lysosomes, and peroxisomes. The common purpose of these membranous partitionings is the creation of cellular compartments that have specific, organized metabolic functions, such as the mitochondrion’s role as the principal site of cellular energy production. Eukaryotic cells also have a cytoskeleton composed of arrays of filaments that give the cell its shape and its capacity to move. Some eukaryotic cells also have long projections on their surface—cilia or flagella—which provide propulsion. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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23

A PLANT CELL

Chloroplast (plant cell only)

Smooth endoplasmic reticulum Dr. Dennis Kunkel/Phototake

Lysosome Nuclear membrane

Mitochondrion

Nucleolus

Vacuole Nucleus

Dr. Dennis Krunkel/Phototake

Golgi body (plant and animal)

Rough endoplasmic reticulum Chloroplast

Golgi body

Plasma membrane Cellulose wall

Cell wall

Pectin Biophoto Associates/Photo Researchers Inc.

Nucleus (plant and animal)

FIGURE 1.23 This figure diagrams a cell in the leaf of a higher plant. The cell wall, membrane, nucleus, chloroplasts, mitochondria, vacuole, endoplasmic reticulum (ER), and other characteristic features are shown.

1.6

What Are Viruses?

Viruses are supramolecular complexes of nucleic acid, either DNA or RNA, encapsulated in a protein coat and, in some instances, surrounded by a membrane envelope (Figure 1.24 on page 25). Viruses are acellular, but they act as cellular parasites in order to reproduce. The bits of nucleic acid in viruses are, in reality, mobile elements of genetic information. The protein coat serves to protect the nucleic acid and allows it to gain entry to the cells that are its specific hosts. Viruses unique for all types of cells are known: viruses infecting bacteria are called bacteriophages (“bacteria eaters”); different viruses infect animal cells and plant cells. Once the nucleic acid of a virus gains access to its specific host, it typically takes over the metabolic machinery of the host cell, diverting it to the production of virus particles. The host metabolic functions are subjugated to the synthesis of viral nucleic acid and proteins. Mature virus particles arise by encapsulating the nucleic acid within a protein coat called the capsid. Thus, viruses are supramolecular assemblies that act as parasites of cells (Figure 1.25 on page 26). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

24

Part 1 Building Blocks of the Cell

TABLE 1.8 Major Features of a Typical Animal Cell Structure

Molecular Composition

Function

Extracellular matrix

The surfaces of animal cells are covered with a flexible and sticky layer of complex carbohydrates, proteins, and lipids.

This complex coating is cell specific, serves in cell–cell recognition and communication, creates cell adhesion, and provides a protective outer layer.

Cell membrane (plasma membrane)

Roughly 50:50 lipid:protein as a 5 nm-thick continuous sheet of lipid bilayer in which a variety of proteins are embedded.

The plasma membrane is a selectively permeable outer boundary of the cell, containing specific systems— pumps, channels, transporters, receptors—for the exchange of materials with the environment and the reception of extracellular information. Important enzymes are also located here.

Nucleus

The nucleus is separated from the cytosol by a double membrane, the nuclear envelope. The DNA is complexed with basic proteins (histones) to form chromatin fibres, the material from which chromosomes are made. A distinct RNA-rich region within the nucleus, the nucleolus is the site of ribosome assembly.

The nucleus is the repository of genetic information encoded in DNA and organized into chromosomes. During mitosis, the chromosomes are replicated and transmitted to the daughter cells. The genetic information of DNA is transcribed into RNA in the nucleus and passes into the cytosol, where it is translated into protein by ribosomes.

Endoplasmic reticulum (ER) and ribosomes

Flattened sacs, tubes, and sheets of internal membrane extend throughout the cytoplasm of the cell and enclose a large interconnecting series of volumes called cisternae. The ER membrane is continuous with the outer membrane of the nuclear envelope. Portions of the sheetlike areas of the ER are studded with ribosomes, giving rise to rough ER. Eukaryotic ribosomes are larger than prokaryotic ribosomes.

The endoplasmic reticulum is a labyrinthine organelle where both membrane proteins and lipids are synthesized. Proteins made by the ribosomes of the rough ER pass through the ER membrane into the cisternae and can be transported via the Golgi to the periphery of the cell. Other ribosomes unassociated with the ER carry on protein synthesis in the cytosol. The nuclear membrane, ER, Golgi, and additional vesicles are all part of a continuous endomembrane system.

Golgi apparatus

The Golgi is an asymmetrical system of flattened membrane-bounded vesicles often stacked into a complex. The face of the complex nearest the ER is the cis face; that most distant from the ER is the trans face. Numerous small vesicles found peripheral to the trans face of the Golgi contain secretory material packaged by the Golgi.

The Golgi is involved in the packaging and processing of macromolecules for secretion and for delivery to other cellular compartments.

Mitochondria

Mitochondria are organelles surrounded by 2 membranes that differ markedly in their protein and lipid composition. The inner membrane and its interior volume—the matrix—contain many important enzymes of energy metabolism. Mitochondria are about the size of bacteria, ≈1 μm. Cells contain hundreds of mitochondria, which collectively occupy about one-fifth of the cell volume.

Mitochondria are the power plants of eukaryotic cells where carbohydrates, fats, and amino acids are oxidized to CO2 and H2O. The energy released is trapped as high-energy phosphate bonds in ATP.

Lysosomes

Lysosomes are vesicles 0.2–0.5 μm in diameter, bounded by a single membrane. They contain hydrolytic enzymes such as proteases and nucleases that act to degrade cell constituents targeted for destruction. They are formed as membrane vesicles budding from the Golgi apparatus.

Lysosomes function in intracellular digestion of materials entering the cell via phagocytosis or pinocytosis. They also function in the controlled degradation of cellular components. Their internal pH is about 5, and the hydrolytic enzymes they contain work best at this pH.

Peroxisomes

Like lysosomes, peroxisomes are 0.2–0.5 μm, singlemembrane–bounded vesicles. They contain a variety of oxidative enzymes that use molecular oxygen and generate peroxides. They are also formed from membrane vesicles budding from the smooth ER.

Peroxisomes act to oxidize certain nutrients, such as amino acids. In doing so, they form potentially toxic hydrogen peroxide, H2O2, and then decompose it to H2O and O2 by way of the peroxide-cleaving enzyme catalase.

Cytoskeleton

The cytoskeleton is composed of a network of protein filaments: actin filaments (or microfilaments), 7 nm in diameter; intermediate filaments, 8–10 nm; and microtubules, 25 nm. These filaments interact in establishing the structure and functions of the cytoskeleton. This interacting network of protein filaments gives structure and organization to the cytoplasm.

The cytoskeleton determines the shape of the cell and gives it its ability to move. It also mediates the internal movements that occur in the cytoplasm, such as the migration of organelles and mitotic movements of chromosomes. The propulsion instruments of cells— cilia and flagella—are constructed of microtubules.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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25

Function

Cell wall

The cell wall is comprised of cellulose fibres embedded in a polysaccharide/protein matrix; it is thick (>0.1 μm), rigid, and porous to small molecules.

The cell wall provides protection against osmotic or mechanical rupture. The walls of neighbouring cells interact in cementing the cells together to form the plant. Channels for fluid circulation and for cell–cell communication pass through the walls. The structural material confers form and strength on plant tissue.

Cell membrane

Plant cell membranes are similar in overall structure and organization to animal cell membranes but differ in lipid and protein composition.

The plasma membrane of plant cells is selectively permeable, containing transport systems for the uptake of essential nutrients and inorganic ions. A number of important enzymes are localized here.

Nucleus

The nucleus, nucleolus, and nuclear envelope of plant cells are like those of animal cells.

Chromosomal organization, DNA replication, transcription, ribosome synthesis, and mitosis in plant cells are generally similar to the analogous features in animals.

Endoplasmic reticulum, Golgi apparatus, ribosomes, lysosomes, peroxisomes, and cytoskeleton

Plant cells also contain all of these characteristic eukaryotic organelles, essentially in the form described for animal cells.

These organelles serve the same purposes in plant cells that they do in animal cells.

Chloroplasts

Chloroplasts have a double-membrane envelope, an inner volume called the stroma, and an internal membrane system rich in thylakoid membranes, which enclose a third compartment, the thylakoid lumen. Chloroplasts are significantly larger than mitochondria. Other plastids are found in specialized structures such as fruits, flower petals, and roots and have specialized roles.

Chloroplasts are the site of photosynthesis, the reactions by which light energy is converted to metabolically useful chemical energy in the form of ATP. These reactions occur on the thylakoid membranes. The formation of carbohydrate from CO2 takes place in the stroma. Oxygen is evolved during photosynthesis. Chloroplasts are the primary source of energy in the light.

Mitochondria

Plant cell mitochondria resemble the mitochondria of other eukaryotes in form and function.

Plant mitochondria are the main source of energy generation in photosynthetic cells in the dark and in non-photosynthetic cells under all conditions.

Vacuole

The vacuole is usually the most obvious compartment in plant cells. It is a very large vesicle enclosed by a single membrane called the tonoplast. Vacuoles tend to be smaller in young cells; but in mature cells, they may occupy more than 50% of the cell’s volume. Vacuoles occupy the centre of the cell, with the cytoplasm being located peripherally around it. They resemble the lysosomes of animal cells.

Vacuoles function in transport and storage of nutrients and cellular waste products. By accumulating water, the vacuole allows the plant cell to grow dramatically in size with no increase in cytoplasmic volume.

(a)

(b)

Eye of Science/Photo Researchers, Inc.

Molecular Composition

BSIP/Photo Researchers, Inc.

Structure

(c)

FIGURE 1.24 Viruses are genetic elements enclosed in a protein coat. Viruses are not free-living organisms and can reproduce only within cells. Viruses show an almost absolute specificity for their particular host cells, infecting and multiplying only within those cells. Viruses are known for virtually every kind of cell. Shown here are examples of (a) an animal virus, adenovirus; (b) bacteriophage T4 on E. coli; and (c) a plant virus, tobacco mosaic virus. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Science Source/Photo Researchers, Inc.

TABLE 1.9 Major Features of a Higher Plant Cell: A Photosynthetic Leaf Cell

26

Part 1 Building Blocks of the Cell

FIGURE 1.25 The virus life cycle. Viruses are mobile bits of genetic information encapsulated in a protein coat. The genetic material can be either DNA or RNA. Once this genetic material gains entry to its host cell, it takes over the host machinery for macromolecular synthesis and subverts it to the synthesis of viral-specific nucleic acids and proteins. These virus components are then assembled into mature virus particles that are released from the cell. Often, this parasitic cycle of virus infection leads to cell death and disease.

Host cell

Release from cell Replication Entry of virus genome into cell

Assembly

Translation Protein coat

Transcription

RNA

Coat proteins

Genetic material (DNA or RNA)

Often, viruses cause disintegration of the cells that they have infected, a process referred to as cell lysis. It is their cytolytic properties that are the basis of viral disease. In certain circumstances, the viral genetic elements may integrate into the host chromosome and become quiescent. Such a state is termed lysogeny. Typically, damage to the host cell activates the replicative capacities of the quiescent viral nucleic acid, leading to viral propagation and release. Some viruses are implicated in transforming cells into a cancerous state, that is, in converting their hosts to an unregulated state of cell division and proliferation. Because all viruses are heavily dependent on their host for the production of viral progeny, viruses must have evolved after cells were established. Presumably, the first viruses were fragments of nucleic acid that developed the ability to replicate independently of the chromosome and then acquired the necessary genes enabling protection, autonomy, and transfer between cells.

1.7

Do Biomolecules Ever Behave as Genetic Agents?

Prions and Viroids Can Act as Genetic Agents DNA is the genetic material in organisms, although some viruses have RNA genomes. The idea that proteins and RNA could carry genetic information was considered early in the history of molecular biology and dismissed for lack of evidence. Prions and viroids may be an exception to this rule. Prions and viroids represent the simplest of pathogenic agents and are composed simply of protein and RNA respectively. Viroids are small (250–400 nucleotides), single-stranded circular RNAs that do not have protein coding capacity and, unlike viruses, are not encapsulated in a protein or membrane shell. They were first discovered in 1971, with the potato spindle tuber viroid (PSTVd) being the first of these “free” RNA pathogens to be uncovered. Since then, other viroids have been found, including the avocado sunblotch viroid (ASBVd), the peach latent mosaic viroid (PLMVd), the coconut cadang-cadang viroid (CCCVd), the citrus exocortis viroid (CEVd), the cucumber pale fruit viroid (CPFVd), and the hop stunt viroid (HSVd). To date, these RNA pathogens are only found in woody (Avsunviroidae) or herbaceous (Pospiviroidae) plants. They may be linear or highly branched dsRNA molecules, but unlike viruses, they lack a protein coat (Figure 1.26). Viroids contain all the genetic information to mediate their own replication within single plant cells, as well as their systematic transmission throughout the entire plant (Figure 1.27 on page 28). Replication may occur in the chloroplast or the nucleus. Viroids replicate by either asymmetric (Pospiviroidae; such as with PSTVd) or symmetric (Avsunviroidae; such as with ASBVd) rolling circle replication, cleavage, and ligation. Pospiviroidae replicate in the plant cell nucleus and nucleolus, whereas Avsunviroidae replicate in the plant chloroplast. Rolling circle replication is a method of RNA replication in which 1 strand of a double-stranded RNA (dsRNA) molecule is nicked and unwound NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 1

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

Transcription initiation (U121)

Cleavage site (C55-U56) (+) ASBVd

(–) ASBVd Cleavage site (C90-G91)

Transcription initiation (U119)

Transcription initiation (C51) in the (+) strand Cleavage site (A48-G49) (+) PLMVd Cleavage site (C289-U290) Transcription initiation (A286) in the (–) strand

Kissing-loop interactions

Cleavage site (G95-96)

(+) PSTVd

GC box HPII′ Transcription initiation(C1 or U359) TL

Loop E

GC box HPII

Pathogenicity

C

Variable

TR

FIGURE 1.26 Secondary structures of representative viroids from the 2 viroid families, Avsunviroidae: ASBVd and PLMVd, and Pospiviroidae: PSTVd. The transcription initiation sites on the viroid genomic RNAs are indicated. Note that for ASBVd and PSTVd, these sites are mapped to terminal loops. The transcription initiation site for the (–)-PSTVd RNA template remains to be determined. For PLMVd, the dashed lines indicate kissing-loop interactions. For PSTVd, the 5 structural domains (Keese et al., 1985) are indicated. TL = left-terminal domain, C = central domain, and TR = right-terminal domain. HPII′ and HPII indicate nucleotide sequences that base pair to form the metastable hairpin II structure. (Ding, B. and Itaya, A. 2007. Viroid: a useful model for studying the basic principles of infection and RNA biology. Molecular Plant-Microbe Interactions 20(1): 7–20.)

from the dsRNA molecule, serving as a template for the synthesis of the second strand. This requires that the dsRNA molecule encodes for the viroid’s own RNA polymerase, RNAse, and RNA ligase enzymes in the case of the asymmetric replication, and RNA polymerase, ribozyme, and RNA ligase in the case of symmetric replication. A ribozyme is a catalytic RNA molecule (acts like an enzyme, but is not a protein) which, in the case of Avsunviroidae, catalyzes its own cleavage. Ribozymes undertake catalysis (bond breaking and bond making) but are comprised of nucleic acids and not amino acids. They may represent the ancestral form of enzymes, which suggests that life based on ribonucleic acid (RNA) predates the current world of life based on deoxyribonucleic acid (DNA) and protein. This forms the basis of the RNA world hypothesis. Another exciting possibility of viroid pathogenicity is the potential for RNA-mediated gene silencing of host defences. Processing of these dsRNA pathogens may produce micro-RNAs NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

27

28

Part 1 Building Blocks of the Cell

ASBVd 7 Vascular exit and invasion of new cells

2 Replication

Chloroplast 1 Organellar import

3 Organellar export

4 Cell-to-cell trafficking

5 Vascular entry

Nucleus 6 Long-distance trafficking PSTVd FIGURE 1.27 Distinct steps of systemic infection of ASBVd and PSTVd, type members of the 2 viroid families. The mechanisms of the different trafficking steps for the family Avsunviroidae remain to be investigated. (Ding, B. and Itaya, A. 2007. Viroid: a useful model for studying the basic principles of infection and RNA biology. Molecular Plant-Microbe Interactions 20(1): 7–20.)

FIGURE 1.28 Speculative models suggest that (a) PrPc is mostly α-helical, whereas (b) PrPsc has both α-helices and β-strands. (Adapted from Figure 1 in Prusiner, S. B., 1996. Molecular biology and the pathogenesis of prion diseases. Trends in Biochemical Sciences 21:482–487.)

(miRNAs) and short interfering RNAs (siRNAs) that may regulate the production of host defence proteins and lead to viroid symptom expression in plants. RNA interference is outlined in Chapter 31 of this book. Prion is an acronym derived from the words proteinaceous infectious particle. The term prion was coined to distinguish such particles, which are pathogenic and thus capable of causing disease, from nucleic acid–containing infectious particles such as viruses and virions. Prions are transmissible agents (genetic material?) that are apparently composed only of a protein that has adopted an abnormal conformation. They produce fatal degenerative diseases of the central nervous system in mammals and are believed to be the agents responsible for the human diseases kuru, Creutzfeldt-Jakob disease, Gerstmann-StrausslerScheinker syndrome, and fatal familial insomnia. Prions also cause diseases in animals, including scrapie (in sheep), “mad cow disease” (bovine spongiform encephalopathy), and chronic wasting disease (in elk and mule deer). All attempts to show that the infectivity of these diseases is due to a nucleic acid–carrying agent have been unsuccessful. Prion diseases are novel in that they are genetic and infectious; their occurrence may be sporadic, dominantly inherited, or acquired by infection. PrP, the prion protein, comes in various forms, (Figure 1.28) such as PrPc, the normal cellular prion protein, and PrPsc, the scrapie form of PrP, a conformational variant of PrPc (a)

(b)

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Chapter 1

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

29

that is protease resistant, sometimes written as PrPres. These two forms are thought to differ only in terms of their secondary and tertiary structure. One model suggests that PrPc is dominated by α-helical elements (Figure 1.28a), whereas PrPsc has both α-helices and β-strands (Figure 1.28b). It has been hypothesized that the presence of PrPsc can cause PrPc to adopt the PrPsc conformation. The various diseases are a consequence of the accumulation of the abnormal PrPsc form, which accumulates as amyloid plaques (amyloid = starchlike) that cause destruction of tissues in the central nervous system. Ironically, recent evidence suggests that PrPc may function as a nucleic acid–binding protein. The 1997 Nobel Prize in Physiology or Medicine was awarded to Stanley B. Prusiner for his discovery of prions.

SUMMARY 1.1 What Are the Distinctive Properties of Living Systems? Living systems display an astounding array of activities that collectively constitute growth, metabolism, response to stimuli, and replication. In accord with their functional diversity, living organisms are complicated and highly organized entities composed of many cells. In turn, cells possess subcellular structures known as organelles, which are complex assemblies of very large polymeric molecules, or macromolecules. The monomeric units of macromolecules are common organic molecules (metabolites). Biological structures play a role in the organism’s existence. From parts of organisms, such as limbs and organs, down to the chemical agents of metabolism, such as enzymes and metabolic intermediates, a biological purpose can be given for each component. Maintenance of the highly organized structure and activity of living systems requires energy that must be obtained from the environment. Energy is required to create and maintain structures and to carry out cellular functions. In terms of the capacity of organisms to self-replicate, the fidelity of self-replication resides ultimately in the chemical nature of DNA, the genetic material. 1.2 What Kinds of Molecules Are Biomolecules? C, H, N, and O are among the lightest elements capable of forming covalent bonds through electron-pair sharing. Because the strength of covalent bonds is inversely proportional to atomic weight, H, C, N, and O form the strongest covalent bonds. Two properties of carbon covalent bonds merit attention: the ability of carbon to form covalent bonds with itself and the tetrahedral nature of the 4 covalent bonds when carbon atoms form only single bonds. Together these properties hold the potential for an incredible variety of structural forms, whose diversity is multiplied further by including N, O, and H atoms. 1.3 What Is the Structural Organization of Complex Biomolecules? Biomolecules are built according to a structural hierarchy: Simple molecules are the units for building complex structures. H2O, CO2, NH4+, NO3–, and N2 are the inorganic precursors for the formation of simple organic compounds from which metabolites are made. These metabolites serve as intermediates in cellular energy transformation and as building blocks (amino acids, sugars, nucleotides, fatty acids, and glycerol) for lipids and for macromolecular synthesis (synthesis of proteins, polysaccharides, DNA, and RNA). The next higher level of structural organization is created when macromolecules come together through non-covalent interactions to form supramolecular complexes, such as multifunctional enzyme complexes, ribosomes, chromosomes, and cytoskeletal elements. The next higher rung in the hierarchical ladder is occupied by the organelles. Organelles are membrane-bounded cellular inclusions dedicated to important cellular tasks, such as the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, and vacuoles, as well as other relatively small cellular inclusions. At the apex of the biomolecular hierarchy is the cell, the unit of life, the smallest entity displaying those attributes associated uniquely with the living state: growth, metabolism, stimulus response, and replication.

1.4 How Do the Properties of Biomolecules Reflect Their Fitness to the Living Condition? Some biomolecules carry the information of life; others translate this information so that the organized structures essential to life are formed. Interactions between such structures are the processes of life. Properties of biomolecules that endow them with the potential for creating the living state include the following: Biological macromolecules and their building blocks have directionality, and thus biological macromolecules are informational; in addition, biomolecules have characteristic 3-dimensional architectures, providing the means for molecular recognition through structural complementarity. Weak forces (H bonds, van der Waals interactions, ionic attractions, and hydrophobic interactions) mediate the interactions between biological molecules and, as a consequence, restrict organisms to the narrow range of environmental conditions where these forces operate. 1.5 What Is the Organization and Structure of Cells? All cells share a common ancestor and fall into 1 of 2 broad categories, either prokaryotic or eukaryotic, depending on whether the cell has a nucleus. Prokaryotes are typically single-celled organisms and have a simple cellular organization. In contrast, eukaryotic cells are structurally more complex, having organelles and various subcellular compartments defined by membranes. Unlike protists, eukaryotes are multicellular. 1.6 What Are Viruses? Viruses are supramolecular complexes of nucleic acid encapsulated in a protein coat and, in some instances, surrounded by a membrane envelope. Viruses are not alive; they are not even cellular. Instead, they are packaged bits of genetic material that can parasitize cells in order to reproduce. Often, they cause disintegration, or lysis, of the cells they have infected. It is these cytolytic properties that are the basis of viral disease. In certain circumstances, the viral nucleic acid may integrate into the host chromosome and become quiescent, creating a state known as lysogeny. If the host cell is damaged, the replicative capacities of the quiescent viral nucleic acid may be activated, leading to viral propagation and release. 1.7 Do Biomolecules Ever Behave as Genetic Agents? DNA is the genetic material in organisms, although some viruses have RNA genomes. The possibility that proteins and RNA carry genetic information was a point of speculation early in the history of molecular biology but was ultimately discounted for lack of evidence. Viroids (RNA infectious particles) and prions (proteinaceous infectious particles) may be an exception. Viroids are naked RNA molecules that cause disease in herbaceous and woody plants. They are capable of self-replication and propagation within the plant. Some viroids have “ribozyme” activity, being capable of catalytically cleaving themselves and acting like enzymes. Prions are devoid of nucleic acid, yet they can transmit disease. Prion diseases are novel because they may be either inherited (like a genetic agent) or acquired by infection. PrP, the prion protein, comes in several forms: PrPc, the normal cellular protein, and a conformational variant of PrPc known as PrPsc or PrPres found in association with prion diseases. The propensity of the PrPsc conformational form to polymerize into cell-destructive aggregates is thought to be the basis of prion diseases.

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30

Part 1 Building Blocks of the Cell

PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. The nutritional requirements of Escherichia coli cells are far simpler than those of humans, yet the macromolecules found in bacteria are about as complex as those of animals. Because bacteria can make all their essential biomolecules while subsisting on a simpler diet, do you think bacteria may have more biosynthetic capacity and hence more metabolic complexity than animals? Organize your thoughts on this question, pro and con, into a rational argument. 2. Without consulting the figures in this chapter, sketch the characteristic prokaryotic and eukaryotic cell types and label their pertinent organelle and membrane systems. 3. Escherichia coli cells are about 2 μm (microns) long and 0.8 μm in diameter. a. How many E. coli cells laid end to end would fit across the diameter of a pinhead? (Assume a pinhead diameter of 0.5 mm.) b. What is the volume of an E. coli cell? (Assume it is a cylinder, with the volume of a cylinder given by V = πr2h, where π = 3.14.) c. What is the surface area of an E. coli cell? What is the surface-tovolume ratio of an E. coli cell? d. Glucose, a major energy-yielding nutrient, is present in bacterial cells at a concentration of about 1 mM. What is the concentration of glucose, expressed as mg/mL? How many glucose molecules are contained in a typical E. coli cell? (Recall that Avogadro’s number = 6.023 × 1023.) e. A number of regulatory proteins are present in E. coli at only 1 or 2 molecules per cell. If we assume that an E. coli cell contains just 1 molecule of a particular protein, what is the molar concentration of this protein in the cell? If the molecular weight of this protein is 40 kD, what is its concentration, expressed as mg/mL? f. An E. coli cell contains about 15 000 ribosomes, which carry out protein synthesis. Assuming ribosomes are spherical and have a diameter of 20 nm (nanometres), what fraction of the E. coli cell volume is occupied by ribosomes? g. The E. coli chromosome is a single DNA molecule whose mass is about 3 × 109 D. This macromolecule is actually a linear array of nucleotide pairs. The average molecular weight of a nucleotide pair is 660 Mr, and each pair imparts 0.34 nm to the length of the DNA molecule. What is the total length of the E. coli chromosome? How does this length compare with the overall dimensions of an E. coli cell? How many nucleotide pairs does this DNA contain? The average E. coli protein is a linear chain of 360 amino acids. If 3 nucleotide pairs in a gene encode 1 amino acid in a protein, how many different proteins can the E. coli chromosome encode? (The answer to this question is a reasonable approximation of the maximum number of different kinds of proteins that can be expected in bacteria.) 4. Assume that mitochondria are cylinders 1.5 μm in length and 0.6 μm in diameter. a. What is the volume of a single mitochondrion? b. Oxaloacetate is an intermediate in the citric acid cycle, an important metabolic pathway localized in the mitochondria of eukaryotic cells. The concentration of oxaloacetate in mitochondria is about 0.03 μM. How many molecules of oxaloacetate are in a single mitochondrion? 5. Assume that liver cells are cuboidal in shape, 20 μm on a side. a. How many liver cells laid end to end would fit across the diameter of a pinhead? (Assume a pinhead diameter of 0.5 mm.) b. What is the volume of a liver cell? (Assume it is a cube.) c. What is the surface area of a liver cell? What is the surface-to-volume ratio of a liver cell? How does this compare to the surface-to-volume ratio of an E. coli cell (compare this answer with that of problem 3c)?

What problems must cells with low surface-to-volume ratios confront that do not occur in cells with high surface-to-volume ratios? d. A human liver cell contains 2 sets of 23 chromosomes, each set being roughly equivalent in information content. The total mass of DNA contained in these 46 enormous DNA molecules is 4 × 1012 D. Because each nucleotide pair contributes 660 D to the mass of DNA and 0.34 nm to the length of DNA, what is the total number of nucleotide pairs and the complete length of the DNA in a liver cell? How does this length compare with the overall dimensions of a liver cell? The maximal information in each set of liver cell chromosomes should be related to the number of nucleotide pairs in the chromosome set’s DNA. This number can be obtained by dividing the total number of nucleotide pairs just calculated by 2. What is this value? If this information is expressed in proteins that average 400 amino acids in length and 3 nucleotide pairs encode 1 amino acid in a protein, how many different kinds of proteins might a liver cell be able to produce? (In reality, liver cell DNA encodes approximately 20 000 different proteins. Thus, a large discrepancy exists between the theoretical information content of DNA in liver cells and the amount of information actually expressed.) 6. Biomolecules interact with one another through molecular surfaces that are structurally complementary. How can various proteins interact with molecules as different as simple ions, hydrophobic lipids, polar but uncharged carbohydrates, and even nucleic acids? 7. What structural features allow biological polymers to be informational macromolecules? Is it possible for polysaccharides to be informational macromolecules? 8. Why is it important that weak forces, not strong forces, mediate biomolecular recognition? 9. What is the distance between the centres of 2 carbon atoms (their limit of approach) that are interacting through van der Waals forces? What is the distance between the centres of 2 carbon atoms joined in a covalent bond? (See Table 1.4.) 10. Why does the central role of weak forces in biomolecular interactions restrict living systems to a narrow range of environmental conditions? 11. Describe what is meant by the phrase “cells are steady-state systems.” 12. The genome of the Mycoplasma genitalium consists of 523 genes, encoding 484 proteins, in just 580 074 base pairs (Table 1.6). What fraction of the M. genitalium genes encode proteins? What do you think the other genes encode? If the fraction of base pairs devoted to protein-coding genes is the same as the fraction of the total genes that they represent, what is the average number of base pairs per proteincoding gene? If it takes 3 base pairs to specify an amino acid in a protein, how many amino acids are found in the average M. genitalium protein? If each amino acid contributes on average 120 D to the mass of a protein, what is the mass of an average M. genitalium protein? 13. Studies of existing cells to determine the minimum number of genes for a living cell have suggested that 206 genes are sufficient. If the ratio of protein-coding genes to non-protein–coding genes is the same in this minimal organism as the genes of Mycoplasma genitalium, how many proteins are represented in these 206 genes? How many base pairs would be required to form the genome of this minimal organism if the genes are the same size as M. genitalium genes? 14. Virus genomes range in size from approximately 3500 nucleotides to approximately 280 000 base pairs. If viral genes are about the same size as M. genitalium genes, what is the minimum and maximum number of genes in viruses? 15. The endoplasmic reticulum (ER) is a site of protein synthesis. Proteins made by ribosomes associated with the ER may pass into the ER membrane or enter the lumen of the ER. Devise a pathway by which a. a plasma membrane protein may reach the plasma membrane; and b. a secreted protein may be deposited outside the cell. NEL

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Chapter 1

The Facts of Life: Chemistry Is the Logic of Biological Phenomena

31

FURTHER READING General Biology Textbooks

Papers on Early Cell Evolution

Campbell, N. A., and Reece, J. B., 2005. Biology, 7th ed. San Francisco: Benjamin/Cummings.

Margulis, L., 1996. Archaeal-eubacterial mergers in the origin of Eukarya: Phylogenetic classification of life. Proceedings of the National Academy of Science, U.S.A. 93:1071–1076.

Solomon, E. P., Berg, L. R., and Martin, D. W., 2004. Biology, 7th ed. Pacific Grove, CA: Brooks/Cole. Cell and Molecular Biology Textbooks Alberts, B., Johnson, A., et al., 2007. Molecular Biology of the Cell, 5th ed. New York: Garland Press. Lewin, B., Cassimeris, L., Lingappa, V. R., and Plopper, G., 2007. Cells. Boston, MA: Jones and Bartlett. Lodish, H., Berk, A., et al., 2007. Molecular Cell Biology, 5th ed. New York: W. H. Freeman. Snyder, L., and Champness, W., 2002. Molecular Genetics of Bacteria, 2nd ed. Herndon, VA: ASM Press. Watson, J. D., Baker, et al., 2007. Molecular Biology of the Gene, 6th ed. Menlo Park, CA: Benjamin/Cummings. Papers on Cell Structure Gil, R., Silva, F. J., Pereto, J., et al., 2004. Determination of the core of a minimal bacterial gene set. Microbiology and Molecular Biology Reviews 68:518–537. Goodsell, D. S., 1991. Inside a living cell. Trends in Biochemical Sciences 16:203–206. Lewis, P. J., 2004. Bacterial subcellular architecture: Recent advances and future prospects. Molecular Microbiology 54:1135–1150. Lloyd, C., Ed., 1986. Cell organization. Trends in Biochemical Sciences 11:437–485. Papers on Genomes Cho, M. K., Magnus, D., et al., 1999. Ethical considerations in synthesizing a minimal genome. Science 286:2087–2090. Kobayashi, K., Ehrlich, S. D., et al., 2003. Essential Bacillus subtilis genes. Proceedings of the National Academy of Science, U.S.A. 100:4678–4683. Lartigue, C., Glass, J. I., et al., 2007. Genome transplantation in bacteria: Changing one species to another. Science 317:632–638.

Pace, N. R., 2006. Time for a change. Nature 441:289. Service, R. F., 1997. Microbiologists explore life’s rich, hidden kingdoms. Science 275:1740–1742. Wald, G., 1964. The origins of life. Proceedings of the National Academy of Science, U.S.A. 52:595–611. Whitfield, J., 2004. Born in a watery commune. Nature 427:674–676. Woese, C. R., 2002. On the creation of cells. Proceedings of the National Academy of Science, U.S.A. 99:8742–8747. Papers on Prions and Virions Cohen, F. E., and Prusiner, S. B., 1998. Pathological conformations of prion proteins. Annual Review of Biochemistry 67:793–819. Diener, T. O., 1971. Potato spindle tuber “virus.” IV. Replicating, low molecular weight RNA. Virology 45:411–428. Ding, B., Itaya, A., et al., 2005. Viroid trafficking: A small RNA makes a big move. Current Opinion in Plant Biology 8:606–612. Ding, B. and Itaya, A., 2007. Viroid: A useful model for studying the basic principles of infection and RNA biology. Molecular Plant-Microbe Interactions 20:7–20. Prusiner, S. B., 1996. Molecular biology and pathogenesis of prion diseases. Trends in Biochemical Sciences 21:482–487. Prusiner, S. B., 1997. Prion diseases and the BSE crisis. Science 278:245–251. Tabler, M, and Tsagris, M., 2004. Viroids: Petite RNA pathogens with distinguished talents. Trends in Plant Science 9:339–348. A Brief History of Life De Duve, C., 2002. Life-Evolving: Molecules, Mind, and Meaning. New York: Oxford University Press. Morowitz, H., and Smith, E., 2007. Energy flow and the organization of life. Complexity 13:51–59.

Szathmary, E., 2005. In search of the simplest cell. Nature 433:469–470.

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2

Water: The Medium of Life

! Where there’s water, there’s life.

If there is magic on this planet, it is contained in water. Loren Eisley (inscribed on the wall of the National Aquarium in Baltimore, Maryland)

2.1

What Are the Properties of Water?

2.2

What Is pH?

2.3

What Are Buffers and What Do They Do?

2.4

What Properties of Water Give It a Unique Role in the Environment?

© Tetra Images/Alamy

KEY QUESTIONS

ESSENTIAL QUESTION Water provided conditions for the origin, evolution, and flourishing of life; water is the medium of life. What are the properties of water that render it so suited to its role as the medium of life?

WHY IT MATTERS

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

Before we begin our discussion of water, the medium of life, we pose the question, “Why is water the ideal medium to support life?” The properties of water make it an ideal medium to support life on Earth. This includes the ability to form hydrogen bonds with any molecule, its polar nature, its high dielectric constant, its support of hydrophobic/ hydrophilic and colligative interactions, and the temperatures at which it boils or freezes. No other liquid medium on the planet can support life in the same way, given that life has evolved in water. Our macromolecules have been designed, over evolutionary time, to fold and function properly in water. Thus the availability of water is crucial for supporting life as we know it on the planet. Canada, which occupies 7% of the world’s land mass, has 9% of the world’s renewable fresh water supply (Federal Water Policy, Environment Canada, 1987). With the world facing water shortages in some areas and water pollution in others, it is crucial to manage our water resources so that it is available to support life for future generations. Water is a major chemical component of Earth’s surface. It is indispensable to life. Indeed, it is the only liquid that most organisms ever encounter. We are prone to take it for granted because of its ubiquity and bland nature, yet we marvel at its many unusual and fascinating properties. At the centre of this fascination is the role of water as the medium of life. Life originated, evolved, and thrives in the seas. Organisms invaded and occupied terrestrial and aerial niches, but none gained true independence from water. Typically, organisms are 70%–90% water. Indeed, normal metabolic activity can occur only when cells are at least 65% H2O. This dependency of life on water is not a simple NEL

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Chapter 2

Top ten countries on index

Bottom ten countries on index

In descending order

In ascending order

Finland Canada Iceland Norway Guyana Suriname Austria Ireland Sweden Switzerland

Haiti Niger Ethiopia Eritrea Malawi Djibouti Chad Benin Rwanada Burundi

0

Water: The Medium of Life

4000 km

Degree of Water Poverty Severe

High

Medium

Medium low

Low

No data

!

The world’s water poverty index. Canada ranks second best of 147 countries in terms of resources, access, capacity, use, and environment. Despite this, Canada ranked low in the “use” category because of wasteful or inefficient water use in the industrial and domestic sectors. (MAP World Population by fresh water availability http://www.ec.gc.ca/eau-water/587612E5-03F4-4B27-885D2DEF07B8F451/didyouknow.pdf Environment Canada. Reproduced with the permission of the Minister of Public Works and Government Services, 2012.)

matter, but it can be grasped by considering the unusual chemical and physical properties of H2O. Subsequent chapters establish that water and its ionization products, hydrogen ions and hydroxide ions, are critical determinants of the structure and function of many biomolecules, including amino acids and proteins, nucleotides and nucleic acids, and even phospholipids and membranes. In yet another essential role, water is an indirect participant: a difference in the concentration of hydrogen ions on opposite sides of a membrane represents an energized condition essential to biological mechanisms of energy transformation. First, let’s review the remarkable properties of water.

2.1

What Are the Properties of Water?

Water Has Unusual Properties Compared with chemical compounds of similar atomic organization and molecular size, water displays unexpected properties. For example, compare water, the hydride of oxygen, with hydrides of oxygen’s nearest neighbours in the periodic table, namely, ammonia (NH3) and hydrogen fluoride (HF), or with the hydride of its nearest congener, sulfur (H2S). Water has a substantially higher boiling point, melting point, heat of vaporization, and surface tension. Indeed, all of these physical properties are anomalously high for a substance of this molecular weight that is neither metallic nor ionic. These properties suggest that intermolecular forces of attraction between H2O molecules are high. Thus, the internal cohesion of this substance is high. Furthermore, water has an unusually high dielectric constant, its maximum density is found in the liquid (not the solid) state, and it has a negative volume of melting (that is, the solid form, ice, occupies more space than does the liquid form, water). It is truly remarkable that so many eccentric properties occur together in this single substance. As chemists, we expect to find an explanation for these apparent eccentricities in the structure of NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

33

34

Part 1 Building Blocks of the Cell Dipole moment H 104.3°

!+ O

H + !

!–

Covalent bond length = 0.095 nm

van der Waals radius of oxygen = 0.14 nm

van der Waals radius of hydrogen = 0.12 nm

FIGURE 2.1 The structure of water. Two lobes of negative charge formed by the lone-pair electrons of the oxygen atom lie above and below the plane of the diagram. This electron density contributes substantially to the large dipole moment of the water molecule. Note that the H i O i H angle is 104.3°, not 109°, which is the angular value found in molecules with tetrahedral symmetry, such as CH4. Many of the important properties of water derive from this angular value, such as the decreased density of its crystalline state, ice.

water. The key to its intermolecular attractions must lie in its atomic constitution. Indeed, water’s unrivalled ability to form hydrogen bonds is the crucial fact to understanding its properties.

Hydrogen Bonding in Water Is Key to Its Properties The 2 hydrogen atoms of water are linked covalently to oxygen, each sharing an electron pair, to give a nonlinear arrangement (Figure 2.1). This “bent” structure of the H2O molecule has enormous influence on its properties. If H2O were linear, it would be a non-polar substance. In the bent configuration, however, the electronegative O atom and the 2 H atoms form a dipole that renders the molecule distinctly polar. Furthermore, this structure is ideally suited to H-bond formation. Water can serve as both a H donor and a H acceptor in H-bond formation. The potential to form 4 H bonds per water molecule is the source of the strong intermolecular attractions that endow this substance with its anomalously high boiling point, melting point, heat of vaporization, and surface tension. In ordinary ice, the common crystalline form of water, each H2O molecule has 4 nearest neighbours to which it is hydrogen bonded: Each H atom donates a H bond to the O of a neighbour, and the O atom serves as a H-bond acceptor from H atoms bound to 2 different water molecules (Figure 2.2). A local tetrahedral symmetry results. Hydrogen bonding in water is cooperative. That is, a H-bonded water molecule serving as an acceptor is a better H-bond donor than an unbonded molecule (and a H2O molecule serving as a H-bond donor becomes a better H-bond acceptor). Thus, participation in H bonding by H2O molecules is a phenomenon of mutual reinforcement. The H bonds between neighbouring molecules are weak (23 kJ/mol each) relative to the H i O covalent bonds (420 kJ/mol). As a consequence, the hydrogen atoms are situated asymmetrically between the 2 oxygen atoms along the O i O axis. There is never any ambiguity about which O atom the H atom is chemically bound to, nor to which O it is H bonded.

The Structure of Ice Is Based on H-Bond Formation In ice, the hydrogen bonds form a space-filling, 3-dimensional network. These bonds are directional and straight; that is, the H atom lies on a direct line between the 2 O atoms. This linearity and directionality mean that the H bonds in ice are strong. In addition, the directional preference of the H bonds leads to an open lattice structure. For example, if the

FIGURE 2.2 The structure of normal ice. The smallest number of H2O molecules in any closed circuit of H-bonded molecules is 6, so this structure bears the name hexagonal ice. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 2

35

Water: The Medium of Life

water molecules are approximated as rigid spheres centred at the positions of the O atoms in the lattice, then the observed density of ice is actually only 57% of that expected for a tightly packed arrangement of such spheres. The H bonds in ice hold the water molecules apart. Melting involves breaking some of the H bonds that maintain the crystal structure of ice so that the molecules of water (now liquid) can actually pack closer together. Thus, the density of ice is slightly less than that of water. Ice floats, a property of great importance to aquatic organisms in cold climates. In liquid water, the rigidity of ice is replaced by fluidity, and the crystalline periodicity of ice gives way to spatial homogeneity. The H2O molecules in liquid water form a disordered H-bonded network, with each molecule having an average of 4.4 close neighbours situated within a centre-to-centre distance of 0.284 nm (2.84 Å). At least half the hydrogen bonds have non-ideal orientations (i.e., they are not perfectly straight); consequently, liquid H2O lacks the regular lattice-like structure of ice. The space about an O atom is not defined by the presence of 4 hydrogens, but can be occupied by other water molecules randomly oriented so that the local environment, over time, is essentially uniform. Nevertheless, the heat of melting for ice is but a small fraction (13%) of the heat of sublimation for ice (the energy needed to go from the solid to the vapour state). This fact indicates that the majority of H bonds between H2O molecules survive the transition from solid to liquid. At 10°C, 2.3 H bonds per H2O molecule remain and the tetrahedral bond order persists even though substantial disorder is now present.

psec

H bond

Molecular Interactions in Liquid Water Are Based on H Bonds The present interpretation of water structure is that water molecules are connected by uninterrupted H-bond paths running in every direction, spanning the whole sample. The participation of each water molecule in an average state of H bonding to its neighbours means that each molecule is connected to every other in a fluid network of H bonds. The average lifetime of a H-bonded connection between 2 H2O molecules in water is 9.5 psec (picoseconds, where 1 psec = 10-12 sec). Thus, about every 10 psec, the average H2O molecule moves, reorients, and interacts with new neighbours, as illustrated in Figure 2.3. In summary, pure liquid water consists of H2O molecules held in a disordered, 3-dimensional network that has a local preference for tetrahedral geometry yet contains a large number of strained or broken hydrogen bonds. The presence of strain creates a kinetic situation in which H2O molecules can switch H-bond allegiances; fluidity ensues.

The Solvent Properties of Water Derive from Its Polar Nature Because of its highly polar nature, water is an excellent solvent for ionic substances such as salts; non-ionic but polar substances such as sugars, simple alcohols, and amines; and carbonyl-containing molecules such as aldehydes and ketones. Although the electrostatic attractions between the positive and negative ions in the crystal lattice of a salt are very strong, water readily dissolves salts. For example, sodium chloride is dissolved because dipolar water molecules participate in strong electrostatic interactions with the Na+ and Cl-ions, leading to the formation of hydration shells surrounding these ions (Figure 2.4). Although hydration shells are stable structures, they are also dynamic. Each water molecule in the inner hydration shell around a Na+ ion is replaced on average every 2–4 nsec (nanoseconds, where 1 nsec = 10-9 sec) by another H2O. Consequently, a water molecule is trapped only several hundred times longer by the electrostatic force field of an ion than it is by the H-bonded network of water. (Recall that the average lifetime of H bonds between water molecules is about 10 psec.) Water Has a High Dielectric Constant The attractions between the water molecules interacting with, or hydrating, ions are much greater than the tendency of oppositely charged ions to attract one another. Water’s ability to surround ions in dipole interactions and diminish their attraction for each other is a measure of its dielectric constant, D. Indeed, ionization in solution depends on the dielectric constant of the solvent; otherwise, the strongly attracted positive and negative ions would unite to form neutral molecules. The

FIGURE 2.3 The fluid network of H bonds linking water molecules in the liquid state. It is revealing to note that, in 10 psec, a photon of light (which travels at 3 : 108 m/sec) would move a distance of only 0.003 m.

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36

Part 1 Building Blocks of the Cell

FIGURE 2.4 Hydration shells surrounding ions in solution. Water molecules orient so that the electrical charge on the ion is sequestered by the water dipole.

+ + + – + + + – + + + – + + + – – – Cl + + – Na+ + – Na+ + Cl– – + + – + + – + + – + + + – + + + + + – – + – + + + Na+ Cl– – + + + – Na – + – + – + + – Na Cl Na Cl + + + – + – + – – – + Cl Na+ Cl– Na Cl– + + + – Na+ Cl– Na+ Cl– Na+ + – + + + + + – + Cl– Na+ Cl– Na+ – Cl– + – + + – + – + + – + – + + – + + + – +

strength of the dielectric constant is related to the force, F, experienced between 2 ions of opposite charge separated by a distance, r, as given in the relationship F =

e1e2 Dr2

,

where e1 and e2 are the charges on the 2 ions. Table 2.1 lists the dielectric constants of some common liquids. Note that the dielectric constant for water is more than twice that of methanol and more than 40 times that of hexane. Water Forms H Bonds with Polar Solutes In the case of non-ionic but polar compounds such as sugars, the excellent solvent properties of water stem from its ability to readily form hydrogen bonds with the polar functional groups on these compounds, such as hydroxyls, amines, and carbonyls (see Figure 1.14 on page 14). These polar interactions between solvent and solute are stronger than the intermolecular attractions between solute molecules caused by van der Waals forces and weaker hydrogen bonding. Thus, the solute molecules readily dissolve in water. Hydrophobic Interactions The behaviour of water toward non-polar solutes is different from the interactions just discussed. Non-polar solutes (or non-polar functional groups on biological macromolecules) do not readily H bond to H2O, and as a result, such compounds tend to be only sparingly soluble in water. The process of dissolving such substances is accompanied by significant reorganization of the water surrounding the solute so that the response of the solvent water to such solutes can be equated to “structure making.” Because non-polar solutes must occupy space, the random H-bonded network of water must reorganize to accommodate them. At the same time, the water molecules participate in as many H-bonded interactions with one another as the temperature permits. Consequently, the H-bonded water

TABLE 2.1 Dielectric Constants* of Some Common Solvents at 25°C Solvent Formamide Water

Dielectric Constant (D) 109 78.5

Methyl alcohol

32.6

Ethyl alcohol

24.3

Acetone

20.7

Acetic acid

6.2

Chloroform

5.0

Benzene

2.3

Hexane

1.9

*The dielectric constant is also referred to as relative permittivity by physical chemists. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 2

Water: The Medium of Life

37

network rearranges toward formation of a local cage-like (clathrate) structure surrounding each solute molecule, as shown for a long-chain fatty acid in Figure 2.5. This fixed orientation of water molecules around a hydrophobic “solute” molecule results in a hydration shell. A major consequence of this rearrangement is that the molecules of H2O participating in the cage layer have markedly reduced options for orientation in 3-dimensional space. Water molecules tend to straddle the non-polar solute such that 2 or 3 tetrahedral directions (H-bonding vectors) are tangential to the space occupied by the inert solute. “Straddling” allows the water molecules to retain their H-bonding possibilities because no H-bond donor or acceptor of the H2O is directed toward the caged solute. The water molecules forming these clathrates are involved in highly ordered structures. That is, clathrate formation is accompanied by significant ordering of structure or negative entropy. Multiple non-polar molecules tend to cluster together because their joint solvation cage involves less total surface area and thus fewer ordered water molecules than in their separate cages. It is as if the non-polar molecules had some net attraction for one another. This apparent affinity of non-polar structures for one another is called hydrophobic interactions (Figure 2.6). In actuality, the “attraction” between non-polar solutes is an entropy-driven process due to a net decrease in order among the H2O molecules. To be specific, hydrophobic interactions between non-polar molecules are maintained not so much by direct interactions between the inert solutes themselves as by the increase in entropy when the water cages coalesce and reorganize. Because interactions between non-polar solute molecules and the water surrounding them are of uncertain stoichiometry and do not share the equality of atom-to-atom participation implicit in chemical bonding, the term hydrophobic interaction is more correct than the misleading expression hydrophobic bond. Amphiphilic Molecules Compounds containing both strongly polar and strongly nonpolar groups are called amphiphilic molecules (from the Greek amphi meaning “both” and philos meaning “loving”). Such compounds are also referred to as amphipathic molecules (from the Greek pathos meaning “passion”). Salts of fatty acids are a typical example that has biological relevance. They have a long non-polar hydrocarbon tail and a strongly polar carboxyl head group, as in the sodium salt of palmitic acid (Figure 2.7). Their behaviour in aqueous solution reflects the combination of the contrasting polar and non-polar nature of these substances. The ionic carboxylate function hydrates readily, whereas the long hydrophobic tail is intrinsically insoluble. Nevertheless, sodium palmitate and other amphiphilic molecules readily disperse in water because the hydrocarbon tails of these substances are joined together in hydrophobic interactions as their polar carboxylate functions are hydrated in typical hydrophilic fashion. Such clusters of amphipathic molecules are termed micelles; Figure 2.7b depicts their structure.

FIGURE 2.5 (left) Disordered network of H-bonded water molecules. (right) Clathrate cage of ordered, H-bonded water molecules around a non-polar solute molecule.

HO

C

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38

Part 1 Building Blocks of the Cell

HO

HO

HO

C

C C

C

C

C

C

HO

O

H C

FIGURE 2.6 Hydrophobic interactions between non-polar molecules (or non-polar regions of molecules) are due to the increase in entropy of solvent water molecules.

Influence of Solutes on Water Properties The presence of dissolved substances disturbs the structure of liquid water, thereby changing its properties. The dynamic H-bonding interactions of water must now accommodate the intruding substance. The net effect is that solutes, regardless of whether they are polar or non-polar, fix nearby water molecules in a more ordered array. Ions, by establishing hydration shells through interactions with the water dipoles, create local order. Hydrophobic substances, for different reasons, make structures within water. To put it another way, by limiting the orientations that neighbouring water molecules can assume, solutes give order to the solvent and diminish the dynamic interplay among H2O molecules that occurs in pure water. FIGURE 2.7 (a) An amphiphilic molecule: sodium palmitate. (b) Micelle formation by amphiphilic molecules in aqueous solution. Because of their negatively charged surfaces, neighbouring micelles repel one another and thereby maintain a relative stability in solution.

(a) The sodium salt of palmitic acid: Sodium palmitate (Na+ –OOC(CH2)14CH3)

(b)

O Na+

– C O

Polar head

CH2 CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

Non-polar tail

CH2

CH2

CH2

CH2 CH2

– – –

–

–

– – –

– – –

–

–

– – –

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Chapter 2 (a)

(b)

Non-permeant solute

(c)

22.4 atm

1m

Semi-permeable membrane

39

Water: The Medium of Life

FIGURE 2.8 The osmotic pressure of a 1 molal (m) solution is equal to 22.4 atmospheres of pressure. (a) If a non-permeant solute is separated from pure water by a semipermeable membrane through which H2O passes freely, (b) water molecules enter the solution (osmosis) and the height of the solution column in the tube rises. The pressure necessary to push water back through the membrane at a rate exactly equalled by the water influx is the osmotic pressure of the solution. (c) For a 1 m solution, this force is equal to 22.4 atm of pressure. Osmotic pressure is directly proportional to the concentration of the non-permeant solute.

H2O

Colligative Properties This influence of the solute on water is reflected in a set of characteristic changes in behaviour termed colligative properties, or properties related by a common principle. These alterations in solvent properties are related in that they all depend only on the number of solute particles per unit volume of solvent and not on the chemical nature of the solute. These effects include freezing point depression, boiling point elevation, vapour pressure lowering, and osmotic pressure effects. For example, 1 mol of an ideal solute dissolved in 1000 g of water (a 1 m, or molal, solution) at 1 atm pressure depresses the freezing point by 1.86°C, raises the boiling point by 0.543°C, lowers the vapour pressure in a temperature-dependent manner, and yields a solution whose osmotic pressure relative to pure water is 22.4 atm (at 25°C). In effect, by imposing local order on the water molecules, solutes make it more difficult for water to assume its crystalline lattice (freeze) or escape into the atmosphere (boil or vaporize). Furthermore, when a solution (such as the 1 m solution discussed here) is separated from a volume of pure water by a semipermeable membrane, the solution draws water molecules across this barrier. The water molecules are moving from a region of higher effective concentration (pure H2O) to a region of lower effective concentration (the solution). This movement of water into the solution dilutes the effects of the solute that is present. The osmotic force exerted by each mole of solute is so strong that it requires the imposition of 22.4 atm of pressure to be negated (Figure 2.8 above). Osmotic pressure from high concentrations of dissolved solutes is a serious problem for cells. Bacterial and plant cells have strong, rigid cell walls to contain these pressures. In contrast, animal cells are bathed in extracellular fluids of comparable osmolality, so no net osmotic gradient exists. Also, to minimize the osmotic pressure created by the contents of their cytosol, cells tend to store substances such as amino acids and sugars in polymeric form. For example, a molecule of glycogen or starch containing 1000 glucose units exerts only 1/1000 the osmotic pressure that 1000 free glucose molecules would.

Water Can Ionize to Form H! and OH" Water shows a small but finite tendency to form ions. This tendency is demonstrated by the electrical conductivity of pure water, a property that clearly establishes the presence of charged species (ions). Water ionizes because the larger, strongly electronegative oxygen atom strips the electron from 1 of its hydrogen atoms, leaving the proton to dissociate (Figure 2.9): H i O i H h H+ + OH-. Two ions are thus formed: protons, or hydrogen ions, H+; and hydroxyl ions, OH-. Free protons are immediately hydrated to form hydronium ions, H3O+: H+ + H2O h H3O+.

H

– O

H

O

H

FIGURE 2.9 The ionization of water.

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+

H

+

40

Part 1 Building Blocks of the Cell

H H

H

+

O

O H

H

H

Indeed, because most hydrogen atoms in liquid water are hydrogen bonded to a neighbouring water molecule, this protonic hydration is an instantaneous process and the ion products of water are H3O+ and OH-:

H

H

O H

H O H+ + OH–.

O H O

H

H

O H

H

FIGURE 2.10 The hydration of H3O+.

The amount of H3O+ or OH- in 1 L (litre) of pure water at 25°C is 1 * 10-7 mol; the concentrations are equal because the dissociation is stoichiometric. Although it is important to keep in mind that the hydronium ion, or hydrated hydrogen ion, represents the true state in solution, the convention is to speak of hydrogen ion concentrations in aqueous solution, even though “naked” protons are virtually non-existent. Indeed, H3O+ itself attracts a hydration shell by H bonding to adjacent water molecules to form an H9O4+ species (Figure 2.10) and even more highly hydrated forms. Similarly, the hydroxyl ion, like all other highly charged species, is also hydrated. Kw, the Ion Product of Water The dissociation of water into hydrogen ions and hydroxyl ions occurs to the extent that 10-7 mol of H+ and 10-7 mol of OH- are present at equilibrium in 1 L of water at 25°C: H2O ∆ H+ + OH-. The equilibrium constant for this process is Keq =

[H+][OH-] , [H2O]

where brackets denote concentration in moles per litre. Because the concentration of H2O in 1 L of pure water is equal to the number of grams in a litre divided by the gram molecular weight of H2O, or 1000/18, the molar concentration of H2O in pure water is 55.5 M (molar). The decrease in H2O concentration as a result of ion formation ([H+], [OH-] = 10-7 M) is negligible in comparison; thus its influence on the overall concentration of H2O can be ignored. Thus, Keq =

(10-7)(10-7) = 1.8 * 10-16 M. 55.5

Because the concentration of H2O in pure water is essentially constant, a new constant, Kw, the ion product of water, can be written as Kw = 55.5 Keq = 10-14 M2 = [H+][H-]. This equation has the virtue of revealing the reciprocal relationship between H+ and OHconcentrations of aqueous solutions. If a solution is acidic (i.e., it has a significant [H+]), then the ion product of water dictates that the OH- concentration is correspondingly less. For example, if [H+] is 10-2 M, [OH-] must be 10-12 M (Kw = 10-14 M2 = [10-2][OH-]; [OH-] = 10-12 M). Similarly, in an alkaline, or basic, solution in which [OH-] is great, [H+] is low.

2.2

What Is pH?

To avoid the cumbersome use of negative exponents to express concentrations that range over 14 orders of magnitude, Søren Sørensen, a Danish biochemist, devised the pH scale by defining pH as the negative logarithm of the hydrogen ion concentration1: pH = -log10 [H+]. 1 To be precise in physical chemical terms, the activities of the various components, not their molar concentrations, should be used in these equations. The activity (a) of a solute component is defined as the product of its molar concentration (c) and an activity coefficient (g): a = [c] g. Most biochemical work involves dilute solutions, and the use of activities instead of molar concentrations is usually neglected. However, the concentration of certain solutes may be very high in living cells.

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Chapter 2

41

Water: The Medium of Life

TABLE 2.2 pH Scale The hydrogen ion and hydroxyl ion concentrations are given in moles per litre at 25°C. pH

[H!]

[OH"]

0

(100)

1.0

0.00000000000001

(10-14)

1

(10-1)

0.1

0.0000000000001

(10-13)

2

(10-2)

0.01

0.000000000001

(10-12)

3

(10-3)

0.001

0.00000000001

(10-11)

4

(10-4)

0.0001

0.0000000001

(10-10)

5

(10-5)

0.00001

0.000000001

(10-9)

6

(10-6)

0.000001

0.00000001

(10-8)

7

(10-7)

0.0000001

0.0000001

(10-7)

8

(10-8)

0.00000001

0.000001

(10-6)

9

(10-9)

0.000000001

0.00001

(10-5)

10

(10-10)

0.0000000001

0.0001

(10-4)

11

(10-11)

0.00000000001

0.001

(10-3)

12

(10-12)

0.000000000001

0.01

(10-2)

13

(10-13)

0.0000000000001

0.1

(10-1)

14

(10-14)

0.00000000000001

1.0

(100)

Table 2.2 above gives the pH scale. Note again the reciprocal relationship between [H+] and [OH-]. Also, because the pH scale is based on negative logarithms, low pH values represent the highest H+ concentrations (and the lowest OH- concentrations, as Kw specifies). Note also that pKw = pH + pOH = 14. The pH scale is used widely in biological applications because hydrogen ion concentrations in biological fluids are very low, about 10-7 M, or 0.0000001 M, a value more easily represented as pH 7. The pH of blood plasma, for example, is 7.4, or 0.00000004 M H+. Certain disease conditions may lower the plasma pH level to 6.8 or less, a situation that may result in death. At pH 6.8, the H+ concentration is 0.00000016 M, 4 times greater than at pH 7.4. At pH 7, [H+] = [OH-]; that is, there is no excess acidity or basicity. The point of neutrality is at pH 7, and solutions having a pH of 7 are said to be at neutral pH. The pH values of various fluids of biological origin or relevance are given in Table 2.3. Because the pH scale is a logarithmic scale, 2 solutions whose pH values differ by 1 pH unit have a 10-fold difference in [H+]. For example, grapefruit juice at pH 3.2 contains more than 12 times as much H+ as orange juice at pH 4.3.

TABLE 2.3 The pH of Various Common Fluids Fluid Household lye

pH 13.6

Bleach

12.6

Household ammonia

11.4

Milk of magnesia

10.3

Baking soda Seawater Pancreatic fluid Blood plasma

8.4 8.0 7.8–8.0 7.4

Intracellular fluids

Strong Electrolytes Dissociate Completely in Water

Liver

6.9

Substances that are almost completely dissociated to form ions in solution are called strong electrolytes. The term electrolyte describes substances capable of generating ions in solution and thereby causing an increase in the electrical conductivity of the solution. Many salts (such as NaCl and K2SO4) fit this category, as do strong acids (such as HCl) and strong bases (such as NaOH). Recall from general chemistry that acids are proton donors and bases are proton acceptors. In effect, the dissociation of a strong acid such as HCl in water can be treated as a proton transfer reaction between the acid HCl and the base H2O to give the conjugate acid H3O+ and the conjugate base Cl-:

Muscle

6.1

HCl + H2O h H3O+ + Cl-. The equilibrium constant for this reaction is K =

[H3O+][Cl-] [H2O][HCl]

.

Saliva

6.6

Urine

5–8

Boric acid

5.0

Beer

4.5

Orange juice

4.3

Grapefruit juice

3.2

Vinegar

2.9

Soft drinks

2.8

Lemon juice

2.3

Gastric juice

1.2–3.0

Battery acid

0.35

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42

Part 1 Building Blocks of the Cell

Customarily, because the term [H2O] is essentially constant in dilute aqueous solutions, it is incorporated into the equilibrium constant K to give a new term, Ka, the acid dissociation constant, where Ka = K [H2O]. Also, the term [H3O+] is often replaced by H+, such that Ka =

[H+][Cl-] . [HCl]

For HCl, the value of Ka is exceedingly large because the concentration of HCl in aqueous solution is vanishingly small. Because this is so, the pH of HCl solutions is readily calculated from the amount of HCl used to make the solution: [H+] in solution = [HCl] added to solution. Thus, a 1 M solution of HCl has a pH of 0; a 1 mM HCl solution has a pH of 3. Similarly, a 0.1 M NaOH solution has a pH of 13. (Because [OH-] = 0.1 M, [H+] must be 10-13 M.) Viewing the dissociation of strong electrolytes another way, we see that the ions formed show little affinity for each other. For example, in HCl in water, Cl- has very little affinity for H+: HCl h H+ + Cl-; and in NaOH solutions, Na+ has little affinity for OH-. The dissociation of these substances in water is effectively complete.

Weak Electrolytes Are Substances That Dissociate Only Slightly in Water Substances with only a slight tendency to dissociate to form ions in solution are called weak electrolytes. Acetic acid, CH3COOH, is a good example: CH3COOH + H2O ∆ CH3COO- + H3O+. The acid dissociation constant Ka for acetic acid is 1.74 × 10-5 M: Ka =

[H+][CH3COO-] [CH3COOH]

= 1.74 * 10-5 M.

Ka is also termed an ionization constant because it states the extent to which a substance forms ions in water. The relatively low value of Ka for acetic acid reveals that the un-ionized form, CH3COOH, predominates over H+ and CH3COO- in aqueous solutions of acetic acid. Viewed another way, CH3COO-, the acetate ion, has a high affinity for H+.

EXAMPLE

What is the pH of a 0.1 M solution of acetic acid? In other words, what is the final pH when 0.1 mol of acetic acid (HAc) is added to water and the volume of the solution is adjusted to equal 1 L? Answer The dissociation of HAc in water can be written simply as HAc ∆ H+ + Ac-, where Ac- represents the acetate ion, CH3COO-. In solution, some amount x of HAc dissociates, generating x amount of Ac- and an equal amount x of H+. Ionic equilibriums characteristically are established very rapidly. At equilibrium, the concentration of HAc + Ac- must equal 0.1 M. So, [HAc] can be represented as (0.1 - x) M, and [Ac-] and [H+] then both equal x molar. From 1.74 × 10-5 M = ([H+][Ac-])/[HAc], we get 1.74 × 10-5 M = x2/[0.1 - x]. The solution to quadratic equations of this form (ax2 + bx + c = 0) is -b ; (b2 - 4ac/2a)1/2. For x2 + (1.74 × 10-5)x - (1.74 × 10-6) = 0, x = 1.319 * 10-3 M, so pH = 2.88. (Note that the calculation of x can be simplified here: because Ka is quite small, x V 0.1 M. Therefore, Ka is essentially equal to x2/0.1. Thus, x2 = 1.74 * 10-6 M2, so x = 1.32 × 10-3 M, and pH = 2.88.) NEL

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Chapter 2

Water: The Medium of Life

The Henderson–Hasselbalch Equation Describes the Dissociation of a Weak Acid in the Presence of Its Conjugate Base Consider the ionization of some weak acid, HA, occurring with an acid dissociation constant, Ka. Then HA ∆ H+ + A-, and Ka =

[H+][A-] . [HA]

Rearranging this expression in terms of the parameter of interest, [H+], we have [H+] =

[Ka][HA] [A-]

.

Taking the logarithm of both sides gives log [H+] = log Ka + log10

[HA] . A-

If we change the signs and define pKa = -log Ka, we have pH = pKa - log10

[HA] , [A-]

pH = pKa + log10

[A-] . [HA]

or

This relationship is known as the Henderson–Hasselbalch equation. Thus, the pH of a solution can be calculated provided Ka and the concentrations of the weak acid HA and its conjugate base A- are known. Note particularly that when [HA] = [A-], pH = pKa. For example, if equal volumes of 0.1 M HAc and 0.1 M sodium acetate are mixed, then pH = pKa = 4.76 pKa = -log Ka = -log10 (1.74 * 10-5) = 4.76. (Sodium acetate, the sodium salt of acetic acid, is a strong electrolyte and dissociates completely in water to yield Na+ and Ac-.) The Henderson–Hasselbalch equation provides a general solution to the quantitative treatment of acid–base equilibriums in biological systems. Table 2.4 gives the acid dissociation constants and pKa values for some weak electrolytes of biochemical interest.

EXAMPLE

If pKa for acetic acid = 4.76, what is the pH when 100 mL of 0.1 N NaOH is added to 150 mL of 0.2 M HAc?

Answer 100 mL 0.1 N NaOH = 0.01 mol OH-, which neutralizes 0.01 mol HAc, giving an equivalent amount of AcOH- + HAc h Ac- + H2O; 0.02 mol of the original 0.03 mol of HAc remains essentially undissociated. The final volume is 250 mL. pH = pKa + log10

[Ac-] = 4.76 + log (0.01 mol/0.02). [HAc]

pH = 4.76 - log10 2 = 4.46 (continued)

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43

44

Part 1 Building Blocks of the Cell

If 150 mL of 0.2 M HAc had merely been diluted with 100 mL of water, this would have left 250 mL of a 0.12 M HAc solution. The pH would be given by Ka =

[H+][Ac-] x2 = = 1.74 * 10-5 M. [HAc] 0.12 M

x = 144 * 10-3 = [H+] pH = 2.84

Titration Curves Illustrate the Progressive Dissociation of a Weak Acid Titration is the analytical method used to determine the amount of acid in a solution. A measured volume of the acid solution is titrated by slowly adding a solution of base, typically NaOH, of known concentration. As incremental amounts of NaOH are added, the pH of the solution is determined and a plot of the pH of the solution versus the amount of OH- added yields a titration curve. The titration curve for acetic acid is shown in Figure 2.11. In considering the progress of this titration, keep in mind 2 important equilibriums: 1. HAc ∆ H+ + Ac2. H+ + OH- ∆ H2O

Low pH 100

Relative abundance

CH3COOH

High pH CH3COO–

As the titration begins, mostly HAc is present, plus some H+ and Ac- in amounts that can be calculated (see the Example on page 42). Addition of a solution of NaOH allows hydroxide ions to neutralize any H+ present. Note that reaction (2) as written is strongly favoured; its apparent equilibrium constant is greater than 1015! As H+ is neutralized, more

TABLE 2.4 Acid Dissociation Constants and pKa Values for Some Weak Electrolytes (at 25°C) 50

0

0

0.5 Equivalents of OH– added

Ka (M)

pKa

HCOOH (formic acid)

1.78 * 10-4

3.75

CH3COOH (acetic acid)

1.74 * 10-5

4.76

CH3CH2COOH (propionic acid)

1.35 * 10-5

4.87

1.38 *

10-4

3.86

6.16 *

10-5

4.21

2.34 *

10-6

5.63 2.15

Acid

pH 4.76

1.0

CH3CHOHCOOH (lactic acid) *

HOOCCH2CH2COOH (succinic acid) pK1 COO-

HOOCCH2CH2

9

5

(succinic acid) pK2

7.08 *

10-3

H2PO4- (phosphoric acid) pK2 HPO42 (phosphoric acid) pK3

6.31 *

10-8

7.20

3.98 *

10-13

12.40

C3N2H+5

1.02 * 10-7

6.99

C6O2N3H-11 (histidine–imidazole group) pKR†

9.12 * 10-7

6.04

H2CO3 (carbonic acid) pK1

1.70 * 10-4

3.77

H3PO4 (phosphoric acid) pK1

CH3COO–

7

pH

Ka = 1.74 * 10-5 H2O K = = 5.55 * 1015. Kw

pH 4.76

3

(imidazole)

-

HCO3 (bicarbonate) pK2

CH3COOH

(HOCH2)3CNH+3

1 0.5 Equivalents of OH– added

1.0

FIGURE 2.11 The titration curve for acetic acid. Note that the titration curve is relatively flat at pH values near the pKa. In other words, the pH changes relatively little as OH- is added in this region of the titration curve.

(tris-hydroxymethyl aminomethane)

NH+4 (ammonium) CH3NH+3 (methylammonium)

5.75 *

10-11

10.24

8.32 *

10-9

8.07

5.62 *

10-10

9.25

2.46 *

10-11

10.62

*The

pK values listed as pK1, pK2, or pK3 are in actuality pKa values for the respective dissociations. This simplification in notation is used throughout this book. R refers to the imidazole ionization of histidine. Data from CRC Handbook of Biochemistry, The Chemical Rubber Co., 1968.

† pK

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Chapter 2

Water: The Medium of Life

HAc dissociates to H+ and Ac-. The stoichiometry of the titration is 1:1—for each increment of OH- added, an equal amount of the weak acid HAc is titrated. As additional NaOH is added, the pH gradually increases as Ac- accumulates at the expense of diminishing HAc and the neutralization of H+. At the point where half the HAc has been neutralized (i.e., where 0.5 equivalent of OH- has been added), the concentrations of HAc and Ac- are equal and pH = pKa for HAc. Thus, we have an experimental method for determining the pKa values of weak electrolytes. These pKa values lie at the midpoint of their respective titration curves. After all the acid has been neutralized (i.e., when 1 equivalent of base has been added), the pH rises exponentially. The shapes of the titration curves of weak electrolytes are identical, as Figure 2.12 reveals. Note, however, that the midpoints of the different curves vary in a way that characterizes the particular electrolytes. The pKa for acetic acid is 4.76, the pKa for imidazole is 6.99, and that for ammonium is 9.25. These pKa values are directly related to the dissociation constants of these substances, or, viewed the other way, to the relative affinities of the conjugate bases for protons. NH3 has a high affinity for protons compared to Ac-; NH+4 is a poor acid compared to HAc.

Phosphoric Acid Has Three Dissociable H! Figure 2.13 shows the titration curve for phosphoric acid, H3PO4. This substance is a polyprotic acid, meaning it has more than 1 dissociable proton. Indeed, it has 3, and thus 3 equivalents of OH- are required to neutralize it, as Figure 2.13 shows. Note that the 3 dissociable H+ are lost in discrete steps, each dissociation showing a characteristic pKa. Note that pK1 occurs at pH = 2.15, and the concentrations of the acid H3PO4 and the conjugate base H2PO-4 are equal. As the next dissociation is approached, H2PO-4 is treated as the acid and HPO24 is its conjugate base. Their concentrations are equal at pH 7.20, so pK2 = 7.20. (Note that at this point, 1.5 equivalents of OH- have been added.) As more OH- is added, the last dissociable hydrogen is titrated, and pK3 occurs at pH = 12.4, where 3HPO24 = [PO4 ]. The shape of the titration curves for weak electrolytes has a biologically relevant property: In the region of the pKa, pH remains relatively unaffected as increments of OH(or H+) are added. The weak acid and its conjugate base are acting as a buffer. Titration midpoint [HA] = [A–] pH = pKa 12 NH3

pKa = 9.25 [NH+ 4 ] = [NH3]

10

N

NH4+ 8 H N N –H + Imidazole H+

pH

N H

Imidazole

[imid.H+] = [imid] pKa = 6.99

6 CH3COOH 4

pKa = 4.76

CH3COO–

[CH3COOH] = [CH3COO–]

2

0.5 Equivalents of OH–

1.0

FIGURE 2.12 The titration curves of several weak electrolytes: acetic acid, imidazole, and ammonium. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

45

46

Part 1 Building Blocks of the Cell [HPO42–] = [PO43–]

14 12

pK3 = 12.4

10

[HPO42–] = [H2PO4–]

8 pH

HPO42–

pK2 = 7.2

6 4

PO43–

[H3PO4] = [H2PO4–]

2 H3PO4

0.5

H2PO4–

pK1 = 2.15

1.0 1.5 2.0 Equivalents OH– added

2.5

3.0

FIGURE 2.13 The titration curve for phosphoric acid.

2.3

What Are Buffers and What Do They Do?

Buffers are solutions that tend to resist changes in their pH as acid or base is added. Typically, a buffer system is composed of a weak acid and its conjugate base. A solution of a weak acid that has a pH nearly equal to its pKa contains, by definition, an amount of the conjugate base nearly equivalent to the weak acid. Note that in this region, the titration curve is relatively flat (Figure 2.14). Addition of H+ then has little effect because it is absorbed by the following reaction: H+ + A- h HA. Similarly, any increase in [OH-] is offset by the process OH- + HA h A- + H2O.

10 A– 8

[HA] = [A–]

pH 6 4

HA

pH = pKa

2

0.5 Equivalents of OH– added

1.0

Buffer action: OH–

H2O

HA

A–

H+ FIGURE 2.14 A buffer system consists of a weak acid, HA, and its conjugate base, A-.

Thus, the pH remains relatively constant. The components of a buffer system are chosen such that the pKa of the weak acid is close to the pH of interest. It is at the pKa that the buffer system shows its greatest buffering capacity. At pH values more than 1 pH unit from the pKa, buffer systems become ineffective because the concentration of 1 of the components is too low to absorb the influx of H+ or OH-. The molarity of a buffer is defined as the sum of the concentrations of the acid and conjugate base forms. Maintenance of pH is vital to all cells. Cellular processes such as metabolism are dependent on the activities of enzymes; in turn, enzyme activity is markedly influenced by pH, as the graphs in Figure 2.15 show. Consequently, changes in pH would be disruptive to metabolism for reasons that become apparent in later chapters. Organisms have a variety of mechanisms to keep the pH of their intracellular and extracellular fluids essentially constant, but the primary protection against harmful pH changes is provided by buffer systems. The buffer systems selected reflect both the need for a pKa value near pH 7 and the compatibility of the buffer components with the metabolic machinery of cells. Two buffer 4systems act to maintain intracellular pH essentially constant: the phosphate (HPO24 /H2PO ) system and the histidine system. The pH of the extracellular fluid that bathes the cells and tissues of animals is maintained by the bicarbonate/carbonic acid (HCO-3 /H2CO3) system.

The Phosphate Buffer System Is a Major Intracellular Buffering System The phosphate system serves to buffer the intracellular fluid of cells at physiological pH because pK2 lies near this pH value. The intracellular pH of most cells is maintained in the range between 6.9 and 7.4. Phosphate is an abundant anion in cells, both in inorganic form and as an important functional group on organic molecules that serve as metabolites or NEL

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Chapter 2

pH = pK2 + log10 7.4 = 7.20 + log10 [HPO24 ] [H2PO-4 ]

[HPO24 ] [H2PO-4 ] [HPO24 ]

(a) Pepsin

Enzyme activity

macromolecular precursors. In both organic and inorganic forms, its characteristic pK2 means that the ionic species present at physiological pH are sufficient to donate or accept hydrogen ions to buffer any changes in pH, as the titration curve for H3PO4 in Figure 2.13 reveals. For example, if the total cellular concentration of phosphate is 20 mM (millimolar) and the pH is 7.4, the distribution of the major phosphate species is given by

[H2PO-4 ]

= 1.58

0

Histidine is 1 of the 20 naturally occurring amino acids commonly found in proteins (see Chapter 4). It possesses as part of its structure an imidazole group, a 5-membered heterocyclic ring possessing 2 nitrogen atoms. The pKa for dissociation of the imidazole hydrogen of histidine is 6.04.

COO– pK a = 6.04

H+

H3+N

N+H

C H

C

6

pH

7

HN

2

3

4

5 pH

6

H

CH

CH2 N

7

8

9

FIGURE 2.15 pH versus enzymatic activity. Pepsin is a protein-digesting enzyme active in the gastric fluid. Fumarase is a metabolic enzyme found in mitochondria. Lysozyme digests the cell walls of bacteria; it is found in tears.

C N

8

N

O–

O

CH2

6

Lysozyme

Not many common substances have pKa values in the range from 6 to 8. Consequently, biochemists conducting in vitro experiments were limited in their choice of buffers effective at or near physiological pH. In 1966, N. E. Good devised a set of synthetic buffers to remedy this problem, and over the years the list has expanded so that a “good” selection is

O

5

CH2

“Good” Buffers Are Buffers Useful within Physiological pH Ranges

CH2

5

(c)

In cells, histidine occurs as the free amino acid, as a constituent of proteins, and as part of dipeptides in combination with other amino acids. Because the concentration of free histidine is low and its imidazole pKa is more than 1 pH unit removed from prevailing intracellular pH, its role in intracellular buffering is minor. However, protein-bound and dipeptide histidine may be the dominant buffering system in some cells. In combination with other amino acids, as in proteins or dipeptides, the imidazole pKa may increase substantially. For example, the imidazole pKa is 7.04 in anserine, a dipeptide containing b-alanine and histidine (Figure 2.16). Thus, this pKa is near physiological pH, and some histidine peptides are well suited for buffering at physiological pH.

+ H3N

4

Enzyme activity

COO– HN

3 pH

Enzyme activity

Dissociation of the Histidine–Imidazole Group Also Serves as an Intracellular Buffering System

CH2

2

Fumarase

[HPO24 ] = 12.25 mM, and [H2PO4 ] = 7.75 mM.

C H

1

(b)

Thus, if [HPO24 ] + [H2PO4 ] = 20 mM, then

H3+N

47

Water: The Medium of Life

N+H

H3C

FIGURE 2.16 Anserine (N-B-alanyl-3-methyl-L-histidine) is an important dipeptide buffer in the maintenance of intracellular pH in some tissues. The structure shown is the predominant ionic species at pH 7. pK1 (COOH) # 2.64; pK2 (imidazole-N!H) # 7.04; pK3 (N!H3) # 9.49. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

48

Part 1 Building Blocks of the Cell

HUMAN BIOCHEMISTRY

The Bicarbonate Buffer System of Blood Plasma The important buffer system of blood plasma is the bicarbonate/carbonic acid couple: H2CO3 ∆ H+ + HCO-3 The relevant pKa, pK1 for carbonic acid, has a value far removed from the normal pH of blood plasma (pH 7.4). [The pK1 for H2CO3 at 25°C is 3.77 (Table 2.4), but at 37°C, pK1 is 3.57.] At pH 7.4, the concentration of H2CO3 is a minuscule fraction of the HCO-3 concentration; thus the plasma appears to be poorly protected against an influx of OH- ions: pH = 7.4 = 3.57 + log10 [HCO-3 ] [H2CO3]

[HCO-3 ] [H2CO3]

= 6761.

[HCO-3 ]

For example, if = 24 mM, then [H2CO3] is only 3.55 μM (3.55 × 10-6 M), and an equivalent amount of OH- (its usual concentration in plasma) would swamp the buffer system, causing a dangerous rise in the plasma pH. How, then, can this bicarbonate system function effectively? The bicarbonate buffer system works well because the critical concentration of H2CO3 is maintained relatively constant through equilibrium with dissolved CO2 produced in the tissues and available as a gaseous CO2 reservoir in the lungs.2 Gaseous CO2 from the lungs and tissues is dissolved in the blood plasma, symbolized as CO2(d), and hydrated to form H2CO3: CO2(g) ∆ CO2(d) CO2(d) + H2O ∆ H2CO3 H2CO3 ∆ H+ + HCO-3 Thus, the concentration of H2CO3 is itself buffered by the available pools of CO2. The hydration of CO2 is actually mediated by an enzyme, carbonic anhydrase, which facilitates the equilibrium by rapidly catalyzing the reaction H2O + CO2(d) ∆ H2CO3. Under the conditions of temperature and ionic strength prevailing in mammalian body fluids, the equilibrium for this reaction lies far to the left, such that more than 300 CO2 molecules are present in solution for every molecule of H2CO3. Because dissolved CO2 and H2CO3 are in equilibrium, the proper expression for H2CO3 availability is [CO2(d)] + [H2CO3], the so-called total carbonic acid pool, which consists primarily of CO2(d). The overall equilibrium for the bicarbonate buffer system then is Kh CO2(d) + H2O ∆ H2CO3 Ka H2CO3 ∆ H+ + HCO-3

An expression for the ionization of H2CO3 under such conditions (i.e., in the presence of dissolved CO2) can be obtained from Kh, the equilibrium constant for the hydration of CO2, and from Ka, the first acid dissociation constant for H2CO3: Kh =

[H2CO3] [CO2(d)]

.

Thus, [H2CO3] = Kh[CO2(d)] Putting this value for [H2CO3] into the expression for the first dissociation of H2CO3 gives Ka = =

[H+][HCO-3 ] [H2CO3] [H+][HCO-3 ] [CO2(d)]

.

Therefore, the overall equilibrium constant for the ionization of H2CO3 in equilibrium with CO2(d) is given by KaKh =

[H+][HCO-3 ] Kh[CO2(d)]

,

and KaKh, the product of 2 constants, can be defined as a new equilibrium constant, Koverall. The value of Kh is 0.003 at 37°C, and Ka, the ionization constant for H2CO3, is 10-3.57 = 0.000269. Therefore Koverall = (0.000269)(0.003) = 8.07 * 10-7 pKoverall = 6.1 , which yields the following Henderson–Hasselbalch relationship: pH = pKoverall + log10

[HCO-3 ] [CO2(d)]

.

Although the prevailing blood pH of 7.4 is more than 1 pH unit away from pKoverall, the bicarbonate system is still an effective buffer. Note that, at blood pH, the concentration of the acid component of the buffer will be less than 10% of the conjugate base component. One might imagine that this buffer component could be overwhelmed by relatively small amounts of alkali, with consequent disastrous rises in blood pH. However, the acid component is the total carbonic acid pool, that is, [CO2(d)] + [H2CO3], which is stabilized by its equilibrium with CO2(g). Gaseous CO2 serves to buffer any losses from the total carbonic acid pool by entering solution as CO2(d), and blood pH is effectively maintained. Thus, the bicarbonate buffer system is an open system. The natural presence of CO2 gas at a partial pressure of 40 mmHg in the alveoli of the lungs and the equilibrium CO2(g) ∆ CO2(d)

2Well-fed

humans exhale about 1 kg of CO2 daily. Imagine the excretory problem if CO2 were not a volatile gas.

keep the concentration of CO2(d) (the principal component of the total carbonic acid pool in blood plasma) in the neighbourhood of 1.2 mM. Plasma [HCO-3 ] is about 24 mM under such conditions.

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Chapter 2

Water: The Medium of Life

49

HUMAN BIOCHEMISTRY

Blood pH and Respiration Hyperventilation, defined as a breathing rate more rapid than necessary for normal CO2 elimination from the body, can result in an inappropriately low [CO2(g)] in the blood. Central nervous system disorders such as meningitis, encephalitis, or cerebral hemorrhage, as well as a number of drug- or hormone-induced physiological changes, can lead to hyperventilation. As [CO2(g)] drops due to excessive exhalation, [H2CO3] in the blood plasma falls, followed by a decline in [H+] and [HCO-3 ] in the blood plasma. Blood pH rises within 20 sec of the onset of hyperventilation, becoming maximal within 15 min. [H+] can change from its normal value of 40 nM

HO

CH2

+

CH2 NH

N

CH2 CH2

(pH = 7.4) to 18 nM (pH = 7.74). This rise in plasma pH (increase in alkalinity) is termed respiratory alkalosis. Hypoventilation is the opposite of hyperventilation and is characterized by an inability to excrete CO2 rapidly enough to meet physiological needs. Hypoventilation can be caused by narcotics, sedatives, anesthetics, and depressant drugs; diseases of the lung also lead to hypoventilation. Hypoventilation results in respiratory acidosis as CO2(g) accumulates, giving rise to H2CO3, which dissociates to form H+ and [HCO-3 ].

SO3H

HEPES

FIGURE 2.17 The structure of HEPES, 4-(2-hydroxy)-1-piperazine ethane sulfonic acid, in its fully protonated form. The pKa of the sulfonic acid group is about 3; the pKa of the piperazine-N!H is 7.55 at 20°C.

available. Some of these compounds are analogs of tris-hydroxymethyl aminomethane (Tris; see end-of-chapter Problem 17), such as triethanolamine (TEA; see end-of-chapter Problem 16) or N,N-bis (2-hydroxyethyl) glycine (Bicine; see end-of-chapter Problems 8 and 18). Others are derivatives of N-ethane sulfonic acids, such as HEPES (Figure 2.17 above).

What Are Common Buffers Used in Biochemistry?

pKa (20°C)

Buffers commonly used in “in vitro” experiments in biochemistry are presented in Figure 2.18. The choice of buffer for any given experiment depends upon the pH that one is trying to achieve, as well as the compatibility of the solutes and ions with other reaction components. For example, a Tris buffer would not be preferred over PIPES if a pH of 6.5 was required. 6.1 (5.2-7.1) 6.3 (5.0-7.4) 6.8 (6.1-7.5) 7.0 (6.5-7.5) 7.2 (6.5-7.9) 7.5 (6.8-8.2) 7.6 (6.8-8.2) 7.6 (7.0-8.2) 8.1 (7.4-8.8) 8.1 (7.5-9.0) 8.3 (7.6-9.0) 8.3 (7.6-8.9) 8.5 (7.7-9.1) 9.5 (8.6-10.0) 9.6 (8.9-10.3) 10.5 (9.7-11.1)

MES Cacodylate PIPES SSC MOPS TES HEPES TAPSO Tricine Tris Bicine Gly-Gly TAPS

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

FIGURE 2.18 Common buffers used in biochemistry and their pKa values at 20°C. The buffering range is presented in brackets after the pKa value.

CHES CAPSO 9.0

CAPS 9.5 10.0 10.5 11.0 11.5

pH (20°C) MES: 2-(N-morpholino)ethanesulfonic acid Cacodylate: dimethylarsinic acid PIPES: piperazine-N,N'-bis(2- ethanesulfonic acid) SSC: saline sodium citrate MOPS: 3-(N-morpholino)propanesulfonic acid TES: 2-{[tris(hydroxymethyl)methyl]amino} ethanesulfonic acid HEPES: 4-2-hydroxyethyl-1 -piperazineethanesulfonic acid TAPSO: 3-[N-tris(hydroxymethyl)methylamino] -2-hydroxypropanesulfonic acid Tricine: N-tris(hydroxymethyl)methylglycine

Tris: tris(hydroxymethyl)methylamine Bicine: N,N,-bis(2-hydroxyethyl)glycine Gly-Gly: N-glycylglycine TAPS: 3-{[tris(hydroxymethyl)methyl]amino} propanesulfonic acid CHES: N-cyclohexyl-2-aminoethanesulfonic acid CAPSO: N-cyclohexyl-2-hydroxyl-3 -aminopropanesulfonic acid [3-(cyclohexylamino)-2-hydroxyl-1 -propanesulphonic acid] CAPS: N-cyclohexyl-3-aminopropanesulfonic acid

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50

Part 1 Building Blocks of the Cell

2.4

What Properties of Water Give It a Unique Role in the Environment?

Water’s physical and chemical properties provide conditions that are advantageous to life on Earth. These are summarized as follows: 1. Water is a “poor” solvent for non-polar substances. Through hydrophobic interactions, membranes form and boundaries and compartments can form, delineating the cell from its environment. Hydrophobic interactions are also a determinant of how a protein folds. 2. Water has a very high dielectric constant, and thus provides a medium for ionization. Ions form an important class of chemical reactants for the organism. 3. Water’s heat capacity, or specific heat (4.1840 J/g °C), is remarkably high and thus is resistant to temperature change. 4. Water’s heat of vaporization (2.24 kJ/g) is exceptionally high. Thus it takes considerable energy to change the state of water from a liquid to a gas. This is an important property utilized by organisms for cooling (through evaporation or conduction). 5. Water’s heat of fusion is 335 J/g, and thus, at 0°C, 335 J is required to convert 1 gram of water to ice. 6. As water cools, its H bonding increases, separating water molecules and making them less dense. Thus, below 4°C, cool water rises and, most important, ice freezes on the surface of bodies of water, forming an insulating layer protecting the liquid water underneath. 7. The thermal conductivity of water is very high compared with that of other liquids. 8. Water has the highest surface tension (75 dyne/cm) of all common liquids (except mercury). Thus it is amenable to capillary action (to be drawn up tubes and vessels) and imparts considerable osmotic capabilities (the bulk movement of water in the direction from a dilute aqueous solution to a more concentrated one across a semipermeable boundary) to water.

SUMMARY 2.1 What Are the Properties of Water? Life depends on the unusual chemical and physical properties of H2O. Its high boiling point, melting point, heat of vaporization, and surface tension indicate that intermolecular forces of attraction between H2O molecules are high. Hydrogen bonds between adjacent water molecules are the basis of these forces. Liquid water consists of H2O molecules held in a random, 3-dimensional network that has a local preference for tetrahedral geometry, yet contains a large number of strained or broken hydrogen bonds. The presence of strain creates a kinetic situation in which H2O molecules can switch H-bond allegiances; fluidity ensues. As kinetic energy decreases (the temperature falls), crystalline water (ice) forms. The solvent properties of water are attributable to the “bent” structure of the water molecule and polar nature of its OOH bonds. Together, these attributes yield a liquid that can form hydration shells around salt ions or dissolve polar solutes through H-bond interactions. Hydrophobic interactions in aqueous environments also arise as a consequence of polar interactions between water molecules. The polarity of the O i H bonds means that water also ionizes to a small but finite extent to release H+ and OH- ions. Kw, the ion product of water, reveals that the concentration of [H+] and [OH-] at 25°C is 10-7 M. 2.2 What Is pH? pH is defined as -log10 [H+]. pH is an important concept in biochemistry because the structure and function of biological molecules depend strongly on functional groups that ionize, or not, depending on small changes in H+ concentration. Weak electrolytes are substances that dissociate incompletely in water. The behaviour of weak electrolytes determines the concentration of H+ and hence, pH. The Henderson–Hasselbalch equation provides a general solution to the quantitative treatment of acid–base equilibriums in biological systems. 2.3 What Are Buffers and What Do They Do? Buffers are solutions composed of a weak acid and its conjugate base. Such solutions can resist changes in pH when acid or base is added to the solution.

Maintenance of pH is vital to all cells, and primary protection against harmful pH changes is provided by buffer systems. The buffer systems used by cells reflect a need for a pKa value near pH 7 and the compatibility of the buffer components with the metabolic apparatus of cells. The phosphate buffer system and the histidine–imidazole system are the 2 prominent intracellular buffers, whereas the bicarbonate buffer system is the principal extracellular buffering system in animals. 2.4 What Properties of Water Give It a Unique Role in the Environment? Life and water are inextricably related. Water is particularly suited to its unique role in living processes and the environment. As a solvent, water is powerful yet innocuous; no other chemically inert solvent compares with water for the substances it can dissolve. Also, water as a “poor” solvent for non-polar substances gives rise to hydrophobic interactions, leading lipids to coalesce, membranes to form, and boundaries delimiting compartments to appear. Water is a medium for ionization. Ions enrich the living environment and introduce an important class of chemical reactions. Ions provide electrical properties to solutions and therefore to organisms. The thermal properties of water are especially relevant to its environmental fitness. It takes substantial changes in heat content to alter the temperature and especially the state of water. Water’s thermal properties allow it to buffer the climate through such processes as condensation, evaporation, melting, and freezing. Furthermore, water’s thermal properties allow effective temperature regulation in living organisms. Osmosis as it relates to water, and in particular, the bulk movement of water in the direction from a dilute aqueous solution to a more concentrated one across semipermeable membranes, determines the shape and form of living things. In large degree, the properties of water define the fitness of the environment. Organisms are aqueous systems in a watery world. NEL

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Chapter 2

Water: The Medium of Life

51

PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

12. At 37°C, if the plasma pH is 7.4 and the plasma concentration of HCO-3 is 15 mM, what is the plasma concentration of H2CO3? What is the plasma concentration of CO2(dissolved)? If metabolic activity changes the concentration of CO2(dissolved) to 3 mM and HCO-3 remains at 15 mM, what is the pH of the plasma?

1. Calculate the pH of the following: a. 5 * 10-4 M HCl b. 7 * 10-5 M NaOH c. 2 mM HCl d. 3 * 10-2 M KOH e. 0.04 mM HCl f. 6 * 10-9 M HCl 2. Calculate the following from the pH values given in Table 2.3: a. [H+] in vinegar b. [H+] in saliva c. [H+] in household ammonia d. [OH-] in milk of magnesia e. [OH-] in beer f. [H+] inside a liver cell 3. The pH of a 0.02 M solution of an acid was measured at 4.6. a. What is the [H+] in this solution? b. Calculate the acid dissociation constant Ka and pKa for this acid. 4. The Ka for formic acid is 1.78 * 10-4 M. a. What is the pH of a 0.1 M solution of formic acid? b. 150 mL of 0.1 M NaOH is added to 200 mL of 0.1 M formic acid, and water is added to give a final volume of 1 L. What is the pH of the final solution? 5. Given 0.1 M solutions of acetic acid and sodium acetate, describe the preparation of 1 L of 0.1 M acetate buffer at a pH of 5.4. 6. If the internal pH of a muscle cell is 6.8, what is the 2[HPO24 ]/[H2PO4 ] ratio in this cell? 7. Given 0.1 M solutions of Na3PO4 and H3PO4, describe the preparation of 1 L of a phosphate buffer at a pH of 7.5. What are the molar concentrations of the ions in the final buffer solution, including Na+ and H+? 8. Bicine is a compound containing a tertiary amino group whose relevant pKa is 8.3 (see Problem 18). Given 1 L of 0.05 M Bicine with its tertiary amino group in the unprotonated form, how much 0.1 N HCl must be added to have a Bicine buffer solution of pH 7.5? What is the molarity of Bicine in the final buffer? What is the concentration of the protonated form of Bicine in this final buffer? 9. What are the approximate fractional concentrations of the following phosphate species at pH values of 0, 2, 4, 6, 8, 10, and 12? a. H3PO4 b. H2PO-4 c. HPO24 d. PO34 10. Citric acid, a tricarboxylic acid important in intermediary metabolism, can be symbolized as H3A. Its dissociation reactions are H3A ∆ H+ + H2A-

H2A- ∆ H+ + HA2HA2-

∆

H+

+

b. If 50 mL of 0.01 M NaOH is added to 100 mL of 0.05 M phosphate buffer at pH 7.2, what is the resultant pH? What are the concentrations of H2PO-4 and HPO-4 in this final solution?

A3-

pK1 = 3.13 pK2 = 4.76 pK3 = 6.40

If the total concentration of the acid and its anion forms is 0.02 M, what  are the individual concentrations of H3A, H2A-, HA2-, and A3- at pH 5.2? 11. a. If 50 mL of 0.01 M HCl is added to 100 mL of 0.05 M phosphate buffer at pH 7.2, what is the resultant pH? What are the concentrations of H2PO-4 and HPO24 in the final solution?

13. Draw the titration curve for anserine (Figure 2.16). The isoelectric point of anserine is the pH where the net charge on the molecule is zero; what is the isoelectric point for anserine? Given a 0.1 M solution of anserine at its isoelectric point and ready access to 0.1 M HCl, 0.1 M NaOH, and distilled water, describe the preparation of 1 L of 0.04 M anserine buffer solution, pH 7.2. 14. Given a solution of 0.1 M HEPES in its fully protonated form, and ready access to 0.1 M HCl, 0.1 M NaOH, and distilled water, describe the preparation of 1 L of 0.025 M HEPES buffer solution, pH 7.8. 15. A 100 g amount of a solute was dissolved in 1000 g of water. The freezing point of this solution was measured accurately and determined to be -1.12°C. What is the molecular weight of the solute? 16. Shown here is the structure of triethanolamine (TEA) in its fully protonated form: CH2CH2OH HOCH2CH2

+ N

CH2CH2OH

H

Its pKa is 7.8. You have available at your lab bench 0.1 M solutions of HCl, NaOH, and the uncharged (free base) form of triethanolamine, as well as ample distilled water. Describe the preparation of a 1 L solution of 0.05 M triethanolamine buffer, pH 7.6. 17. Tris-hydroxymethyl aminomethane (Tris) is widely used for the preparation of buffers in biochemical research. Shown here is the structure of Tris in its protonated form: + HOCH2

NH3

C

CH2OH

CH2OH

Its acid dissociation constant, Ka, is 8.32 * 10-9 M. You have available at your lab bench a 0.1 M solution of Tris in its protonated form, 0.1 M solutions of HCl and NaOH, and ample distilled water. Describe the preparation of a 1 L solution of 0.02 M Tris buffer, pH 7.8. 18. Bicine [N, N-bis (2-hydroxyethyl) glycine] is another commonly used buffer in biochemistry labs (see Problem 8). The structure of Bicine in its fully protonated form is shown below: CH2CH2OH H+N

CH2COOH

CH2CH2OH

a. Draw the titration curve for Bicine, assuming the pKa for its free COOH group is 2.3 and the pKa for its tertiary amino group is 8.3. b. Draw the structure of the fully deprotonated form (completely dissociated form) of Bicine. c. You have available a 0.1 M solution of Bicine at its isoelectric point (pHI), 0.1 M solutions of HCl and NaOH, and ample distilled H2O. Describe the preparation of 1 L of 0.04 M Bicine buffer, pH 7.5. d. What is the concentration of fully protonated form of Bicine in your final buffer solution?

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52

Part 1 Building Blocks of the Cell

19. Hydrochloric acid is a significant component of gastric juice. What is the concentration of chloride ion in gastric juice if pH = 1.2? a. From the pKa for lactic acid given in Table 2.4, calculate the concentration of lactate in blood plasma (pH = 7.4) if the concentration of lactic acid is 1.5 mM. b. When a 0.1 M solution of a weak acid was titrated with base, the following results were obtained:

Equivalents of base added

pH observed

0.05

3.4

0.15

3.9

0.25

4.2

0.40

4.5

0.60

4.9

0.75

5.2

0.85

5.4

0.95

6.0

20. The enzyme alcohol dehydrogenase catalyzes the oxidation of ethyl alcohol by NAD+ to give acetaldehyde plus NADH and a proton: CH3CH2OH + NAD+ h CH3CHO = NADH + H+ The rate of this reaction can be measured by following the change in pH. The reaction is run in 1 mL 10 mM Tris buffer at pH 8.6. If the pH of the reaction solution falls to 8.4 after 10 minutes, what is the rate of alcohol oxidation, expressed as nanomoles of ethanol oxidized per mL per sec of reaction mixture? 21. In light of the Human Biochemistry box on page 49, what would be the effect on blood pH if cellular metabolism produced a sudden burst of carbon dioxide? 22. On the basis of Figure 2.12, what will be the pH of the acetate–acetic acid solution when the ratio of [acetate]/[acetic acid] is 10? a. 3.76 b. 4.76 c. 5.76 d. 14.76

Plot the results of this titration and determine the pKa of the weak acid from your graph.

FURTHER READING Properties of Water Cooke, R., and Kuntz, I. D., 1974. The properties of water in biological systems. Annual Review of Biophysics and Bioengineering 3:95–126.

Edsall, J. T., and Wyman, J., 1958. Carbon dioxide and carbonic acid, in Biophysical Chemistry, Vol. 1, Chap. 10. New York: Academic Press.

Finney, J. L., 2004. Water? What’s so special about it? Philosophical Transactions of the Royal Society, London, Series B 359:1145–1165.

Gillies R. J., and Lynch R. M., 2001. Frontiers in the measurement of cell and tissue pH. Novartis Foundation Symposium 240:7–19.

Franks, F., Ed., 1982. The Biophysics of Water. New York: John Wiley & Sons.

Kelly, J. A., 2000. Determinants of blood pH in health and disease. Critical Care 4:6–14.

Stillinger, F. H., 1980. Water revisited. Science 209:451–457.

Masoro, E. J., and Siegel, P. D., 1971. Acid-Base Regulation: Its Physiology and Pathophysiology. Philadelphia: W.B. Saunders.

Tokmakoff, A., 2007. Shining light on the rapidly evolving structure of water. Science 317:54–55. Properties of Solutions Cooper, T. G., 1977. The Tools of Biochemistry, Chap. 1. New York: John Wiley & Sons. Segel, I. H., 1976. Biochemical Calculations, 2nd ed., Chap. 1. New York: John Wiley & Sons. Titration Curves Darvey, I. G., and Ralston, G. B., 1993. Titration curves—misshapen or mislabeled? Trends in Biochemical Sciences 18:69–71. pH and Buffers

Norby, J. G., 2000. The origin and meaning of the little p in pH. Trends in Biochemical Sciences 25:36–37. Perrin, D. D., 1982. Ionization Constants of Inorganic Acids and Bases in Aqueous Solution. New York: Pergamon Press. Rose, B. D., 1994. Clinical Physiology of Acid–Base and Electrolyte Disorders, 4th ed. New York: McGraw-Hill. The Fitness of the Environment Henderson, L. J., 1913. The Fitness of the Environment. New York: Macmillan. (Republished 1970. Gloucester, MA: P. Smith.) Hille, B., 1992. Ionic Channels of Excitable Membranes, 2nd ed., Chap. 10. Sunderland, MA: Sinauer Associates.

Beynon, R. J., and Easterby, J. S., 1996. Buffer Solutions: The Basics. New York: IRL Press: Oxford University Press.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

3

Thermodynamics of Biological Systems

! The sun is the source of energy for virtually

Publicphoto/Photo Researchers Inc.

all life. We even harvest its energy in the form of electricity using windmills driven by air heated by the sun.

ESSENTIAL QUESTION Living things require energy. Movement, growth, synthesis of biomolecules, and the transport of ions and molecules across membranes all demand energy input. All organisms must acquire energy from their surroundings and must utilize that energy efficiently to carry out life processes. To study such bioenergetic phenomena requires familiarity with thermodynamics. Thermodynamics also allows us to determine whether chemical processes and reactions occur spontaneously. The student should appreciate the power and practical value of thermodynamic reasoning and realize that this is well worth the effort needed to understand it. What are the laws and principles of thermodynamics that allow us to describe the flows and interchanges of heat, energy, and matter in biochemical systems?

A theory is the more impressive the greater is the simplicity of its premises, the more different are the kinds of things it relates and the more extended is its range of applicability. Therefore, the deep impression which classical thermodynamics made upon me. It is the only physical theory of universal content which I am convinced, that within the framework of applicability of its basic concepts, will never be overthrown. Albert Einstein

KEY QUESTIONS 3.1

What Are the Basic Concepts of Thermodynamics?

3.2

What Can Thermodynamic Parameters Tell Us about Biochemical Events?

3.3

What Are the Characteristics of High-Energy Biomolecules?

3.4

What Are the Complex Equilibria Involved in ATP Hydrolysis?

3.5

Why Are Coupled Processes Important to Living Things?

3.6

What Is the Daily Human Requirement for ATP?

Even the most complicated aspects of thermodynamics are based ultimately on 3 simple and straightforward laws. These laws and their extensions sometimes run counter to our intuition. However, once truly understood, the basic principles of thermodynamics become powerful devices for sorting out complicated chemical and biochemical problems. Once we reach this milestone in our scientific development, thermodynamic thinking becomes an enjoyable and satisfying activity. Several basic thermodynamic principles are presented in this chapter, including the analysis of heat flow, entropy production, and free energy functions and the relationship between entropy and information. In addition, some ancillary concepts are considered, including the concept of standard states, the effect of pH on standard-state free energies, the effect of concentration on the net free energy change of a reaction, and the importance of coupled processes in living things. The chapter concludes with a discussion of ATP and other energy-rich compounds.

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

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54

Part 1 Building Blocks of the Cell

" William Francis Giauque was born in Niagara

Bettmann/CORBIS

Falls, Ontario, Canada in 1895 of parents who were U.S. citizens and became an instructor in Chemistry at the University of California, Berkeley in 1922 and then a Full Professor at Berkeley in 1934. He won the Nobel Prize in Chemistry in 1949 for his studies in the third law of thermodynamics with the study of the properties of matter at temperatures close to absolute zero.

WHY IT MATTERS Before we begin our discussion on the thermodynamics of biological systems, we pose the question, “Why is thermodynamics important to macromolecular function?” Thermodynamics controls the energy flow for biochemical processes and must be regulated in a controlled manner. Unregulated energy flow results in net loss of energy from a system, the production of unusable energy forms (such as heat), and potential detrimental effects on the cell (e.g., macromolecular unfolding due to excess heat production). Thus thermodynamics determines whether specific reactions, crucial for life processes, proceed or not.

3.1

What Are the Basic Concepts of Thermodynamics?

In any consideration of thermodynamics, a distinction must be made between the system and the surroundings. The system is that portion of the universe with which we are concerned. It might be a mixture of chemicals in a test tube, or a single cell, or an entire organism. The surroundings include everything else in the universe (Figure 3.1). The nature of the system must also be specified. There are 3 basic kinds of systems: isolated, closed, and open. An isolated system cannot exchange matter or energy with its surroundings. A  closed system may exchange energy, but not matter, with the surroundings. An open system may exchange matter, energy, or both with the surroundings. Living things are typically open systems that exchange matter (nutrients and waste products) and energy (e.g., heat from metabolism) with their surroundings.

The First Law: The Total Energy of an Isolated System Is Conserved It was realized early in the development of thermodynamics that heat could be converted into other forms of energy, and moreover that all forms of energy could ultimately be converted to some other form. The first law of thermodynamics states that the total energy of an isolated NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 3 Isolated system: No exchange of matter or energy

Open system

Closed system

Energy Surroundings

Matter

Energy

Surroundings

Surroundings

FIGURE 3.1 The characteristics of isolated, closed, and open systems. Isolated systems exchange neither matter nor energy with their surroundings. Closed systems may exchange energy, but not matter, with their surroundings. Open systems may exchange either matter or energy with the surroundings.

system is conserved. Thermodynamicists have formulated a mathematical function for keeping track of heat transfers and work expenditures in thermodynamic systems. This function is called the internal energy, commonly designated E or U, and it includes all the energies that might be exchanged in physical or chemical processes, including rotational, vibrational, and translational energies of molecules and also the energy stored in covalent and non-covalent bonds. The internal energy depends only on the present state of a system and hence is referred to as a state function. The internal energy does not depend on how the system got there and is thus independent of path. An extension of this thinking is that we can manipulate the system through any possible pathway of changes, and as long as the system returns to the original state, the internal energy, E, will not have been changed by these manipulations. The internal energy, E, of any system can change only if energy flows in or out of the system in the form of heat or work. For any process that converts one state (state 1) into another (state 2), the change in internal energy, !E, is given as !E = E2 - E1 = q + w,

55

Open system: Energy exchange and/or matter exchange may occur

Closed system: Energy exchange may occur

Isolated system

Thermodynamics of Biological Systems

(3.1)

where the quantity q is the heat absorbed by the system from the surroundings, and w is the work done on the system by the surroundings. Mechanical work is defined as movement through some distance caused by the application of a force. Both movement and force are required for work to have occurred. Examples of work done in biological systems include the flight of insects and birds, the circulation of blood by a pumping heart, the transmission of an impulse along a nerve, and the lifting of a weight by someone who is exercising. On the other hand, if a person strains to lift a heavy weight but fails to move the weight at all, then, in the thermodynamic sense, no work has been done. (The energy expended in the muscles of the would-be weight lifter is given off in the form of heat.) In chemical and biochemical systems, work is often concerned with the pressure and volume of the system under study. The mechanical work done on the system is defined as w = -P!V, where P is the pressure and !V is the volume change and is equal to V2 - V1. When work is defined in this way, the sign on the right side of Equation 3.1 is positive. (Sometimes w is defined as work done by the system; in this case, the equation is !E = q - w.) Work may occur in many forms, such as mechanical, electrical, magnetic, and chemical. !E, q, and w must all have the same units. The calorie, abbreviated cal, and kilocalorie (kcal) have been traditional choices of chemists and biochemists, but the SI unit, the joule, is now recommended.

Enthalpy Is a More Useful Function for Biological Systems If the definition of work is limited to mechanical work (w = -P!V) and no change in volume occurs, an interesting simplification is possible. In this case, !E is merely the heat exchanged at constant volume. This is so because if the volume is constant, no mechanical work can be done on or by the system. Then !E = q. Thus !E is a very useful quantity in constant volume processes. However, chemical and especially biochemical processes and reactions are much more likely to be carried out at constant pressure. In constant pressure processes, !E is not necessarily equal to the heat transferred. For this reason, chemists and NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

56

Part 1 Building Blocks of the Cell

biochemists have defined a function that is especially suitable for constant pressure processes. It is called the enthalpy, H, and it is defined as H = E + PV.

(3.2)

The clever nature of this definition is not immediately apparent. However, if the pressure is constant, then we have !H = !E + P!V = q + w + P!V = q - P!V + P!V = q.

(3.3)

So, !E is the heat transferred in a constant volume process, and !H is the heat transferred in a constant pressure process. Often, because biochemical reactions normally occur in liquids or solids rather than in gases, volume changes are typically small, and enthalpy and internal energy are often essentially equal. In order to compare the thermodynamic parameters of different reactions, it is convenient to define a standard state. For solutes in a solution, the standard state is normally unit activity (often simplified to 1 M concentration). Enthalpy, internal energy, and other thermodynamic quantities are often given or determined for standard-state conditions and are then denoted by a superscript degree sign (“°”), as in !H°, !E°, and so on. Enthalpy changes for biochemical processes can be determined experimentally by measuring the heat absorbed (or given off) by the process in a calorimeter (a reaction vessel that can be used to measure the heat evolved by a reaction). Alternatively, for any process A ∆ B at equilibrium, the standard-state enthalpy change for the process can be determined from the temperature dependence of the equilibrium constant: !H" = -R

d(ln keq) d(1 /T )

.

(3.4)

Here R is the gas constant, defined as R = 8.314 J/mol # K. A plot of R(ln Keq) versus 1/T is called a van’t Hoff plot. The example below demonstrates how a van’t Hoff plot is constructed and how the enthalpy change for a reaction can be determined from the plot itself.

EXAMPLE

In a study1 of the temperature-induced reversible denaturation of the protein chymotrypsinogen,

R ln Keq

30

native state (N) ∆ denatured state (D)

20

Keq = [D]/[N].

10

John F. Brandts measured the equilibrium constants for the denaturation over a range of pH and temperatures. The data for pH are

0 –10

T (K): Keq:

54.5°C –3.21–(–17.63) = 14.42

3.04–3.067 = –0.027 2.98 3.00 3.02 3.04 3.06 1000 –1 (K ) T

326.1 0.12

327.5 0.27

329.0 0.68

330.7 1.9

332.0 5.0

333.8 21

A plot of R(ln Keq) versus 1/T (a van’t Hoff plot) is shown in Figure 3.2. !H° for the denaturation process at any temperature is the negative of the slope of the plot at that temperature. As shown,

–20 –30

324.4 0.041

3.08

3.10

FIGURE 3.2 The enthalpy change, !H°, for a reaction can be determined from the slope of a plot of R ln Keq versus 1/T. To illustrate the method, the values of the data points on either side of the 327.5 K (54.5°C) data point have been used to calculate !H° at 54.5°C. Regression analysis would normally be preferable. (Adapted from Brandts, J. F., 1964. The thermodynamics of protein denaturation. I. The denaturation of chymotrypsinogen. Journal of the American Chemical Society 86:4291–4301.)

!H" = -

[-3.2 - (-17.6)] [(3.04 - 3.067) * 10-3]

= +533 kJ/mol.

What does this value of !H° mean for the unfolding of the protein? Positive values of !H° would be expected for the breaking of hydrogen bonds as well as for the exposure of hydrophobic groups from the interior of the native, folded protein during the unfolding process. Such events would raise the energy of the protein solution. The magnitude of this enthalpy change (533 kJ/mol) at 54.5°C is large, compared to similar values of !H° for other proteins and for this same protein at 25°C (Table 3.1). If we consider only this positive enthalpy change for the unfolding process, the native, folded state is strongly favoured. As we shall see, however, other parameters must be taken into account. 1 Brandts, J. F., 1964. The thermodynamics of protein denaturation. I. The denaturation of chymotrypsinogen. Journal of the American Chemical Society 86:4291–4301.

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Chapter 3

TABLE 3.1

Thermodynamic Parameters for Protein Denaturation !H° kJ/mol

!S° kJ/mol # K

!G° kJ/mol

!Cp kJ/mol # K

Chymotrypsinogen (pH 3, 25°C)

164

0.440

31.0

10.9

b-Lactoglobulin (5 M urea, pH 3, 25°C)

-88

-0.300

2.5

9.0

Myoglobin (pH 9, 25°C)

180

0.400

57.0

5.9

Ribonuclease (pH 2.5, 30°C)

240

0.780

3.8

8.4

Protein (and conditions)

Thermodynamics of Biological Systems

Adapted from Cantor, C., and Schimmel, P., 1980. Biophysical Chemistry. San Francisco: W.H. Freeman; and Tanford, C., 1968. Protein denaturation. Advances in Protein Chemistry 23:121–282.

The Second Law: Systems Tend Toward Disorder and Randomness The second law of thermodynamics has been described and expressed in many different ways, including the following: 1. Systems tend to proceed from ordered (low-entropy or low-probability) states to disordered (high-entropy or high-probability) states. 2. The entropy of the system plus surroundings is unchanged by reversible processes; the entropy of the system plus surroundings increases for irreversible processes. 3. All naturally occurring processes proceed toward equilibrium, that is, to a state of minimum potential energy. Energy flows spontaneously so as to become diffused or dispersed or spread out. Energy dispersal results in entropy increase. Several of these statements of the second law invoke the concept of entropy, which is a measure of disorder and randomness in the system (or the surroundings). An organized or ordered state is a low-entropy state, whereas a disordered state is a high-entropy state. All else being equal, reactions involving large, positive entropy changes, !S, are more likely to occur than reactions for which !S is not large and positive. S = k ln W

(3.5)

!S = k ln Wfinal - k ln Winitial,

(3.6)

and

where Wfinal and Winitial are the final and initial number of microstates, respectively, and where k is Boltzmann’s constant (k = 1.38 × 10-23 J/K). Seen in this way, entropy represents energy dispersion—the dispersion of energy among a large number of molecular motions relatable to quantized states (microstates). An increase in entropy is just an increase in the number of microstates in any macrostate. On the other hand, if only one microstate corresponds to a given macrostate, then the system has no freedom to choose its microstate—and it has zero entropy. This definition is useful for statistical calculations (in fact, it is a foundation of statistical thermodynamics), but a more common form relates entropy to the heat transferred in a process: dSreversible =

dq T

(3.7)

where dSreversible is the entropy change of the system in a reversible2 process, q is the heat transferred, and T is the temperature at which the heat transfer occurs. Equation 3.6 says that entropy change measures the dispersal of energy in a process. When Winitial is less than Wfinal, energy is dispersed from a small number of microstates (Winitial) to a larger number of microstates (Wfinal), and !S is a positive quantity. That is, dispersal of energy into a larger number of microstates results in an increase in entropy. 2

A reversible process is one that can be reversed by an infinitesimal modification of a variable.

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58

Part 1 Building Blocks of the Cell

A DEEPER LOOK

Entropy, Information, and the Importance of “Negentropy”

p

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s r l e o a A e t f e p a p r i h e r o i s e e o m s s i r t n i r a d s t m o e ch t s t t a t e s e e n o h o e f y b e n i a n d

i

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r e i a i f x d e t s

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When a thermodynamic system undergoes an increase in entropy, it becomes more disordered. On the other hand, a decrease in entropy reflects an increase in order. A more ordered system is more highly organized and possesses a greater information content. To appreciate the implications of decreasing the entropy of a system, consider the random collection of letters in the figure. This disorganized array of letters possesses no inherent information content, and nothing can be learned by its perusal. On the other hand, this particular array of letters can be systematically arranged to construct the first sentence of the Einstein quotation that opened this chapter: “A theory is the more impressive the greater is the simplicity of its premises, the more different are the kinds of things it relates and the more extended is its range of applicability.” Arranged in this way, this same collection of 151 letters possesses enormous information content—the profound words of a great scientist. Just as it would have required significant effort to rearrange these 151 letters in this way, so large amounts of energy are required to construct and maintain living organisms. Energy input is required to produce information-rich, organized structures such as proteins and nucleic acids. Information content can be thought of as negative entropy. In 1945 Erwin Schrödinger took time out from his studies of quantum mechanics to publish a delightful book titled What Is Life? In it, Schrödinger coined the term negentropy to describe the negative entropy changes that confer organization and information content to living organisms. Schrödinger pointed out that organisms must “acquire negentropy” to sustain life.

The Third Law: Why Is “Absolute Zero” So Important? The third law of thermodynamics states that the entropy of any crystalline, perfectly ordered substance must approach zero as the temperature approaches 0 K, and, at T = 0 K, entropy is exactly zero. Based on this, it is possible to establish a quantitative, absolute entropy scale for any substance as S =

L0

T

CPd ln T

(3.8)

where CP is the heat capacity at constant pressure. The heat capacity of any substance is the amount of heat 1 mole of it can store as the temperature of that substance is raised by 1 degree. For a constant pressure process, this is described mathematically as Cp =

dH . dT

(3.9)

If the heat capacity can be evaluated at all temperatures between 0 K and the temperature of interest, an absolute entropy can be calculated. For biological processes, entropy changes are more useful than absolute entropies. The entropy change for a process can be calculated if the enthalpy change and the free energy change are known.

Free Energy Provides a Simple Criterion for Equilibrium An important question for chemists, and particularly for biochemists, is, “Will the reaction proceed in the direction written?” J. Willard Gibbs, one of the founders of thermodynamics, realized that the answer to this question lay in a comparison of the enthalpy change and the entropy change for a reaction at a given temperature. The Gibbs free energy, G, is defined as G = H - TS.

(3.10) NEL

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Chapter 3

59

Thermodynamics of Biological Systems

For any process A ∆ B at constant pressure and temperature, the free energy change is given by !G = !H - T!S.

(3.11)

If !G is equal to 0, the process is at equilibrium and there is no net flow in either the forward or the reverse direction. When !G = 0, !S = !H/T and the enthalpic and entropic changes are exactly balanced. Any process with a non-zero !G proceeds spontaneously to a final state of lower free energy. If !G is negative, the process proceeds spontaneously in the direction written. If !G is positive, the reaction or process proceeds spontaneously in the reverse direction. (The sign and value of !G do not allow us to determine how fast the process will go.) If the process has a negative !G, it is said to be exergonic, whereas processes with positive !G values are endergonic. The Standard-State Free Energy Change The free energy change, !G, for any reaction depends upon the nature of the reactants and products, but it is also affected by the conditions of the reaction, including temperature, pressure, pH, and the concentrations of the reactants and products. As explained earlier, it is useful to define a standard state for such processes. If the free energy change for a reaction is sensitive to solution conditions, what is the particular significance of the standard-state free energy change? To answer this question, consider a reaction between 2 reactants A and B to produce the products C and D. A + B ∆ C + D.

(3.12)

The free energy change for non–standard-state concentrations is given by !G = !G " + RT ln

[C] [D] . [A] [B]

(3.13)

At equilibrium, !G = 0 and [C] [D]/[A] [B] = Keq. We then have !G ° = -RT ln Keq,

(3.14)

!G ° = -2.3RT log10Keq,

(3.15)

Keq = 10-!G°/2.3RT.

(3.16)

or, in base 10 logarithms,

This can be rearranged to

In any of these forms, this relationship allows the standard-state free energy change for any process to be determined if the equilibrium constant is known. More important, it states that the point of equilibrium for a reaction in solution is a function of the standard-state free energy change for the process. That is, !G° is another way of writing an equilibrium constant.

10 8

EXAMPLE

The equilibrium constants determined by Brandts at several temperatures for the denaturation of chymotrypsinogen (see previous example) can be used to calculate the free energy changes for the denaturation process. For example, the equilibrium constant at 54.5°C is 0.27, so

!G° (kJ/mol)

6 4 2 0 –2 –4

!G ° = -(8.314 J/mol · K)(327.5 K) ln (0.27)

–6

!G ° = -(2.72 kJ/mol) ln (0.27)

–8 –10

!G ° = 3.56 kJ/mol. The positive sign of !G ° means that the unfolding process is unfavourable; that is, the stable form of the protein at 54.5°C is the folded form. On the other hand, the relatively small magnitude of !G ° means that the folded form is only slightly favoured. Figure 3.3 shows the dependence of !G ° on temperature for the denaturation data at pH 3 (from the data given in the Example on page 56). (continued)

50

52

54 56 58 Temperature (°C)

60

62

FIGURE 3.3 The dependence of !G° on temperature for the denaturation of chymotrypsinogen. (Adapted from Brandts, J. F., 1964. The thermodynamics of protein denaturation. I. The denaturation of chymotrypsinogen. Journal of the American Chemical Society 86:4291–4301.)

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60

Part 1 Building Blocks of the Cell 2.4

Having calculated both !H ° and !G ° for the denaturation of chymotrypsinogen, we can also calculate !S°. Using Equation 3.11

2.3

!S° (kJ/mol • K)

2.2 2.1

!S" = -

2.0

(!G - !H") . T

(3.17)

At 54.5°C (327.5 K),

1.9 1.8

!S° = -(3560 - 533 000 J/mol)/327.5 K !S° = 1620 J/mol # K.

1.7 1.6 1.5 1.4 52

54 56 58 Temperature (°C)

60

FIGURE 3.4 The dependence of !S° on temperature for the denaturation of chymotrypsinogen. (Adapted from Brandts, J. F., 1964. The thermodynamics of protein denaturation. I. The denaturation of chymotrypsinogen. Journal of the American Chemical Society 86:4291–4301.)

Figure 3.4 presents the dependence of !S ° on temperature for chymotrypsinogen denaturation at pH 3. A positive !S ° indicates that the protein solution has become more disordered as the protein unfolds. Comparison of the value of 1.62 kJ/mol · K with the values of !S ° in Table 3.1 shows that the present value (for chymotrypsinogen at 54.5°C) is large. The physical significance of the thermodynamic parameters for the unfolding of chymotrypsinogen becomes clear later in this chapter.

3.2

What Can Thermodynamic Parameters Tell Us about Biochemical Events?

For biochemical reactions in which hydrogen ions (H+) are consumed or produced, the usual definition of the standard state is awkward. Standard state for the H+ ion is 1 M, which corresponds to pH 0. At this pH, nearly all enzymes would be denatured and biological reactions could not occur. It makes more sense to use free energies and equilibrium constants determined at pH 7. Biochemists have thus adopted a modified standard state, designated with prime (#) symbols, as in !G°#, Keq #, !H°#, and so on. For values determined in this way, a standard state of 10-7 M H+ and unit activity (1 M for solutions and 1 atm for gases and pure solids defined as unit activity) for all other components (in the ionic forms that exist at pH 7) is assumed. For biochemical events, a single parameter (e.g., !H or !S) is not very meaningful. A positive !H° for the unfolding of a protein might reflect either the breaking of hydrogen bonds within the protein or the exposure of hydrophobic groups to water (Figure 3.5). However, comparison of several thermodynamic parameters can provide meaningful insights about a process. For example, the transfer of Na+ and Cl- ions from the gas phase to aqueous solution involves a very large negative !H° (thus a very favourable stabilization of the ions) and a comparatively small !S° (Table 3.2). The negative entropy term reflects the ordering of water molecules in the hydration shells of the Na+ and Cl- ions. The

Folded

Unfolded

FIGURE 3.5 Unfolding of a soluble protein exposes significant numbers of non-polar groups to water, forcing order on the solvent and resulting in a negative !S° for the unfolding process. Orange spheres represent non-polar groups; blue spheres are polar and/or charged groups. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 3

TABLE 3.2 Thermodynamic Parameters for Several Simple Processes* Process Hydration of ions† Na+(g) + Cl-(g) h Na+(g)Cl-(aq)

Dissociation of ions in solution‡ H2O + CH3COOH h H3O + CH3COOTransfer of hydrocarbon from pure liquid to water‡ Toluene (in pure toluene) h toluene (aqueous)

Thermodynamics of Biological Systems

!H° kJ/mol

!S° kJ/mol # K

!G° kJ/mol

- 760.0

- 0.185

- 705.0

- 10.3

- 0.126

27.26

- 0.071

22.7

1.72

!CP kJ/mol # K

- 0.143

* All data collected for 25°C. † Berry, R. S., Rice, S. A., and Ross, J., 1980. Physical Chemistry. New York: John Wiley. ‡ Tanford, C., 1980. The Hydrophobic Effect. New York: John Wiley.

unfavourable -T!S contribution is more than offset by the large heat of hydration, which makes the hydration of ions a very favourable process overall. The negative entropy change for the dissociation of acetic acid in water also reflects the ordering of water molecules in the ion hydration shells. In this case, however, the enthalpy change is much smaller in magnitude. As a result, !G° for dissociation of acetic acid in water is positive, and acetic acid is thus a weak (largely undissociated) acid. The transfer of a non-polar hydrocarbon molecule from its pure liquid to water is an appropriate model for the exposure of protein hydrophobic groups to solvent when a protein unfolds. The transfer of toluene from liquid toluene to water involves a negative !S°, a positive !G°, and a !H° that is small compared to !G° (a pattern similar to that observed for the dissociation of acetic acid). What distinguishes these two very different processes is the change in heat capacity (Table 3.2). A positive heat capacity change for a process indicates that the molecules have acquired new ways to move (and thus to store heat energy). A negative !CP means that the process has resulted in less freedom of motion for the molecules involved. !CP is negative for the dissociation of acetic acid and positive for the transfer of toluene to water. The explanation is that polar and non-polar molecules both induce organization of nearby water molecules, but in different ways. The water molecules near a non-polar solute are organized but labile. Hydrogen bonds formed by water molecules near non-polar solutes rearrange more rapidly than the hydrogen bonds of pure water. On the other hand, the hydrogen bonds formed between water molecules near an ion are less labile (rearrange more slowly) than they would be in pure water. This means that !CP should be negative for the dissociation of ions in solution, as observed for acetic acid (Table 3.2).

3.3

What Are the Characteristics of High-Energy Biomolecules?

Virtually all life on earth depends on energy from the sun. Among life forms, there is a hierarchy of energetics: Certain organisms capture solar energy directly, whereas others derive their energy from this group in subsequent processes. Organisms that absorb light energy directly are called phototrophic organisms. These organisms store solar energy in the form of various organic molecules. Organisms that feed on these latter molecules, releasing the stored energy in a series of oxidative reactions, are called chemotrophic organisms. Despite these differences, both types of organisms share common mechanisms for generating a useful form of chemical energy. Once captured in chemical form, energy can be released in controlled exergonic reactions to drive a variety of life processes (which require energy). A small family of universal biomolecules mediates the flow of energy from exergonic reactions to the energy-requiring processes of life. These molecules are the reduced coenzymes and the high-energy phosphate compounds. Phosphate compounds are considered high energy if they exhibit large negative free energies of hydrolysis (i.e., if !G°# is more negative than -25 kJ/mol). Table 3.3 lists the most important members of the high-energy phosphate compounds. Such molecules include phosphoric anhydrides (ATP, ADP), an enol phosphate (PEP), NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

0.265

61

62

Part 1 Building Blocks of the Cell

TABLE 3.3 Free Energies of Hydrolysis of Some High-Energy Compounds* Compound and Hydrolysis Reaction

!G°! (kJ/mol) Structure

Phosphoenolpyruvate h pyruvate + Pi

-62.2

Figure 3.13

1,3-Bisphosphoglycerate h 3-phosphoglycerate + Pi

-49.6

Figure 3.12

Creatine phosphate h creatine + Pi

-43.3

Figure 13.21

Acetyl phosphate h acetate + Pi

-43.3

Figure 3.12

Adenosine-5#-triphosphate h ADP + Pi

-35.7†

Figure 3.9

-30.5

Figure 3.9

-35.7

Figure 3.11

Adenosine-5#-triphosphate h ADP + Pi (with excess

Mg2+)

Adenosine-5#-diphosphate h AMP + Pi Pyrophosphate h Pi + Pi (in 5 mM

Mg2+)

-33.6

Figure 3.10

Adenosine-5#-triphosphate h AMP + PPi (excess Mg2+)

-32.3

Figure 10.11

Uridine diphosphoglucose h UDP + glucose

-31.9

Figure 22.10

Acetyl-coenzyme A h acetate + CoA

-31.5

page 599

S-Adenosylmethionine h methionine + adenosine

-25.6‡

Figure 25.28

Glucose-1-phosphate h glucose + Pi

-21.0

Figure 7.13

Sn-Glycerol-3-phosphate h glycerol + Pi

-9.2

Figure 8.5

Adenosine-5#-monophosphate h adenosine + Pi

-9.2

Figure 10.11

* Adapted primarily from Handbook of Biochemistry and Molecular Biology, 1976, 3rd ed. In Physical and Chemical Data, G. Fasman, ed., Vol. 1, pp. 296–304. Boca Raton, FL: CRC Press. † From Gwynn, R. W., and Veech, R. L., 1973. The equilibrium constants of the adenosine triphosphate hydrolysis and the adenosine triphosphatecitrate lyase reactions. Journal of Biological Chemistry 248:6966–6972. ‡ From Mudd, H., and Mann, J., 1963. Activation of methionine for transmethylation. Journal of Biological Chemistry 238:2164–2170.

acyl phosphates (such as acetyl phosphate), and guanidino phosphates (such as creatine phosphate). Also included are thioesters, such as acetyl-CoA, which do not contain phosphorus, but which have a large negative free energy of hydrolysis. As noted earlier, the exact amount of chemical free energy available from the hydrolysis of such compounds depends on concentration, pH, temperature, and so on, but the !G°# values for hydrolysis of these substances are substantially more negative than those for most other metabolic species. Two important points: First, high-energy phosphate compounds are not long-term energy storage substances. They are transient forms of stored energy, meant to carry energy from point to point, from one enzyme system to another, in the minute-to-minute existence of the cell. (As we shall see in subsequent chapters, other molecules bear the responsibility for long-term storage of energy supplies.) Second, the term high-energy compound should not be construed to imply that these molecules are unstable and hydrolyze or decompose unpredictably. ATP, for example, is a very stable molecule. A substantial activation energy must be delivered to ATP to hydrolyze the terminal, or g, phosphate group. In fact, as shown in Figure 3.6, the activation energy that must be absorbed by the molecule to break the O i Pgbond is normally 200–400 kJ/mol, which is substantially larger than the net 30.5 kJ/mol released in the hydrolysis reaction. Biochemists are much more concerned with the net release of 30.5 kJ/mol than with the activation energy for the reaction (because suitable enzymes cope with the latter). The net release of large quantities of free energy distinguishes the high-energy phosphoric anhydrides from their “low-energy” ester cousins, such as glycerol-3-phosphate (Table 3.3). The next section provides a quantitative framework for understanding these comparisons.

ATP Is an Intermediate Energy-Shuttle Molecule One last point about Table 3.3 deserves mention. Given the central importance of ATP as a high-energy phosphate in biology, students are sometimes surprised to find that ATP holds an intermediate place in the rank of high-energy phosphates. PEP, 1,3-BPG, creatine phosphate, acetyl phosphate, and pyrophosphate all exhibit higher values of !G°´. This is not a biological anomaly. ATP is uniquely situated between the very-high-energy phosphates synthesized in the breakdown of fuel molecules and the numerous lower-energy NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 3

Thermodynamics of Biological Systems FIGURE 3.6 The activation energies for phosphoryl group transfer reactions (200–400 kJ/ mol) are substantially larger than the free energy of hydrolysis of ATP (–30.5 kJ/mol).

Transition state

Activation energy kJ ! 200–400 mol

ATP Reactants

ADP + Pi Phosphoryl group transfer potential ! –30.5 kJ/mol

Products

acceptor molecules that are phosphorylated in the course of further metabolic reactions. ADP can accept both phosphates and energy from the higher-energy phosphates, and the ATP thus formed can donate both phosphates and energy to the lower-energy molecules of metabolism. The ATP/ADP pair is an intermediately placed acceptor/donor system among high-energy phosphates. In this context, ATP functions as a very versatile but intermediate energy-shuttle device that interacts with many different energy-coupling enzymes of metabolism.

Group Transfer Potentials Quantify the Reactivity of Functional Groups Many reactions in biochemistry involve the transfer of a functional group from a donor molecule to a specific receptor molecule or to water. The concept of group transfer potential explains the tendency for such reactions to occur. Biochemists define the group transfer potential as the free energy change that occurs upon hydrolysis, that is, upon transfer of the particular group to water. This concept and its terminology are preferable to the more qualitative notion of high-energy bonds. The concept of group transfer potential is not particularly novel. Other kinds of transfer (e.g., of hydrogen ions and electrons) are commonly characterized in terms of appropriate measures of transfer potential (pKa and reduction potential, Eo, respectively). As shown in Table 3.4, the notion of group transfer is fully analogous to those of ionization potential and reduction potential. The similarity is anything but coincidental because all of these are really specific instances of free energy changes. If we write AH h A- + H+

(3.18a)

we really don’t mean that a proton has literally been removed from the acid AH. In the gas phase at least, this would require the input of approximately 1200 kJ/mol! What we really mean is that the proton has been transferred to a suitable acceptor molecule, usually water: AH + H2O h A- + H3O+.

(3.18b)

The appropriate free energy relationship is of course !G . 2.303 RT Similarly, in the case of an oxidation–reduction reaction pKa =

A h A+ + e-

(3.19)

(3.20a)

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63

64

Part 1 Building Blocks of the Cell

TABLE 3.4 Types of Transfer Potential

Simple equation Equation including acceptor Measure of transfer potential

Proton Transfer Potential (Acidity)

Standard Reduction Potential (Electron Transfer Potential)

Group Transfer Potential (High-Energy Bond)

AH ∆ A- + H+

A ∆ A+ + e-

A " P ∆ A + Pi

AH + H2O ∆ pKa =

A-

+ H3

O+

!G" 2.303 RT

Free energy change of transfer !G° per mole of H+ is given by: transferred

A +

H+

!eo =

∆

A+

+

1 2

H2

-!G" nf

2A " PO24 + H2O ∆ A i H + HPO4

ln Keq =

!G° per mole of etransferred

-!G" RT

!G° per mole of phosphate transferred

Adapted from: Klotz, I. M., 1986. Introduction to Biomolecular Energetics. New York: Academic Press.

we don’t really mean that A oxidizes independently. What we really mean (and what is much more likely in biochemical systems) is that the electron is transferred to a suitable acceptor: A + H+ h A+ +

1 H 2 2

(3.20b)

and the relevant free energy relationship is -!G" (3.21) nf where n is the number of equivalents of electrons transferred and F is Faraday’s constant. Similarly, the release of free energy that occurs upon the hydrolysis of ATP and other “high-energy phosphates” can be treated quantitatively in terms of group transfer. It is common to write for the hydrolysis of ATP !eo =

ATP + H2O h ADP + Pi.

(3.22)

The free energy change, which we henceforth call the group transfer potential, is given by !G° = -RT ln Keq

(3.23)

where Keq is the equilibrium constant for the group transfer, which is normally written as Keq =

[ADP] [Pi] [ATP] [H2O]

.

(3.24)

Even this set of equations represents an approximation because ATP, ADP, and Pi all exist in solutions as a mixture of ionic species. This problem is discussed in a later section. For now, it is enough to note that the free energy changes listed in Table 3.3 are the group transfer potentials observed for transfers to water.

The Hydrolysis of Phosphoric Acid Anhydrides Is Highly Favourable ATP contains 2 pyrophosphoryl or phosphoric acid anhydride linkages, as shown in Figure 3.7. Other common biomolecules possessing phosphoric acid anhydride linkages include ADP, GTP, GDP and the other nucleoside diphosphates and triphosphates, sugar nucleotides such as UDP–glucose, and inorganic pyrophosphate itself. All exhibit large negative free energies of hydrolysis, as shown in Table 3.3. The chemical reasons for the large negative !G°#values for the hydrolysis reactions include destabilization of the reactant due to bond strain caused by electrostatic repulsion, stabilization of the products by ionization and resonance, and entropy factors due to hydrolysis and subsequent ionization. Destabilization Due to Electrostatic Repulsion Electrostatic repulsion in the reactants is best understood by comparing these phosphoric anhydrides with other reactive anhydrides, such as acetic anhydride. As shown in Figure 3.8a, the electronegative carbonyl oxygen atoms withdraw electrons from the C “ O bonds, producing partial negative charges on the oxygens and partial positive charges on the carbonyl carbons. Each of these electrophilic carbonyl NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 3

N

N

N

N –O

P

O

O

O O

O–

O

P

P

O

CH2

O–

O–

O

OH OH ATP (adenosine-5'-triphosphate)

carbons is further destabilized by the other acetyl group, which is also electron-withdrawing in nature. As a result, acetic anhydride is unstable with respect to the products of hydrolysis. The situation with phosphoric anhydrides is similar. The phosphorus atoms of the pyrophosphate anion are electron-withdrawing and destabilize PPi with respect to its hydrolysis products. Furthermore, the reverse reaction, reformation of the anhydride bond from the 2 anionic products, requires that the electrostatic repulsion between these anions be overcome (see following). Stabilization of Hydrolysis Products by Ionization and Resonance The pyro-phosphoryl moiety possesses 2 negative charges at pH values above 7.5 or so (Figure 3.8a). The hydrolysis products, 2 phosphate esters, each carry about 2 negative charges at pH values above 7.2. The increased ionization of the hydrolysis products helps stabilize the electrophilic phosphorus nuclei. (a) Acetic anhydride:

H2O

$– O $+ C O

+ H3C

$– O $+ C CH3

Phosphoric anhydrides: O

O

O O–

2 CH3C

RO

P

O

OR'

O–

O–

2 H+

P

O

H2O RO

P O–

O O–

+

–O

P O–

(b) Competing resonance in acetic anhydride: O– H3C

C

O

O O +

C

H3C

CH3

C

O O

C

O–

O CH3

H3C

C

O +

C

CH3

These can only occur alternately

Simultaneous resonance in the hydrolysis products: O–

O H3C

C

O–

H3C

C

–O

O O

–O

C

CH3

65

FIGURE 3.7 The triphosphate chain of ATP contains 2 pyrophosphate linkages, both of which release large amounts of energy upon hydrolysis.

NH2 Phosphoric anhydride linkages

Thermodynamics of Biological Systems

O

C

CH3

These resonances can occur simultaneously FIGURE 3.8 (a) Electrostatic repulsion between adjacent partial positive charges (on carbon and phosphorus respectively) is relieved upon hydrolysis of the anhydride bonds of acetic anhydride and phosphoric anhydrides. (b) The competing resonances of acetic anhydride and the simultaneous resonance forms of the hydrolysis product, acetate. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

OR'

66

Part 1 Building Blocks of the Cell

Resonance stabilization in the products is best illustrated by the reactant anhydrides (Figure 3.8b). The unpaired electrons of the bridging oxygen atom in acetic anhydride (and phosphoric anhydride) cannot participate in resonance structures with both electrophilic centres at once. This competing resonance situation is relieved in the product acetate or phosphate molecules. Entropy Factors Arising from Hydrolysis and Ionization For the phosphoric anhydrides, and for most of the high-energy compounds discussed here, there is an additional “entropic” contribution to the free energy of hydrolysis. Most of the hydrolysis reactions of Table 3.3 result in an increase in the number of molecules in solution. As shown in Figure 3.9, the hydrolysis of ATP (at pH values above 7) creates 3 species—ADP, inorganic phosphate (Pi), and a hydrogen ion—from only 2 reactants (ATP and H2O). The entropy of the solution increases because the more particles, the more disordered the system.3 (This effect is ionization-dependent because, at low pH, the hydrogen ion created in many of these reactions simply protonates 1 of the phosphate oxygens, and 1 fewer “particle” results from the hydrolysis.)

The Hydrolysis "G°! of ATP and ADP Is Greater Than That of AMP The concepts of destabilization of reactants and stabilization of products described for pyrophosphate also apply for ATP and other phosphoric anhydrides (Figure 3.9). ATP and ADP are destabilized relative to the hydrolysis products by electrostatic repulsion, NH2 N

N –

–

O$ –O

+

P$

–

O$ O

O–

+

P$

O$

+

P$

O

O–

N O

CH2

N

O

O– OH OH ATP

NH2

H2O –

H+

+

–O

P

–

O$

O OH

+

–O

O–

$+

P

O$ O

N

N

$+

P

N O

CH2

N

O

O–

O–

OH OH ADP NH2

H2O –

O$

O H+

+

–O

P O–

OH

+

–O

N

N

+

P$

N O

CH2

N

O

O– OH OH AMP

FIGURE 3.9 Hydrolysis of ATP to ADP (and/or of ADP to AMP) leads to relief of electrostatic repulsion. 3

Imagine the “disorder” created by hitting a crystal with a hammer and breaking it into many small pieces. NEL

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Chapter 3

Thermodynamics of Biological Systems

competing resonance, and entropy. AMP, on the other hand, is a phosphate ester (not an anhydride) possessing only a single phosphoryl group and is not markedly different from the product inorganic phosphate in terms of electrostatic repulsion and resonance stabilization. Thus, the !G°# for hydrolysis of AMP is much smaller than the corresponding values for ATP and ADP.

Acetyl Phosphate and 1,3-Bisphosphoglycerate Are Phosphoric-Carboxylic Anhydrides The mixed anhydrides of phosphoric and carboxylic acids, frequently called acyl phosphates, are also energy-rich. Two biologically important acyl phosphates are acetyl phosphate and 1,3-bisphosphoglycerate. Hydrolysis of these species yields acetate and 3-phosphoglycerate, respectively, in addition to inorganic phosphate (Figure 3.10). Once again, the large !G°# values indicate that the reactants are destabilized relative to products. This arises from bond strain, which can be traced to the partial positive charges on the carbonyl carbon and phosphorus atoms of these structures. The energy stored in the mixed anhydride bond (which is required to overcome the charge–charge repulsion) is released upon hydrolysis. Increased resonance possibilities in the products relative to the reactants also contribute to the large negative !G°# values. The value of !G°# depends on the pKa values of the starting anhydride and the product phosphoric and carboxylic acids, and of course also on the pH of the medium.

Enol Phosphates Are Potent Phosphorylating Agents The largest value of !G°# in Table 3.3 belongs to phosphoenolpyruvate or PEP, an example of an enolic phosphate. This molecule is an important intermediate in carbohydrate metabolism, and due to its large negative !G°#, it is a potent phosphorylating agent. PEP is formed via dehydration of 2-phosphoglycerate by enolase during fermentation and glycolysis. PEP is subsequently transformed into pyruvate upon transfer of its phosphate to ADP by pyruvate kinase (Figure 3.11). The very large negative value of !G°# for the latter reaction is to a large extent the result of a secondary reaction of the enol form of pyruvate. Upon hydrolysis, the unstable enolic form of pyruvate immediately converts to the keto form with a resulting large negative !G°# (Figure 3.12). Together, the hydrolysis and subsequent tautomerization result in an overall !G°# of -62.2 kJ/mol.

O–

O C

CH3

O

P

O–

O O–

+

H2O

!G°' = –43.3 kJ/mol

CH3

C

O–

+

HO

O

P

O–

+

H+

O

Acetyl phosphate

O–

O C

O

HCOH

P

O–

O O–

+

O

H2O

!G°' = –49.6 kJ/mol

C

O–

+

HCOH

O

P

P

O–

+

H+

O

O– CH2

HO

O– O–

CH2

O

O 1,3-Bisphosphoglycerate

P

O–

O 3-Phosphoglycerate

FIGURE 3.10 The hydrolysis reactions of acetyl phosphate and 1,3-bisphosphoglycerate. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

67

68

Part 1 Building Blocks of the Cell

FIGURE 3.11 Phosphoenolpyruvate (PEP) is produced by the enolase reaction (in glycolysis; see Chapter 17) and in turn drives the phosphorylation of ADP to form ATP in the pyruvate kinase reaction.

O –O

O O–

P

OH

–O

H2O

O

O–

O

Enolase

COO–

P

H2C C COO– Mg2+ Phosphoenolpyruvate (PEP) !G°' = +1.8 kJ/mol

H2C CH 2-Phosphoglycerate

O –O

P

O– ADP

O H2C

C

–O

P

2+,

+

H2O

!G = –28.6 kJ/mol

–O

H2C

C

OH O–

P

O

K+

+

H2C

C

COO–

Pyruvate (unstable enol form)

OH

H3C

Mg !G°' = –31.7 kJ/mol

O O–

O

Pyruvate kinase

COO–

Phosphoenolpyruvate PEP

O

ATP

H+

C

COO–

Pyruvate

Tautomerization !G = –33.6 kJ/mol

O H3C

C

COO–

Pyruvate (stable keto)

COO–

PEP FIGURE 3.12 Hydrolysis and the subsequent tautomerization account for the very large !G°# of PEP.

Guanidino Phosphates Are Energy Reserve Compounds Some tissues (skeletal and smooth muscle, brain, photoreceptor cells, retina cells, hair cells of the inner ear, and spermatozoa) consume ATP so rapidly that the reserves of ATP are insufficient to provide for this energy demand. Phosphocreatine serves as an energy reservoir for the rapid buffering and regeneration of ATP in situ, as well as for intracellular energy transport by the phosphocreatine shuttle (Figure 3.13). Conversion of creatine (Cr) FIGURE 3.13 The phosphocreatine shuttle. Mitochondrial creatine kinases (sMtCK, uMtCK) produce phosphocreatine (PCr) from creatine (Cr), which is shuttled to the cytosolic creatine kinases (MM-CK, BB-CK, MB-CK). PCr is built up in the cytosol from ATPdependent processes, such as oxidative phosphorylation or glycolysis. The large cytosolic PCr pool (up to 30 mM in the cell) is utilized to maintain the cytosolic ATP/ADP ratio or to regenerate ATP for ATP-consuming processes. (Adapted from Schlattner, U. et al., 2006. Mitochondrial creatine kinase in human health and disease. Biochimica et Biophysica Acta 1762: 164–180).

Mitochondria

Cytosol

Cr ATP

CK ADP

ATP

ATP

ATP

CK

CK

ADP

ADP ADP

PCr Glycolysis

CK

Cytosolic ATP/ADP ratio

Cytosolic ATP consumption

Oxidative phosphorylation CK

Mitochondrial creatine kinase (sMICK, uMICK)

CK

Cytosolic creatine kinase (MM-CK, BB-CK, MB-CK) ATPases (transporters, pumps, enzymes) NEL

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Chapter 3

Thermodynamics of Biological Systems

69

to phosphocreatine (PCr) is effected by creatine kinase (CK), of which there are numerous isoforms. Mitochondrial isozymes of creatine kinases (MtCK) are present in vertebrates where sarcomeric MtCK (sMtCK) occurs in striated muscle and ubiquitous MtCK (uMtCK) occurs in most other tissues. The cytosolic isoforms of CK consist of combinations of 2 subunits: the brain-type subunit (B) and the muscle-type subunit (M). Thus the 3 isoforms of cytosolic CK are BB-CK, MB-CK, and MM-CK. PCr, generated by MtCKs is shuttled to the cytosolic CKs, which are coupled to ATP-dependent processes. Cytosolic CKs convert PCr to ATP, thus regenerating the ATP utilized as an energy source by ATPases. PCr is not only an energy buffer for ATP utilization, but also a shuttle of energy between subcellular sites of energy (ATP) production (mitochondria and glycolysis) and those of energy utilization (ATPases).

3.4

What Are the Complex Equilibria Involved in ATP Hydrolysis?

So far, as in Equation 3.25, the hydrolyses of ATP and other high-energy phosphates have been portrayed as simple processes. The situation in a real biological system is far more complex, owing to the operation of several ionic equilibria. First, ATP, ADP, and the other species in Table 3.3 can exist in several different ionization states that must be accounted for in any quantitative analysis. Second, phosphate compounds bind a variety of divalent and monovalent cations with substantial affinity, and the various metal complexes must also be considered in such analyses. Consideration of these special cases makes the quantitative analysis far more realistic. The importance of these multiple equilibria in group transfer reactions is illustrated for the hydrolysis of ATP, but the principles and methods presented are general and can be applied to any similar hydrolysis reaction.

The "G°! of Hydrolysis for ATP Is pH Dependent

NH2

HO

P

O O

OH

P OH

O O

P

CH2

–50

–40

N

N O

–60

N

N O

–70

!G° (kJ/mol)

ATP has 4 dissociable protons, as indicated in Figure 3.14. Three of the protons on the triphosphate chain dissociate at very low pH. The last proton to dissociate from the triphosphate chain possesses a pKa of 6.95. At higher pH values, ATP is completely deprotonated. ADP and phosphoric acid also undergo multiple ionizations. These multiple ionizations make the equilibrium constant for ATP hydrolysis more complicated than the simple expression in Equation 3.24. Multiple ionizations must also be taken into account when the pH dependence of !G° is considered. The calculations are beyond the scope of this text, but Figure 3.15 shows the variation of !G° as a function of pH. The free energy of hydrolysis is nearly constant from pH 4 to pH 6. At higher values of pH, !G° varies linearly with pH, becoming more negative by 5.7 kJ/mol for every pH unit of increase at 37°C. Because the pH of most biological tissues and fluids is near neutrality, the effect on !G° is relatively small, but it must be taken into account in certain situations.

–35.7

O –30

OH HO

Colour indicates the locations of the dissociable protons of ATP FIGURE 3.14 Adenosine-5#-triphosphate (ATP).

OH

4 5 6 7 8 9 10 11 12 13 pH

FIGURE 3.15 The pH dependence of the free energy of hydrolysis of ATP. Because pH varies only slightly in biological environments, the effect on !G is usually small.

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70

Part 1 Building Blocks of the Cell

Metal Ions Affect the Free Energy of Hydrolysis of ATP

–36.0

!G°' (kJ/mol)

–35.0 –34.0 –33.0 –32.0 –31.0 –30.0 1

2

3 4 5 –Log10 [Mg2+]

6

FIGURE 3.16 The free energy of hydrolysis of ATP as a function of total Mg2+ ion concentration at 38°C and pH 7.0. (Adapted from Gwynn, R.W., and Veech, R. L., 1973. The equilibrium constants of the adenosine triphosphate hydrolysis and the adenosine triphosphate-citrate lyase reactions. Journal of Biological Chemistry 248:6966–6972.)

Most biological environments contain substantial amounts of divalent and monovalent metal ions, including Mg2+, Ca2+, Na+, K+, and so on. What effect do metal ions have on the equilibrium constant for ATP hydrolysis and the associated free energy change? Figure 3.16 shows the change in !G°# with pMg (i.e., -log10[Mg2+]) at pH 7.0 and 38°C. The free energy of hydrolysis of ATP at zero Mg2+ is -35.7 kJ/mol, and at 5 mM total Mg2+ (the minimum in the plot) the !Gobs° is approximately -31 kJ/mol. Thus, in most real biological environments (with pH near 7 and Mg2+ concentrations of 5 mM or more) the free energy of hydrolysis of ATP is altered more by metal ions than by protons. A widely used “consensus value” for !G°# of ATP in biological systems is -30.5 kJ/mol (Table 3.3). This value, cited in the 1976 Handbook of Biochemistry and Molecular Biology (3rd ed., Physical and Chemical Data, Vol. 1, pp. 296–304, Boca Raton, FL: CRC Press), was determined in the presence of “excess Mg2+.” This is the value we use for metabolic calculations in the balance of this text. Why does the G°# of ATP hydrolysis depend so strongly on Mg2+ concentration? The answer lies in the strong binding of Mg2+ by the triphosphate oxygens of ATP. As shown in Figure 3.17, the binding of Mg2+ to ATP is dependent on Mg2+ ion concentration and also on pH. At pH 7 and 1 mM [Mg2+], approximately 1 Mg2+ ion is bound to each ATP. The decrease in binding of Mg2+ at low pH is the result of competition by H+ and Mg2+ for the negatively charged oxygen atoms of ATP.

Concentration Affects the Free Energy of Hydrolysis of ATP Through all these calculations of the effect of pH and metal ions on the ATP hydrolysis equilibrium, we have assumed “standard conditions” with respect to concentrations of all species except for protons. The levels of ATP, ADP, and other high-energy metabolites never even begin to approach the standard state of 1 M. In most cells, the concentrations of these species are more typically 1–5 mM, or even less. Earlier, we described the effect of concentration on equilibrium constants and free energies in the form of Equation 3.13. For the present case, we can rewrite this as !G = !G" + RT ln

[%ADP] [%Pi]

(3.25)

[%ATP]

where the terms in brackets represent the sum (%) of the concentrations of all the ionic forms of ATP, ADP, and Pi. It is clear that changes in the concentrations of these species can have large effects on !G. The concentrations of ATP, ADP, and Pi may, of course, vary rather independently in real biological environments, but if, for the sake of some model calculations, we assume that all 3 concentrations are equal, then the effect of concentration on !G is as shown in Figure 3.18. The free energy of hydrolysis of ATP, which is -35.7 kJ/mol at 1 M, becomes -49.4 kJ/mol at 5 mM (i.e., the concentration for which pC = -2.3 in Figure 3.18). At 1 mM

Number of Mg2+ bound per ATP

1.5 1

6

0.5 0

5 4

4

3

6 pH

8

pMg

2 1

FIGURE 3.17 Number of Mg2+ ions bound per ATP as a function of pH and [Mg2+]. pMg = -log10 [Mg2+]. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

ATP, ADP, and Pi, the free energy change becomes even more negative, at -53.6 kJ/mol. Clearly, the effects of concentration are much greater than the effects of protons or metal ions under physiological conditions. Does the “concentration effect” change ATP’s position in the energy hierarchy (in Table 3.3)? Not really. All the other high- and low-energy phosphates experience roughly similar changes in concentration under physiological conditions and thus similar changes in their free energies of hydrolysis. The roles of the very-high-energy phosphates (PEP, 1,3-bisphosphoglycerate, and creatine phosphate) in the synthesis and maintenance of ATP in the cell are considered in our discussions of metabolic pathways. In the meantime, several of the problems at the end of this chapter address some of the more interesting cases.

3.5

–53.5 –50

–45

–40

–35.7

Why Are Coupled Processes Important to Living Things?

0

Many of the reactions necessary to keep cells and organisms alive must run against their thermodynamic potential, that is, in the direction of positive !G. Among these are the synthesis of adenosine triphosphate (ATP) and other high-energy molecules and the creation of ion gradients in all mammalian cells. These processes are driven in the thermodynamically unfavourable direction via coupling with highly favourable processes. Many such coupled processes are discussed later in this text. They are crucially important in intermediary metabolism, oxidative phosphorylation, and membrane transport, as we shall see. We can predict whether pairs of coupled reactions will proceed spontaneously by simply summing the free energy changes for each reaction. For example, consider the reaction from glycolysis (discussed in Chapter 17) involving the conversion of phosphoenolpyruvate (PEP) to pyruvate (Figure 3.19). The hydrolysis of PEP is energetically very favourable, and it is used to drive phosphorylation of adenosine diphosphate (ADP) to form ATP, a process that is energetically unfavourable. Using values of !G that would be typical for a human erythrocyte, PEP + H2O h pyruvate + Pi

!G = -78 kJ/mol

(3.26)

ADP + Pi h ATP + H20

!G = +55 kJ/mol

(3.27)

Total !G = -23 kJ/mol

(3.28)

PEP + ADP h pyruvate + ATP

71

Thermodynamics of Biological Systems

!G (kJ/mol)

Chapter 3

1.0 2.0 –Log10 [C] Where C = concentration of ATP, ADP, and Pi

3.0

FIGURE 3.18 The free energy of hydrolysis of ATP as a function of concentration at 38°C, pH 7.0. The plot follows the relationship described in Equation 3.25, with the concentrations [C] of ATP, ADP, and Pi assumed to be equal.

The net reaction catalyzed by this enzyme depends upon coupling between the 2 reactions shown in Equations 3.26 and 3.27 to produce the net reaction shown in Equation 3.28 with a net negative !G. Many other examples of coupled reactions are considered in our discussions of intermediary metabolism (see Part III). In addition, many of the complex biochemical systems discussed in the later chapters of this text involve reactions and processes with positive !G values that are driven forward by coupling to reactions with a negative !G.

3.6

What Is the Daily Human Requirement for ATP?

We can end this discussion of ATP and the other important high-energy compounds in biology by discussing the daily metabolic consumption of ATP by humans. An approximate calculation gives a somewhat surprising and impressive result. Assume that the average adult human consumes approximately 11 700 kJ (2800 kcal, i.e., 2800 calories) per day. Assume also that the metabolic pathways leading to ATP synthesis operate at a thermodynamic efficiency of approximately 50%. Thus, of the 11 700 kJ a person consumes as food, about 5860 kJ end up in the form of synthesized ATP. As indicated earlier, the hydrolysis of 1 mole of ATP yields approximately 50 kJ of free energy under cellular conditions. This means that the body cycles through 5860/50 = 117 moles of ATP each day. The disodium salt of ATP has a molecular weight of 551 g/mol, so an average person hydrolyzes about (117 moles)(551 g/mole) = 64 467 g of ATP per day.

COO– C

OPO32 –

ADP + Pi

ATP

COO– C

O

CH2

CH3

PEP

Pyruvate

FIGURE 3.19 The pyruvate kinase reaction.

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72

Part 1 Building Blocks of the Cell

The average adult human, with a typical weight of 70 kg or so, thus consumes approximately 65 kg of ATP per day, an amount nearly equal to his or her own body weight! Fortunately, we have a highly efficient recycling system for ATP/ADP utilization. The energy released from food is stored transiently in the form of ATP. Once ATP energy is used and ADP and phosphate are released, our bodies recycle it to ATP through intermediary metabolism so that it may be reused. The typical 70 kg body contains only about 50 grams of ATP/ADP total. Therefore, each ATP molecule in our bodies must be recycled nearly 1300 times each day! Were it not for this fact, at current commercial prices of about $20 per gram, our ATP “habit” would cost more than $1 million per day! In these terms, the ability of biochemistry to sustain the marvellous activity and vigour of organisms gains our respect and fascination.

A DEEPER LOOK

ATP Changes the Keq by a Factor of 108 Consider a process, A ∆ B. It could be a biochemical reaction, or the transport of an ion against a concentration gradient, or even a mechanical process (such as muscle contraction). Assume that it is a thermodynamically unfavourable reaction. Let’s say, for purposes of illustration, that !G°# = +13.8 kJ/mol. From the equation,

equilibrium in the presence of typical prevailing concentrations of ATP, ADP, and Pi.* Keq =

!G°# = -RT ln Keq

850 =

+13 800 = -(8.31 J/K # mol)(298 K) ln Keq

850,000 = 2.2 * 108 0.0038

ln Keq = -5.57. Therefore, Keq = 0.0038 = [Beq]/[Aeq]. This reaction is clearly unfavourable (as we could have foreseen from its positive !G°#). At equilibrium, there is 1 molecule of product B for every 263 molecules of reactant A. Not much A was transformed to B. Now suppose the reaction A ∆ B is coupled to ATP hydrolysis, as is often the case in metabolism: A + ATP ∆ B + ADP +Pi. The thermodynamic properties of this coupled reaction are the same as the sum of the thermodynamic properties of the partial reactions:

A + ATP + H2O ∆ B + ADP + Pi

[Beq] [8 * 10-3] [10-3]

Comparison of the [Beq]/[Aeq] ratio for the simple A ∆ B reaction with the coupling of this reaction to ATP hydrolysis gives

which yields

ATP + H2O ∆ ADP + Pi

[Aeq] [ATP]

[Aeq] [8 * 10-3] [Beq] / [Aeq] = 850,000

we have

A ∆ B

[Beq] [ADP] [Pi]

The equilibrium ratio of B to A is more than 108 greater when the reaction is coupled to ATP hydrolysis. A reaction that was clearly unfavourable (Keq = 0.0038) has become emphatically spontaneous! The involvement of ATP has raised the equilibrium ratio of B/A by more than 200 million–fold. It is informative to realize that this multiplication factor does not depend on the nature of the reaction. Recall that we defined A ∆ B in the most general terms. Also, the value of this equilibrium constant ratio, some 2.2 * 108, is not at all dependent on the particular reaction chosen or its standard free energy change, !G°#. You can satisfy yourself on this point by choosing some value for !G°# other than +13.8 kJ/mol and repeating these calculations (keeping the concentrations of ATP, ADP, and Pi at 8, 8, and 1 mM, as before). NH2

!G"# = +13.8

kJ mol

Phosphoric anhydride linkages

!G"# = -30.5

kJ mol

O

!G"# = -16.7 kJ/mol

N –O

P O–

That is,

N

N

O

P O–

N

O

O O

P O–

O

CH2

O

!G°#overall = -16.7 kJ/mol. OH OH

So -16,700 = -RT ln Keq = - (8.31)(298) ln Keq ln Keq = -16,700/ - 2476 = 6.75 keq = 850 Using this equilibrium constant, let’s now consider the cellular situation in which the concentrations of A and B are brought to

ATP (adenosine-5'-triphosphate) *The concentrations of ATP, ADP, and Pi in a normal, healthy bacterial cell growing at 25°C are maintained at roughly 8 mM, 8 mM, and 1 mM, respectively. Therefore, the ratio [ADP][Pi]/[ATP] is about 10-3. Under these conditions, !G for ATP hydrolysis is approximately -47.6 kJ/mol. NEL

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Chapter 3

Thermodynamics of Biological Systems

73

SUMMARY The activities of living things require energy. Movement, growth, synthesis of biomolecules, and the transport of ions and molecules across membranes all demand energy input. All organisms must acquire energy from their surroundings and must utilize that energy efficiently to carry out life processes. To study such bioenergetic phenomena requires familiarity with thermodynamics. Thermodynamics also allows us to determine whether chemical processes and reactions occur spontaneously. 3.1 What Are the Basic Concepts of Thermodynamics? The system is that portion of the universe with which we are concerned. The surroundings include everything else in the universe. An isolated system cannot exchange matter or energy with its surroundings. A closed system may exchange energy, but not matter, with the surroundings. An open system may exchange matter, energy, or both with the surroundings. Living things are typically open systems. The first law of thermodynamics states that the total energy of an isolated system is conserved. Enthalpy, H, is defined as H = E + PV. !H is equal to the heat transferred in a constant pressure process. For biochemical reactions in liquids, volume changes are typically small, and enthalpy and internal energy are often essentially equal. There are several statements of the second law of thermodynamics, including the following: (1) Systems tend to proceed from ordered (low-entropy or low-probability) states to disordered (high entropy or high probability) states. (2) The entropy of the system plus surroundings is unchanged by reversible processes; the entropy of the system plus surroundings increases for irreversible processes. (3) All naturally occurring processes proceed toward equilibrium, that is, to a state of minimum potential energy. The third law of thermodynamics states that the entropy of any crystalline, perfectly ordered substance must approach zero as the temperature approaches 0 K, and, at T = 0 K, entropy is exactly zero. The Gibbs free energy, G, defined as G = H TS, provides a simple criterion for equilibrium. The free energy change for a reaction can be very different from the standard-state value if the concentrations of reactants and products differ significantly from unit activity (1 M for solutions). For the reaction A + B ∆ C + D, the free energy change for non–standard-state concentrations is given by !G = !G" + RT ln

[C] [D] . [A] [B]

3.2 What Can Thermodynamic Parameters Tell Us about Biochemical Events? For biochemical reactions in which hydrogen ions (H+) are consumed or produced, a modified standard state, designated with

prime (#) symbols, as in !G°#, Keq #, !H°#, may be employed. For a reaction in which H+ is produced, !G°# is given by !G°# = !G° + RT ln [H+] For biochemical events, a single parameter (e.g., !H or !S) is not very meaningful, but comparison of several thermodynamic parameters can provide meaningful insights about a process. Thermodynamic parameters can be used to predict whether a given reaction will occur as written and to calculate the relative contributions of molecular phenomena (e.g., hydrogen bonding or hydrophobic interactions) to an overall process. 3.3 What Are the Characteristics of High-Energy Biomolecules? A small family of universal biomolecules mediates the flow of energy from exergonic reactions to the energy-requiring processes of life. These molecules are the reduced coenzymes and the high-energy phosphate compounds. High-energy phosphates are not long-term energy storage substances, but rather transient forms of stored energy. 3.4 What Are the Complex Equilibria Involved in ATP Hydrolysis? ATP, ADP, and similar species can exist in several different ionization states that must be accounted for in any quantitative analysis. Also, phosphate compounds bind a variety of divalent and monovalent cations with substantial affinity, and the various metal complexes must also be considered in such analyses. 3.5 Why Are Coupled Processes Important to Living Things? Many of the reactions necessary to keep cells and organisms alive must run against their thermodynamic potential, that is, in the direction of positive !G. These processes are driven in the thermodynamically unfavourable direction via coupling with highly favourable processes. Many such coupled processes are crucially important in intermediary metabolism, oxidative phosphorylation, and membrane transport. 3.6 What Is the Daily Human Requirement for ATP? The average adult human, with a typical weight of 70 kg or so, consumes approximately 2800 calories per day. The energy released from food is stored transiently in the form of ATP. Once ATP energy is used and ADP and phosphate are released, our bodies recycle it to ATP through intermediary metabolism so that it may be reused. The typical 70 kg body contains only about 50 grams of ATP/ADP total. Therefore, each ATP molecule in our bodies must be recycled nearly 1300 times each day.

PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. An enzymatic hydrolysis of fructose-1-P, fructose-1-P + H2O ∆ fructose + Pi, was allowed to proceed to equilibrium at 25°C. The original concentration of fructose-1-P was 0.2 M, but when the system had reached equilibrium the concentration of fructose-1-P was only 6.52 * 10-5 M. Calculate the equilibrium constant for this reaction and the free energy of hydrolysis of fructose-1-P. 2. The equilibrium constant for some process A ∆ B is 0.5 at 20°C and 10 at 30°C. Assuming that !H° is independent of temperature, calculate !H° for this reaction. Determine !G° and !S° at 20°C and at 30°C. Why is it important in this problem to assume that !H° is independent of temperature? 3. The standard-state free energy of hydrolysis for acetyl phosphate is !G° = -42.3 kJ/mol. Acetyl-P + H2O h acetate + Pi

Calculate the free energy change for acetyl phosphate hydrolysis in a solution of 2 mM acetate, 2 mM phosphate, and 3 nM acetyl phosphate. 4. Define a state function. Name 3 thermodynamic quantities that are state functions and 3 that are not. 5. ATP hydrolysis at pH 7.0 is accompanied by release of a hydrogen ion to the medium: ATP4- + H2O ∆ ADP3- + HPO42- + H+. If the !G°# for this reaction is -30.5 kJ/mol, what is !G° (i.e., the free energy change for the same reaction with all components, including H+, at a standard state of 1 M)? 6. For the process A ∆ B, Keq (AB) is 0.02 at 37°C. For the process B ∆ C, Keq (BC) = 1000 at 37°C. a. Determine Keq (AC), the equilibrium constant for the overall process A ∆ C, from Keq (AB) and Keq (BC). b. Determine standard-state free energy changes for all 3 processes, and use !G° (AC) to determine Keq (AC). Make sure this value agrees with that determined in part a of this problem.

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74

Part 1 Building Blocks of the Cell

7. Draw all possible resonance structures for creatine phosphate and discuss their possible effects on resonance stabilization of the molecule. 8. Write the equilibrium constant, Keq, for the hydrolysis of creatine phosphate and calculate a value for Keq at 25°C from the value of !G°# in Table 3.3. 9. Imagine that creatine phosphate, rather than ATP, is the universal energy-carrier molecule in the human body. Repeat the calculation presented in Section 3.6, calculating the weight of creatine phosphate that would need to be consumed each day by a typical adult human if creatine phosphate could not be recycled. If recycling of creatine phosphate were possible, and if the typical adult human body contained 20 grams of creatine phosphate, how many times would each creatine phosphate molecule need to be turned over or recycled each day? Repeat the calculation assuming that glycerol-3-phosphate is the universal energy carrier and that the body contains 20 grams of glycerol-3-phosphate. 10. Calculate the free energy of hydrolysis of ATP in a rat liver cell in which the ATP, ADP, and Pi concentrations are 3.4, 1.3, and 4.8 mM, respectively. 11. Hexokinase catalyzes the phosphorylation of glucose from ATP, yielding glucose-6-P and ADP. Using the values of Table 3.3, calculate the standard-state free energy change and equilibrium constant for the hexokinase reaction. 12. Would you expect the free energy of hydrolysis of acetoacetylcoenzyme A (see diagram) to be greater than, equal to, or less than that of acetyl-coenzyme A? Provide a chemical rationale for your answer. O C

CH3

O CH2

C

S

CoA

13. Consider carbamoyl phosphate, a precursor in the biosynthesis of pyrimidines: O + H3N

C O

2–

PO3

Based on the discussion of high-energy phosphates in this chapter, would you expect carbamoyl phosphate to possess a high free energy of hydrolysis? Provide a chemical rationale for your answer. 14. You are studying the various components of the venom of a poisonous lizard. One of the venom components is a protein that appears to be

temperature sensitive. When heated, it denatures and is no longer toxic. The process can be described by the following simple equation: T (toxic) ∆ N (nontoxic). There is only enough protein from this venom to carry out 2 equilibrium measurements. At 298 K, you find that 98% of the protein is in its toxic form. However, when you raise the temperature to 320 K, you find that only 10% of the protein is in its toxic form. a. Calculate the equilibrium constants for the T-to-N conversion at these 2 temperatures. b. Use the data to determine the !H°, !S°, and !G° for this process. 15. Consider the data in Figures 3.3 and 3.4. Is the denaturation of chymotrypsinogen spontaneous at 58°C? What is the temperature at which the native and denatured forms of chymotrypsinogen are in equilibrium? 16. Consider Tables 3.1 and 3.2, as well as the discussion of Table 3.2 in the text, and discuss the meaning of the positive !CP in Table 3.1. 17. The difference between !G° and !G°# was discussed in Section 3.2. Consider the hydrolysis of acetyl phosphate (Figure 3.10) and determine the value of !G° for this reaction at each of pH 2, 7, and 12. The value of !G°# for the enolase reaction (Figure 3.11) is 1.8 kJ/mol. What is the value of !G° for enolase at pH 2, 7, and 12? Why is this case different from that of acetyl phosphate? 18. What is the significance of the magnitude of !G°# for ATP in the calculations in the box on page 72? Repeat these calculations for the case of coupling of a reaction to 1,3-bisphosphoglycerate hydrolysis to see what effect this reaction would have on the equilibrium ratio for components A and B under the conditions stated on that page. 19. The hydrolysis of 1,3-bisphosphoglycerate is favourable, due in part to the increased resonance stabilization of the products of the reaction. Draw resonance structures for the reactant and the products of this reaction to establish that this statement is true. 20. The acyl-CoA synthetase reaction activates fatty acids for oxidation in cells: R-COO- + CoASH + ATP h R-COSCoA + AMP + pyrophosphate The reaction is driven forward in part by hydrolysis of ATP to AMP and pyrophosphate. However, pyrophosphate undergoes further cleavage to yield 2 phosphate anions. Discuss the energetics of this reaction in both the presence and the absence of pyrophosphate cleavage.

FURTHER READING General Readings on Thermodynamics

Chemistry of Adenosine-5 #-Triphosphate

Alberty, R. A., 2003. Thermodynamics of Biochemical Reactions. New York: John Wiley. Cantor, C. R., and Schimmel, P. R., 1980. Biophysical Chemistry. San Francisco: W. H. Freeman. Dickerson, R. E., 1969. Molecular Thermodynamics. New York: Benjamin Co. Edsall, J. T., and Gutfreund, H., 1983. Biothermodynamics: The Study of Biochemical Processes at Equilibrium. New York: John Wiley. Edsall, J. T., and Wyman, J., 1958. Biophysical Chemistry. New York: Academic Press. Klotz, L. M., 1967. Energy Changes in Biochemical Reactions. New York: Academic Press. Lambert, F. L., 2002. Disorder: A cracked crutch for supporting entropy discussions. Journal of Chemical Education 79:187–192. Lambert, F.L., 2002. Entropy is simple, qualitatively. Journal of Chemical Education 79:1241–1246. [See also www.entropysite.com/entropy_is_ simple/index.html/ for a revision of this paper.] Lehninger, A. L., 1972. Bioenergetics, 2nd ed. New York: Benjamin Co. Morris, J. G., 1968. A Biologist’s Physical Chemistry. Reading, MA: Addison-Wesley.

Alberty, R. A., 1968. Effect of pH and metal ion concentration on the equilibrium hydrolysis of adenosine triphosphate to adenosine diphosphate. Journal of Biological Chemistry 243:1337–1343. Alberty, R. A., 1969. Standard Gibbs free energy, enthalpy, and entropy changes as a function of pH and pMg for reactions involving adenosine phosphates. Journal of Biological Chemistry 244:3290–3302. Gwynn, R. W., Veech, R. L., 1973. The equilibrium constants of the adenosine triphosphate hydrolysis and the adenosine triphosphate-citrate lyase reactions. Journal of Biological Chemistry 248:6966–6972. Special Topics Brandts, J. F., 1964. The thermodynamics of protein denaturation. I. The denaturation of chymotrypsinogen. Journal of the American Chemical Society 86:4291–4301. Schneider, E. D., and Sagan, D., 2005. Into the Cool: Energy Flow, Thermodynamics, and Life. Chicago: University of Chicago Press. Schrödinger, E., 1945. What Is Life? New York: Macmillan. Segel, I. H., 1976. Biochemical Calculations, 2nd ed. New York: John Wiley. Tanford, C., 1980. The Hydrophobic Effect, 2nd ed. New York: John Wiley. NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

4

Amino Acids

! All objects have mirror images. Like many

molecules, amino acids exist in mirror-image forms (stereoisomers) that are not superimposable. Only the l-isomers of amino acids commonly occur in nature. (Three Sisters Wilderness, central Oregon. The Middle Sister, reflected in an alpine lake.)

To hold, as ’twere, the mirror up to nature. William Shakespeare

irisphoto1/Shutterstock

Hamlet

ESSENTIAL QUESTION Proteins are the indispensable agents of biological function, and amino acids are the building blocks of proteins. The stunning diversity of the thousands of proteins found in nature arises from the intrinsic properties of only 20 commonly occurring amino acids. These features include (1) the capacity to polymerize, (2) novel acid–base properties, (3) varied structure and chemical functionality in the amino acid side chains, and (4) chirality. This chapter describes each of these properties, laying a foundation for discussions of protein structure (Chapters 5 and 6), enzyme function (Chapters 12–14), and many other subjects in later chapters.

KEY QUESTIONS 4.1

What Are the Structures and Properties of Amino Acids?

4.2

What Are the Acid–Base Properties of Amino Acids?

4.3

What are the Post-translational Modifications of Amino Acids?

4.4

What Are the Optical and Stereochemical Properties of Amino Acids?

4.5

What Are the Spectroscopic Properties of Amino Acids?

4.6

What Is the Fundamental Structural Pattern in Proteins?

Why are amino acids uniquely suited to their role as the building blocks of proteins?

WHY IT MATTERS? Before we begin our discussion of amino acids and their properties, we pose the question, “Why do amino acids matter?” As we will demonstrate in upcoming paragraphs and subsequent chapters, they are vitally important for various reasons, the first and most significant being that they are critically essential for life. Most notably, amino acids are intuitively associated with metabolism and nutrition, key variables necessary for survival. They encroach on our lives daily due to the well-known fact that amino acids are widely used in the food industry [e.g., the food flavour enhancer monosodium glutamate (MSG) is derived from glutamic acid] and also in the pharmaceutical field for potential drug development.

Online homework and a Student Self Assessment for this chapter may be assigned in OWL NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

76

Part 1 Building Blocks of the Cell

" Canadian bodybuilder Joe Weider—Amino

© Robert Galbraith/Reuters/CORBIS

acids are the building blocks of proteins and also of bodybuilders.

4.1

What Are the Structures and Properties of Amino Acids?

Typical Amino Acids Contain a Central Tetrahedral Carbon Atom The structure of a single, typical, amino acid is shown in Figure 4.1. Central to this structure is the tetrahedral alpha (a) carbon (Ca), which is covalently linked to both the amino group and the carboxyl group. Also bonded to this a-carbon are a hydrogen and a variable side chain. It is the side chain, the so-called R group, that gives each amino acid its identity. The detailed acid–base properties of amino acids are discussed in the following sections. It is sufficient for now to realize that, in neutral solution (pH 7), the carboxyl group exists as ⎯COO- and the amino group as i NH3+. Because the resulting amino acid contains 1 positive and 1 negative charge, it is a neutral molecule called a zwitterion. Amino acids are also chiral molecules. With 4 different groups attached to it, the a-carbon is said to be asymmetric. The 2 possible configurations for the a-carbon constitute non-identical mirror-image isomers, or enantiomers. Details of amino acid stereochemistry are discussed in Section 4.4.

Amino Acids Can Join via Peptide Bonds The crucial feature of amino acids that allows them to polymerize to form peptides and proteins is the existence of their 2 identifying chemical groups: the amino ( i NH3+) and carboxyl (⎯COO-) groups, as shown in Figure 4.2. The amino and carboxyl groups of amino acids can react in a head-to-tail fashion, eliminating a water molecule and forming a covalent amide linkage, which, in the case of peptides and proteins, is typically referred to as a peptide bond. The equilibrium for this reaction in aqueous solution favours peptide bond hydrolysis. For this reason, biological systems as well as peptide chemists in the laboratory must couple peptide bond formation in an indirect manner or with energy input.

!-Carbon

H + H3N Amino group

R

R

Side chain

R

C! COO– Carboxyl group

+ NH3

COO– COO–

Ball-and-stick model

+ NH3

Amino acids are tetrahedral structures

FIGURE 4.1 Anatomy of an amino acid. Except for proline and its derivatives, all amino acids commonly found in proteins possess this type of structure. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 4

Amino Acids

R H H

+

Ca

N

O

–

C

H

H

+ Ca

O

–

+

O

–

C

N

Two amino acids

O

+

–

Removal of a water molecule...

H2O

Peptide bond

–

+ Amino end

...formation of the CO NH bond

Carboxyl end

FIGURE 4.2 The a-COOH and a-NH3+ groups of 2 amino acids can react with the resulting loss of a water molecule to form a covalent amide bond.

Repetition of the reaction shown in Figure 4.2 produces polypeptides and proteins. The remarkable properties of proteins, which we shall discover and come to appreciate in later chapters, all depend in one way or another on the unique properties and chemical diversity of the 20 common amino acids found in proteins.

There Are 20 Common Amino Acids The structures and abbreviations for the 20 amino acids commonly found in proteins are shown in Figure 4.3. All the amino acids except proline have both free a-amino and free a-carboxyl groups (Figure 4.1). There are several ways to classify the common amino acids. The most useful of these classifications is based on the polarity of the side chains. Thus, the structures shown in Figure 4.3 are grouped into the following categories: (1) non-polar or hydrophobic amino acids, (2) neutral (uncharged) but polar amino acids, (3) acidic amino acids (which have a net negative charge at pH 7.0), and (4) basic amino acids (which have a net positive charge at neutral pH). In later chapters, the importance of this classification system for predicting protein properties becomes clear. Also shown in Figure 4.3 (see Table 4.1 as well) are the 3-letter and 1-letter codes used to represent the amino acids. These codes are useful when displaying and comparing the sequences of proteins in shorthand form. (Note that several of the 1-letter abbreviations are phonetic in origin: arginine = “Rginine” = R, phenyl-alanine = “Fenylalanine” = F, aspartic acid = “asparDic” = D.) Non-polar Amino Acids The non-polar amino acids (Figure 4.3a) are critically important for the processes that drive protein chains to “fold,” that is to form their natural (and functional) structures, as shown in Chapter 6. Amino acids termed non-polar include all those with alkyl chain R groups (alanine, valine, leucine, and isoleucine); as well as proline (with its unusual cyclic structure); methionine (1 of the 2 sulfur-containing amino acids); and 2  aromatic amino acids, phenylalanine and tryptophan. Tryptophan is sometimes NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

77

78 (a)

Part 1 Building Blocks of the Cell Nonpolar (hydrophobic) COOH H3N+

C

H

CH2

COOH + H2N

C

H2C

CH2 CH2

CH H3C

CH3

Leucine (Leu, L)

Proline (Pro, P)

COOH H3N+

H

H

C

COOH H3N+

H

C CH

CH3

CH3

Alanine (Ala, A) (b)

CH3

Valine (Val, V)

Polar, uncharged COOH

COOH H3N+

H

C

H3N+

H

C

H

CH2 OH

Glycine (Gly, G)

Serine (Ser, S) COOH

COOH H3N+

C

H3N+

H

CH2

C

C

NH2

Asparagine (Asn, N)

(c)

H

CH2

CH2

O

C

O

NH2

Glutamine (Gln, Q)

Acidic COOH COOH N+

H3

C

H

H3N+

C

H

CH2

CH2

CH2

COOH

COOH

Aspartic acid (Asp, D)

Glutamic acid (Glu, E)

FIGURE 4.3 The 20 amino acids that are the building blocks of most proteins can be classified as (a) non-polar (hydrophobic); (b) polar, neutral; (c) acidic; or (d) basic. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be produced without permission.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 4

COOH H3N+

C

COOH

H

H3N+

CH2

CH2 S

N H

CH3 Methionine (Met, M)

C

C CH

Tryptophan (Trp, W)

COOH H3

H

C

CH2

N+

Amino Acids

COOH

H

CH2

H3N+

C

H

H3C

C

H

CH2 CH3 Phenylalanine (Phe, F)

Isoleucine (Ile, I)

COOH H3N+

C

H

H

C

OH

COOH H3N+

C

H

CH2

CH3

SH

Threonine (Thr, T)

Cysteine (Cys, C) COOH

COOH H3N+

C

H

H3N+

C CH2

CH2 HC

C

H+N

NH C H

OH Tyrosine (Tyr, Y) (d)

H

Histidine (His, H)

Basic COOH

COOH H3N+

C

H

H3N+

CH2

CH2

CH2

CH2 CH2

CH2

NH

CH2 Lysine (Lys, K)

H

C

NH3+

C H2+N

NH2

Arginine (Arg, R)

FIGURE 4.3 continued

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79

80

Part 1 Building Blocks of the Cell

considered a borderline member of this group because it can interact favourably with water via the N⎯H moiety of the indole ring. Proline, strictly speaking, is not an amino acid but an a-imino acid. Polar, Uncharged Amino Acids The polar, uncharged amino acids (Figure 4.3b), except for glycine, contain R groups that can form hydrogen bonds with water and play a variety of nucleophilic roles in enzyme reactions. These amino acids are usually more soluble in water than the non-polar amino acids. The amide groups of asparagine and glutamine; the hydroxyl groups of tyrosine, threonine, and serine; and the sulfhydryl group of cysteine are all good hydrogen bond–forming moieties. Glycine, the simplest amino acid, has only a single hydrogen for an R group, and this hydrogen is not a good hydrogen bond former. Glycine’s solubility properties are influenced mainly by its polar amino and carboxyl groups, and thus glycine is best considered a member of the polar, uncharged group. It should be noted that tyrosine has significant non-polar characteristics due to its aromatic ring and could arguably be placed in the non-polar group. However, with a pKa of 10.1, tyrosine’s phenolic hydroxyl is a charged, polar entity at high pH. Acidic Amino Acids There are 2 acidic amino acids, aspartic acid and glutamic acid, whose R groups contain a carboxyl group (Figure 4.3c). These side-chain carboxyl groups are weaker acids than the a-COOH group but are sufficiently acidic to exist as ⎯COO− at neutral pH. Aspartic acid and glutamic acid thus have a net negative charge at pH 7. These forms are appropriately referred to as aspartate and glutamate. These negatively charged amino acids play several important roles in proteins. Many proteins that bind metal ions for structural or functional purposes possess metal-binding sites containing 1 or more aspartate and glutamate side chains. The acid–base chemistry of such groups is considered in detail in Section 4.2. Basic Amino Acids Three of the common amino acids have side chains with net positive charges at neutral pH: histidine, arginine, and lysine (Figure 4.3d). Histidine contains an imidazole group, arginine contains a guanidino group, and lysine contains a protonated alkyl amino group. The side chains of the latter 2 amino acids are fully protonated at pH 7, but histidine, with a side-chain pKa of 6.0, is only 10% protonated at pH 7. With a pKa near neutrality, histidine side chains play important roles as proton donors and acceptors in many enzyme reactions. Histidine-containing peptides are important biological buffers, as discussed in Chapter 2. Arginine and lysine side chains, which are protonated under physiological conditions, participate in electrostatic interactions in proteins.

Are There Other Ways to Classify Amino Acids? There are alternative ways to classify the 20 common amino acids. For example, it would be reasonable to imagine that the amino acids could be described as hydrophobic, hydrophilic, or amphipathic: Hydrophobic:

Hydrophilic:

Amphipathic:

alanine

proline

arginine

glutamine

lysine

glycine

valine

asparagine

histidine

methionine

isoleucine

aspartic acid

serine

tryptophan

leucine

cysteine

threonine

tyrosine

phenylalanine

glutamic acid

Lysine can be considered amphipathic because its R group consists of an aliphatic side chain, which can interact with hydrophobic amino acids in proteins, and an amino group, which is normally charged at neutral pH. Methionine is the least polar of the amphipathic amino acids, but its thioether sulfur can be an effective metal ligand in proteins. Cysteine can deprotonate at pH values greater than 7; and the thiolate anion is the most potent nucleophile that can be generated among the 20 common acids. The imidazole ring of NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 4

81

Amino Acids

histidine has 2 nitrogen atoms, each with an H. The pKa for dissociation of the first of these 2 Hs is around 6. However, once 1 N⎯H has dissociated, the pKa value for the other becomes greater than 10.

Amino Acids 21 and 22—and More? Although uncommon, natural amino acids beyond the well-known 20 actually do occur. Selenocysteine (Figure 4.4a) was first identified in 1986 (see Chapter 30, page 1012), and it has since been found in a variety of organisms. More recently, Joseph Krzycki and his colleagues at Ohio State University discovered a lysine derivative—pyrrolysine—in several archaeal species, including Methanosarcina barkeri, found as a bottom-dwelling microbe of freshwater lakes. Pyrrolysine (Figure 4.4a) and selenocysteine both are incorporated naturally into proteins thanks to specially adapted RNA molecules. Both selenocysteine and pyrrolysine bring novel structural and chemical features to the proteins that contain them. How many more unusual amino acids might be incorporated in proteins in a similar manner?

Several Amino Acids Occur Only Rarely in Proteins There are several amino acids that occur only rarely in proteins and are produced by modifications of 1 of the 20 amino acids already incorporated into a protein (Figure 4.4b), including hydroxylysine and hydroxyproline, which are found mainly in the collagen and gelatin proteins, pyroglutamic acid, which is found in a light-driven proton-pumping protein called bacteriorhodopsin, and G-carboxyglutamic acid, which is found in calciumbinding proteins. Certain amino acids and their derivatives, although not found in proteins, nonetheless are biochemically important. A few of the more notable examples are shown in Figure 4.4c. G-Aminobutyric acid, or GABA, is produced by the decarboxylation of glutamic acid (b)

(a) COOH + H3N

C

H

5-Hydroxylysine

COOH + H3N

C

+ H3N

C

SeH

CH2

CH2

CH2

CH2

CH2

CHOH

N

(c) CH3

NH + 3

(CH2)3

NH+2

CH2

NH + 3

CH2

CH2

HO

C

H

NH N

OH OH Epinephrine

H2C

H CH2

COOH

COOH + H3N

C

OH

HN

H

CH2

C H

Pyroglutamic acid

C

C O

H CH2

C H2

CH HOOC

COOH

NH+3

CH3 Pyrrolysine

"-Aminobutyric acid (GABA)

C

"-Carboxyglutamic acid

CH2

C O

COOH

HN

H

CH2

HN

COOH

COOH

H

CH2

Selenocysteine

4-Hydroxyproline

Histamine

HO N H

CH2 CH2

Serotonin

NH + 3 FIGURE 4.4 The structures of several amino acids that are less common but nevertheless found in certain proteins. Hydroxylysine and hydroxyproline are found in connective-tissue proteins; pyroglutamic acid is found in bacteriorhodopsin (a protein in Halobacterium halobium). Epinephrine, histamine, and serotonin, although not amino acids, are derived from and closely related to amino acids.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

82

Part 1 Building Blocks of the Cell O

HO OH HO

NH2 L -DOPA

FIGURE 4.5 The structure of L-DOPA. L-DOPA is derived from the amino acid L-tyrosine and is used in the treatment of Parkinson’s disease. (Knowles Nobel Lecture Adapted from Knowles, Asymmetric Hydrogenations, 2001.)

and is a potent neurotransmitter. Histamine, which is synthesized by decarboxylation of histidine, and serotonin, which is derived from tryptophan, similarly function as neurotransmitters and regulators. Epinephrine (also known as adrenaline), derived from tyrosine, is an important hormone.

Amino Acid Derivatives The chemical synthesis of amino acid derivatives has become an important and extremely demanding field in organic chemistry. These peptide intermediates are obtained from amino acid modifications and retain many of the amino acid structural characteristics in the final product. Many amino acids are synthetically used in the production of potential drugs, inhibitors of enzymes, and antagonists of receptors and small peptides (for example a tryptophan-derived ketone is a potent antagonist of Substance P). The importance of this field is highlighted in the awarding of the 2001 Nobel Prize in Chemistry to American Chemist William S. Knowles for his notable studies on the catalytic synthesis of L-DOPA (Figure 4.5), the widely used drug for the treatment of Parkinsonism. Taken together, these findings clearly signify the relevance of amino acids and their derivatives.

4.2

What Are the Acid–Base Properties of Amino Acids?

Amino Acids Are Weak Polyprotic Acids From a chemical point of view, the common amino acids are all weak polyprotic acids. The ionizable groups are not strongly dissociating ones, and the degree of dissociation thus depends on the pH of the medium. All the amino acids contain at least 2 dissociable hydrogens. Consider the acid–base behaviour of glycine, the simplest amino acid. At low pH, both the amino and the carboxyl groups are protonated and the molecule has a net positive charge. If the counter-ion in solution is a chloride ion, this form is referred to as glycine hydrochloride. If the pH is increased, the carboxyl group is the first to dissociate, yielding the neutral zwitterionic species Gly0 (Figure 4.6). A further increase in pH eventually results in dissociation of the amino group to yield the negatively charged glycinate. If we denote these 3 forms as Gly+, Gly0, and Gly-, we can write the first dissociation of Gly+ as

Gly+ + H2O ∆ Gly0 + H3O+ and the dissociation constant K1 as

K1 = pH 1 Net charge +1

[Gly+]

pH 13 Net charge –1

R

Cα

R

!

O

N

C

O

Cα

COOH C

H

"

C

O

+ H3N

.

pH 7 Net charge 0

R

! N

[Gly0][H3O+]

N

O

Cα

O

COO–

H+

+ H3N

C

H

"

C O

COO–

H+

H2N

C

H

R

R

R

Cationic form

Zwitterion (neutral)

Anionic form

FIGURE 4.6 The ionic forms of the amino acids, shown without consideration of any ionizations on the side chain. The cationic form is the low pH form, and the titration of the cationic species with base yields the zwitterion and finally the anionic form. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 4

Amino Acids

TABLE 4.1 pKa Values of Common Amino Acids A-COOH pKa

A-NH3+ pKa

Alanine (Ala, A)

2.4

9.7

Arginine (Arg, R)

2.2

9.0

Asparagine (Asn, N)

2.0

8.8

Aspartic acid (Asp, D)

2.1

9.8

Cysteine (Cys, C)

1.7

10.8

8.3

Glutamic acid (Glu, E)

2.2

9.7

4.3

Glutamine (Gln, Q)

2.2

9.1

Glycine (Gly, G)

2.3

9.6

Histidine (His, H)

1.8

9.2

Isoleucine (Ile, I)

2.4

9.7

Leucine (Leu, L)

2.4

9.6

Lysine (Lys, K)

2.2

9.0

Methionine (Met, M)

2.3

9.2

Phenylalanine (Phe, F)

1.8

9.1

Proline (Pro, P)

2.1

10.6

Serine (Ser, S)

2.2

9.2

~13

Threonine (Thr, T)

2.6

10.4

~13

Tryptophan (Trp, W)

2.4

9.4

Tyrosine (Tyr, Y)

2.2

9.1

Valine (Val, V)

2.3

9.6

Amino Acid

R group pKa 12.5 3.9

6.0

10.5

10.1

Values for K1 for the common amino acids are typically 0.4 to 1.0 * 10−2 M, so that typical values of pK1 centre on values of 2.0 to 2.4 (Table 4.1). In a similar manner, we can write the second dissociation reaction as

Gly0 + H2O ∆ Gly- + H3O+ and the dissociation constant K2 as

K2 =

[Gly-][H3O+] [Gly0]

.

Typical values for pK2 are in the range of 9.0 to 9.8. At physiological pH, the a-carboxyl group of a simple amino acid (with no ionizable side chains) is completely dissociated, whereas the a-amino group has not really begun its dissociation. The titration curve for such an amino acid is shown in Figure 4.7. Remember, the dissociation constant, Ka, of an acid or base defines or measures the position of its equilibrium. In the case of the dissociation constant for acetic acid (CH3COOH), the Ka would be equivalent to

Ka = [H+][CH3COO-]>[CH3COOH] = 1.8 * 10-5 The larger the Ka value, the stronger the acid. Likewise, the smaller the Ka value, the weaker the acid. The term pKa is a simple logarithmic transformation of Ka defined as

pKa = -log Ka Thus, the pKa for CH3COOH = 4.76 (a constant value). To describe the relationship between the ratio of conjugate base (A-) to conjugate acid (HA) and pH, the Henderson–Hasselbalch equation is used:

pH = pKa + log [A-]>[HA]. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

FIGURE 4.7 Titration of glycine, a simple amino acid. The isoelectric point, pI, the pH where glycine has a net charge of 0, can be calculated as (pK1 + pK2)/2.

Gly+

Gly–

Gly0 COO–

COOH H3N+ CH2 14

COO–

H3N+ CH2

H2N

CH2

12 pK 2

10 pH

8 Isoelectric point

6 4 2 0

pK 1 1.0

Equivalents of H+

0

Equivalents of OH–

1.0

0

1.0 Equivalents of OH– added

2.0

2.0

1.0 Equivalents of H+ added

0

EXAMPLE

What is the pH of a glycine solution in which the a-NH3+ group is onethird dissociated? Answer The appropriate Henderson–Hasselbalch equation is pH = pKa + log 10

[Gly- ] Gly0

.

If the a-amino group is one-third dissociated, there is 1 part Gly- for every 2 parts Gly0. The important pKa is the pKa for the amino group. The glycine a-amino group has a pKa of 9.6. The result is pH = 9.6 + log 10(1/2) pH = 9.3 Note that the dissociation constants of both the a-carboxyl and the a-amino groups are affected by the presence of the other group. The adjacent a-amino group makes the a-COOH group more acidic (i.e., it lowers the pKa), so it gives up a proton more readily than simple alkyl carboxylic acids. Thus, the pK1 of 2.0–2.1 for a-carboxyl groups of amino acids is substantially lower than that of acetic acid (pKa = 4.76), for example. What is the chemical basis for the low pKa of the a-COOH group of amino acids? The a-NH3+ (ammonium) group is strongly electron-withdrawing, and the positive charge of the amino group exerts a strong field effect and stabilizes the carboxylate anion. (The effect of the a-COOgroup on the pKa of the a-NH3+ group is the basis for Problem 4 at the end of this chapter.)

Side Chains of Amino Acids Undergo Characteristic Ionizations As we have seen, the side chains of several of the amino acids (Arg, Lys, Tyr, His, Asp, Glu) also contain dissociable groups. Thus, aspartic and glutamic acids contain an additional carboxyl function, and lysine possesses an aliphatic amino function. Histidine contains an ionizable imidazolium proton, and arginine carries a guanidinium function. Typical pKa values of these groups are shown in Table 4.1. The b-carboxyl group of aspartic acid and the g-carboxyl side chain of glutamic acid exhibit pKa values intermediate to the a-COOH NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 4 Lys2+

Lys+

Glu+

COO–

COOH H3N+ C

Glu–

Glu0

H3N+ C

H

COO– H3N+ C

H

H3N+ C

Glu2–

H

H2N

H

H3N+ C

Lys–

Lys0

COO–

COOH

H

COO– H2N

C

Amino Acids

H

COO– H2N

C

COO–

CH2

CH2

CH2

CH2

C

CH2

CH2

CH2

CH2

H

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

COOH

COO–

COO–

NH3+

NH3+

NH3+

NH2

COOH 14

14

12

12 pK 3

10

pK 3 pK 2

10

8

H

Isoelectric point

8

pH

pH 6

6 pK 2

4 pK 1

2 0

0

4

Isoelectric point

1.0 2.0 Equivalents of OH– added

pK 1

2

3.0

0

0

1.0 2.0 Equivalents of OH– added

3.0

FIGURE 4.8 Titrations of glutamic acid and lysine.

on one hand and typical aliphatic carboxyl groups on the other hand. In a similar fashion, the e-amino group of lysine exhibits a pKa that is higher than that of the a-amino group but similar to that for a typical aliphatic amino group. These intermediate side-chain pKa values reflect the slightly diminished effect of the a-carbon dissociable groups that lie several carbons removed from the side-chain functional groups. Figure 4.8 shows typical titration curves for glutamic acid and lysine, along with the ionic species that predominate at various points in the titration. The only other side-chain groups that exhibit any significant degree of dissociation are the para-OH group of tyrosine and the -SH group of cysteine. The pKa of the cysteine sulfhydryl is 8.32, so it is about 5% dissociated at pH 7. The tyrosine para-OH group is a very weakly acidic group, with a pKa of about 10.1. This group is essentially fully protonated and uncharged at pH 7. It is important to note that side-chain pKa values for amino acids in proteins can be different from the values shown in Table 4.1. On average, values for side chains in proteins are 1 pH unit closer to neutrality compared to the free amino acid values. Moreover, environmental effects in the protein can change pKa values dramatically.

Isoelectric Points of Amino Acids The isoelectric point (pI) of an amino acid is the pH at which it will have an overall net charge of zero and hence no net electrophoretic mobility. In general, the pIs for amino acids will lie between the pH values at which the amino acids have net charges of +1 and -1, respectively. The pIs of 2 amino acids are shown in Figures 4.7 and 4.8 respectively. While the pI for amino acids with no ionizable side chains (such as Gly) can be determined by (a-COOH pKa + a-NH3+ pKa)>2, this rule cannot be applied to the acidic and basic amino acids. For the case of an acidic amino acid, the pI can be calculated using (a-COOH pKa + R group pKa)>2, while for a basic amino acid, the pI can be determined using (R group pKa + a-NH3+ pKa)>2. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

W X

C

4.3

W Z

Z

C

X

Y

Y

Perspective drawing W X

W Z

Z

X

What Are the Post-translational Modifications of Amino Acids?

Inherently, most of the functional groups of individual amino acids can undergo modification. These modifications are carried out by specific enzymatic processes once a protein has been synthesized. Amino acid side chain post-translational modifications include but are not limited to carboxylation, hydroxylation, acetylation, methylation, sulfation, formylation, deamidation, glycosylation, and phosphorylation. We elaborate in more detail on some of the more prominent post-translational modifications found in proteins in Chapter 5, Table 5.5.

Y Y Fischer projections

4.4 FIGURE 4.9 Enantiomeric molecules based on a chiral carbon atom. Enantiomers are non-superimposable mirror images of each other.

What Are the Optical and Stereochemical Properties of Amino Acids?

Amino Acids Are Chiral Molecules Except for glycine, all the amino acids isolated from proteins have 4 different groups attached to the a-carbon atom. In such a case, the a-carbon is said to be asymmetric, or chiral (from the Greek cheir, meaning “hand”), and the 2 possible configurations for the a-carbon constitute non-superimposable mirror-image isomers, or enantiomers (Figure 4.9). Enantiomeric molecules display a special property called optical activity—the ability to rotate the plane of polarization of plane-polarized light. Clockwise rotation of incident light is referred to as dextrorotatory behaviour, and counterclockwise rotation is called levorotatory behaviour. The magnitude and direction of the optical rotation depend on the nature of the amino acid side chain. Some protein-derived amino acids at a given pH are dextrorotatory and others are levorotatory, even though all are of the L-configuration. The direction of optical rotation can be specified in the name by using a (+) for dextrorotatory compounds and a (-) for levorotatory compounds, as in L(-)-leucine.

Chiral Molecules Are Described by the D,L and R,S Naming Conventions The discoveries of optical activity and enantiomeric structures (see Critical Developments in Biochemistry, page 88) made it important to develop suitable nomenclature for chiral molecules. Two systems are in common use today: the so-called D,L system and the (R,S) system. In the D,L system of nomenclature, the (+) and (-) isomers of glyceraldehyde are denoted as D-glyceraldehyde and L-glyceraldehyde respectively (see Critical Developments in Biochemistry, page 91). Absolute configurations of all other carbon-based molecules are referenced to D- and L-glyceraldehyde. When sufficient care is taken to avoid racemization of the amino acids during hydrolysis of proteins, it is found that all the amino acids derived from natural proteins are of the L-configuration. Amino acids of the D-configuration are nonetheless found in nature, especially as components of certain peptide antibiotics, such as valinomycin, gramicidin, and actinomycin D, and in the cell walls of certain microorganisms. Despite its widespread acceptance, problems exist with the D,L system of nomenclature. For example, this system can be ambiguous for molecules with 2 or more chiral centres. To address such problems, the (R,S) system of nomenclature for chiral molecules was proposed in 1956 by Robert Cahn, Sir Christopher Ingold, and Vladimir Prelog. In this more versatile system, priorities are assigned to each of the groups attached to a chiral centre on the basis of atomic number, the atoms with higher atomic numbers having higher priorities. The newer (R,S) system of nomenclature is superior to the older D,L system in one important way: The configuration of molecules with more than one chiral centre can be more easily, completely, and unambiguously described with (R,S) notation. Several amino NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 4

Amino Acids

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

Green Fluorescent Protein—The “Light Fantastic” from Jellyfish to Gene Expression Aequorea victoria, a species of jellyfish found in the northwest Pacific Ocean, contains a green fluorescent protein (GFP) that works together with another protein, aequorin, to provide a defence mechanism for the jellyfish. When the jellyfish is attacked or shaken, aequorin produces a blue light. This light energy is captured by GFP, which then emits a bright green flash that presumably blinds or startles the attacker. Remarkably, the fluorescence of GFP occurs without the assistance of a prosthetic group—a “helper molecule” that would mediate GFP’s fluorescence. Instead, the light-transducing capability of GFP is the result of a reaction between 3 amino acids in the protein itself. As shown below, adjacent serine, tyrosine, and glycine in the sequence of the protein react to form the pigment complex—termed a chromophore. No enzymes are required; the reaction is autocatalytic. Because the light-transducing talents of GFP depend only on the protein itself (upper photo, chromophore highlighted), GFP has quickly become a darling of genetic engineering laboratories. The promoter of any gene whose cellular expression is of interest can be fused to the DNA sequence coding for GFP. Telltale green fluorescence tells the researcher when this fused gene has been expressed (see lower photo and also Chapter 27). O Phe-Ser-Tyr-Gly-Val-Gln 69 64

O2 HO

N H

N Phe

Gln Val

N O O

H

" Amino acid substitutions in GFP can tune the colour of emitted light; examples include YFP, CFP, and BFP (yellow, cyan, and blue fluorescent protein). Shown here is an image of African green monkey kidney cells expressing YFP fused to a-tubulin, a major cytoskeletal protein. (Image courtesy of Michelle E. King and George S. Bloom, University of Virginia.)

acids, including isoleucine, threonine, hydroxyproline, and hydroxylysine, have 2 chiral centres. In the (R,S) system, L-threonine is (2S,3R)-threonine.

4.5

What Are the Spectroscopic Properties of Amino Acids?

One of the most important and exciting advances in modern biochemistry has been the application of spectroscopic methods, which measure the absorption and emission of energy of different frequencies by molecules and atoms. Spectroscopic studies of proteins, nucleic acids, and other biomolecules are providing many new insights into the structure of and dynamic processes in these molecules. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

Discovery of Optically Active Molecules and Determination of Absolute Configuration The optical activity of quartz and certain other materials was first discovered by Jean-Baptiste Biot in 1815 in France, and in 1848 a young chemist in Paris named Louis Pasteur made a related and remarkable discovery. Pasteur noticed that preparations of optically inactive sodium ammonium tartrate contained 2 visibly different kinds of crystals that were mirror images of each other. Pasteur carefully separated the 2 types of crystals, dissolved each in water, and found that each solution was optically active. Even more intriguing, the specific rotations of these 2 solutions were equal in magnitude and of opposite sign. Because these differences in optical rotation were apparent properties of the dissolved molecules, Pasteur eventually proposed that the molecules themselves were mirror images of each other, just like their respective crystals. Based on this and other related evidence, van’t Hoff and LeBel proposed the tetrahedral arrangement of valence bonds to carbon. In 1888, Emil Fischer decided that it should be possible to determine the relative configuration of (+)-glucose, a 6-carbon sugar with 4 asymmetric centres (see figure). Because each of the 4 C could be either of 2 configurations, glucose conceivably could exist in any 1 of 16 possible isomeric structures. It took 3 years to complete the solution of an elaborate chemical and logical puzzle. By 1891, Fischer had reduced his puzzle to a choice between 2 enantiomeric structures. (Methods for determining absolute configuration were not yet available, so Fischer made a simple guess, selecting the structure shown in the figure.) For this remarkable feat, Fischer received the Nobel Prize in Chemistry in 1902. In 1951, J. M. Bijvoet

in Utrecht, the Netherlands, used a new X-ray diffraction technique to show that Emil Fischer’s arbitrary guess 60 years earlier had been correct. It was M. A. Rosanoff, a chemist and instructor at New York University, who first proposed (in 1906) that the isomers of glyceraldehyde be the standards for denoting the stereochemistry of sugars and other molecules. Later, when experiments showed that the configuration of (+)-glyceraldehyde was related to (+)-glucose, (+)-glyceraldehyde was given the designation D. Emil Fischer rejected the Rosanoff convention, but it was otherwise universally accepted. Ironically, this nomenclature system is often mistakenly referred to as the Fischer convention. CHO H

C

OH

HO

C

H

H

C

OH

H

C

OH

CH2OH

# The absolute configuration of (+)-glucose.

Phenylalanine, Tyrosine, and Tryptophan Absorb Ultraviolet Light Many details of the structure and chemistry of the amino acids have been elucidated or at least confirmed by spectroscopic measurements. None of the amino acids absorbs light in the visible region of the electromagnetic spectrum. Several of the amino acids, however, do absorb ultraviolet radiation, and all absorb in the infrared region. The absorption of energy by electrons as they rise to higher energy states occurs in the ultraviolet/visible region of the energy spectrum. Only the aromatic amino acids phenylalanine, tyrosine, and tryptophan exhibit significant ultraviolet absorption above 250 nm, as shown in Figure 4.10. These strong absorptions can be used for spectroscopic determinations of protein concentration. The aromatic amino acids also exhibit relatively weak fluorescence, and it has recently been shown that tryptophan can exhibit phosphorescence—a relatively long-lived emission of

FIGURE 4.10 The ultraviolet absorption spectra of the aromatic amino acids at pH 6. (Wetlaufer, D. B., 1962. Ultraviolet spectra of proteins and amino acids. Advances in Protein Chemistry 17:303–390.)

40,000 20,000

Molar absorptivity,

10,000 5,000 Trp

2,000 1,000

Tyr

500 200 100

Phe

50 20 10

200

220 240 260 280 Wavelength (nm)

300

320

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Chapter 4

89

Amino Acids

light. These fluorescence and phosphorescence properties are especially useful in the study of protein structure and dynamics. The ability to quantify proteins during sequential purification steps is advantageous as it allows for target protein concentrations to be determined. Various spectrophotometric methods exist for protein quantification such as Biuret, Lowry, and UV absorption. When employing a UV absorption assay as the basis for determining protein concentration, appropriate standards are typically included in the procedure. Since this method exploits aromatic amino acid UV light absorbance, changes in UV spectrophotometric readings among different solutions allow for the precise calculation of target protein concentrations. Remember, according to the Beer-Lambert Law (see below), the concentration of a given target protein solution is directly related to its UV absorption reading:

A = e cl, where A = absorbance, e = molar extinction coefficient, c = concentration of the target protein, and l = length of the optical path.

Amino Acids Can Be Characterized by Nuclear Magnetic Resonance The development in the 1950s of nuclear magnetic resonance (NMR), a spectroscopic technique that involves the absorption of radio frequency energy by certain nuclei in the presence of a magnetic field, played an important part in the chemical characterization of amino acids and proteins. Several important principles emerged from these studies. First, the chemical shift1 of amino acid protons depends on their particular chemical environment and thus on the state of ionization of the amino acid. Second, the change in electron density during a titration is transmitted throughout the carbon chain in the aliphatic amino acids and the aliphatic portions of aromatic amino acids, as evidenced by changes in the chemical shifts of relevant protons. Finally, the magnitude of the coupling constants between protons on adjacent carbons depends in some cases on the ionization state of the amino acid. This apparently reflects differences in the preferred conformations in different ionization states. Proton NMR spectra of 2 amino acids are shown in Figure 4.11. Because they are highly sensitive to their environment, the chemical shifts of individual NMR signals can detect the

L-Alanine

COOH

L-Tyrosine

+ H3N

COOH C

H

Relative intensity

Relative intensity

+ H3N

CH3

10

9

8

7

6

5 ppm

4

3

2

1

0

C

H

CH2

OH

10

9

8

7

6

5 ppm

4

3

2

1

FIGURE 4.11 Proton NMR spectra of several amino acids. Zero on the chemical shift scale is defined by the resonance of tetramethylsilane (TMS). (The large resonance at approximately 5 ppm is due to the normal HDO impurity in the D2O solvent.) (Adapted from Aldrich Library of NMR Spectra)

1The chemical shift for any NMR signal is the difference in resonance frequency between the observed signal and a suitable reference signal. If 2 nuclei are magneti-

cally coupled, the NMR signals of these nuclei split, and the separation between such split signals—known as the coupling constant—is likewise dependent on the structural relationship between the 2 nuclei. NEL

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0

90

Part 1 Building Blocks of the Cell

pH-dependent ionizations of amino acids. Figure 4.12 shows the 13C chemical shifts occurring in a titration of lysine. Note that the chemical shifts of the carboxyl C, Ca, and Cb carbons of lysine are sensitive to dissociation of the nearby a-COOH and a-NH3+ protons (with pKa values of about 2 and 9 respectively), whereas the Cd and Ce carbons are sensitive to dissociation of the a-NH3+ group. Such measurements have been very useful for studies of the ionization behaviour of amino acid residues in proteins. More sophisticated NMR measurements at very high magnetic fields are also used to determine the 3-dimensional structures of peptides and proteins.

14 pK 3

12

pH

10 8

pK 2 Carboxyl C

6

!

"

#

$

%

4 2

pK 1

4700

4500

4300 1400 1200 1000 800 Chemical shift in Hz (vs. TMS)

600

FIGURE 4.12 A plot of chemical shifts versus pH for the carbons of lysine. Changes in chemical shift are most pronounced for atoms near the titrating groups. Note the correspondence between the pKa values and the particular chemical shift changes. All chemical shifts are defined relative to tetramethylsilane (TMS). (Suprenant, H., et al., 1980. Carbon-13 NMR studies of amino acids: Chemical shifts, protonation shifts, microscopic protonation behavior. Journal of Magnetic Resonance 40:231–243.)

A DEEPER LOOK

The Murchison Meteorite—Discovery of Extraterrestrial Handedness The predominance of L-amino acids in biological systems is one of life’s intriguing features. Prebiotic syntheses of amino acids would be expected to produce equal amounts of L- and D-enantiomers. Some kind of enantiomeric selection process must have intervened to select L-amino acids over their D-counterparts as the constituents of proteins. Was it random chance that chose L- over D-isomers? Analysis of carbon compounds—even amino acids—from extraterrestrial sources might provide deeper insights into this mystery. John Cronin and Sandra Pizzarello examined the enantiomeric distribution of unusual amino acids obtained from the Murchison meteorite, which struck the earth on September 28, 1969, near Murchison, Australia. NH3+ CH3

CH2

CH

C

CH3

CH3

NH3+

COOH

2-Amino-2,3-dimethylpentanoic

(By selecting unusual amino acids for their studies, Cronin and Pizzarello ensured that they were examining materials that were native to the meteorite and not earth-derived contaminants.) Four a-dialkyl amino acids— a-methyl-isoleucine, a-methyl-alloisoleucine, a-methyl-norvaline, and isovaline—were found to have an L-enantiomeric excess of 2%–9%. This may be the first demonstration that a natural L-enantiomer enrichment occurs in certain cosmological environments. Could these observations be relevant to the emergence of L-enantiomers as the dominant amino acids on Earth? And, if so, could there be life elsewhere in the universe that is based upon the same amino acid handedness?

CH3

CH2

C

COOH

CH3 acid*

Isovaline

NH3+ CH3

CH2

CH2

C CH3

COOH

! Amino acids found in the Murchison meteorite.

!-Methylnorvaline

*The 4 stereoisomers of this amino acid include the D- and L-forms of a-methyl-isoleucine and a-methyl-alloisoleucine. (Cronin, J. R., and Pizzarello, S., 1997. Enantiomeric excesses in meteoritic amino acids. Science 275:951–955.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 4

Amino Acids

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

Rules for Description of Chiral Centres in the (R,S) System Naming a chiral centre in the (R,S) system is accomplished by viewing the molecule from the chiral centre to the atom with the lowest priority. If the other 3 atoms facing the viewer then decrease in priority in a clockwise direction, the centre is said to have the (R) configuration (where R is from the Latin rectus, meaning “right”). If the 3 atoms in question decrease in priority in a counterclockwise fashion, the chiral centre is of the (S) configuration (where S is from the Latin sinistrus, meaning “left”). If 2 of the atoms coordinated to a chiral centre are identical, the atoms bound to these 2 are considered for

C

SH 7 OH 7 NH2 7 COOH 7 CHO 7 CH2OH 7 CH3.

From this, it is clear that D-glyceraldehyde is (R)-glyceraldehyde and L-alanine is (S)-alanine (see figure). Interestingly, the a-carbon configuration of all the L-amino acids except for cysteine is (S). Cysteine, by virtue of its thiol group, is in fact (R)-cysteine.

OH

CHO HO

priorities. For such purposes, the priorities of certain functional groups found in amino acids and related molecules are in the following order

H

H

H

CH2OH L-Glyceraldehyde

C

CH2OH

OHC

OH

CHO

CH2OH

(S)-Glyceraldehyde

D-Glyceraldehyde

C

HOH2C

CHO

(R)-Glyceraldehyde

+ NH3

COOH + H 3N

H

OH

H

H –OOC

CH3

CH3

(S)-Alanine

L-Alanine

# The assignment of (R) and (S) notation for glyceraldehyde and l-alanine.

4.6

What Is the Fundamental Structural Pattern in Proteins?

Chemically, proteins are unbranched polymers of amino acids linked head to tail, from carboxyl group to amino group, through formation of covalent peptide bonds, which are a type of amide linkage (Figure 4.13, see also Figure 4.2). Peptide bond formation results in the release of H2O. The peptide “backbone” of a protein consists of the repeated sequence ⎯N⎯Ca⎯Co⎯, where the N represents the amide nitrogen, the Ca is the a-carbon atom of an amino acid in the polymer chain, and the final Co is the carbonyl carbon of the amino acid, which in turn is linked to the amide N of the next amino acid down the line. The geometry of the peptide backbone is shown in Figure 4.14. Note that the carbonyl oxygen and the amide hydrogen are trans to each other in this figure. This conformation is favoured energetically because it results in less steric hindrance between non-bonded atoms in neighbouring amino acids. Because the a-carbon atom of the amino acid is a chiral centre (in all amino acids except glycine), the polypeptide chain is inherently asymmetric. Only L-amino acids are found in proteins.

R1 + H3N

CH

R2

O C

Amino acid 1

O–

+

+ H3N

CH

O C

Amino acid 2

O–

+ H 3N H2O

R1

O

CH

C

R2 N H

CH

O C

O–

Dipeptide

FIGURE 4.13 Peptide formation is the creation of an amide bond between the carboxyl group of one amino acid and the amino group of another amino acid. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

91

92

Part 1 Building Blocks of the Cell

O H

0.123 nm 121.1° m

2n

5 0.1

123.2°

C 115.6°

C!

0.133 nm 5 nm C 0.14 ! 121.9° 119.5°

R

R

N

H

118.2°

0.1 nm H

FIGURE 4.14 The peptide bond is shown in its usual trans conformation of carbonyl O and amide H. The Ca atoms are the a-carbons of 2 adjacent amino acids joined in peptide linkage. The dimensions and angles are the average values observed by crystallographic analysis of amino acids and small peptides. The peptide bond is the light-coloured bond between C and N. (Adapted from Ramachandran, G. N., et al., 1974. The mean geometry of the peptide unit from crystal structure data. Biochimica et Biophysica Acta 359:298–302.)

The Peptide Bond Has Partial Double-Bond Character The peptide linkage is usually portrayed by a single bond between the carbonyl carbon and the amide nitrogen (Figure 4.15a). Therefore, in principle, rotation may occur about any covalent bond in the polypeptide backbone because all 3 kinds of bonds (N i Ca, Ca i Co, and the Co i N peptide bond) are single bonds. In this representation, the Co and N atoms of the peptide grouping are both in planar sp2 hybridization and the Co and O atoms are linked by a π bond, leaving the nitrogen with a lone pair of electrons in a 2p orbital. However, another resonance form for the peptide bond is feasible in which the Co and N atoms participate in a π bond, leaving a lone e− pair on the oxygen (Figure 4.15b). This structure prevents free rotation about the Co i N peptide bond because it becomes a double bond. The real nature of the peptide bond lies somewhere between these extremes; that is, it has  partial double-bond character, as represented by the intermediate form shown in Figure 4.15c. Peptide bond resonance has several important consequences. First, it restricts free rotation around the peptide bond and leaves the peptide backbone with only 2 degrees of freedom per amino acid group: rotation around the N i Ca bond and rotation around the Ca i Co bond.2 Second, the 6 atoms composing the peptide bond group tend to be coplanar, forming the so-called amide plane of the polypeptide backbone (Figure 4.16). Third, the Co i N bond length is 0.133 nm, which is shorter than normal C i N bond lengths (e.g., the Ca i N bond of 0.145 nm) but longer than typical C “ N bonds (0.125 nm). The peptide bond is estimated to have 40% double-bond character.

The Polypeptide Backbone Is Relatively Polar Peptide bond resonance also causes the peptide backbone to be relatively polar. As shown in Figure 4.15b, the amide nitrogen is in a protonated or positively charged form, and the carbonyl oxygen is a negatively charged atom in this double-bonded resonance state. In actuality, the hybrid state of the partially double-bonded peptide arrangement gives a net positive charge of 0.28 on the amide N and an equivalent net negative charge of 0.28 on the carbonyl O. The presence of these partial charges means that the peptide bond has a permanent dipole. Nevertheless, the peptide backbone is relatively unreactive chemically, and protons are gained or lost by the peptide groups only at extreme pH conditions. 2The angle of rotation about the N i C

a bond is designated

f (phi), whereas the Ca i Co angle of rotation is designated c (psi). NEL

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Chapter 4

Amino Acids

(a) C

Cα

H C

O

N

O

C

N

H Cα

C A pure double bond between C and O would permit free rotation around the C N bond.

(b) C –O

C

+ N

Cα

H

C

O C

H

N Cα

The other extreme would prohibit C N bond rotation but would place too great a charge on O and N. (c) C $ –O

C

$+

H

Cα

N

C

C

H N

O

Cα

The true electron density is intermediate. The barrier to C N bond rotation of about 88 kJ/mol is enough to keep the amide group planar. FIGURE 4.15 The partial double-bond character of the peptide bond. Resonance interactions among the carbon, oxygen, and nitrogen atoms of the peptide group can be represented by 2 resonance extremes (a and b). (a) The usual way the peptide atoms are drawn. (b) In an equally feasible form, the peptide bond is now a double bond; the amide N bears a positive charge and the carbonyl O has a negative charge. (c) The actual peptide bond is best described as a resonance hybrid of the forms in (a) and (b). Significantly, all the atoms associated with the peptide group are coplanar, rotation about Co⎯N is restricted, and the peptide is distinctly polar. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.) H

O

R

C

-carbon

C N

-carbon

C

H H

R FIGURE 4.16 The coplanar relationship of the atoms in the amide group is highlighted as an imaginary shaded plane lying between 2 successive a-carbon atoms in the peptide backbone. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

93

94

Part 1 Building Blocks of the Cell

Peptides Can Be Classified According to How Many Amino Acids They Contain Peptide is the name assigned to short polymers of amino acids. Peptides are classified according to the number of amino acid units in the chain. Each unit is called an amino acid residue, the word residue denoting what is left after the release of H2O when an amino acid forms a peptide link upon joining the peptide chain. Dipeptides have 2 amino acid residues, tripeptides have 3, tetrapeptides 4, and so on. After about 12 residues, this terminology becomes cumbersome, so peptide chains of more than 12 and less than about 20 amino acid residues are usually referred to as oligopeptides, and when the chain exceeds several dozen amino acids in length, the term polypeptide is used. The distinctions in this terminology are not precise.

Proteins Are Composed of One or More Polypeptide Chains The terms polypeptide and protein are used interchangeably in discussing single polypeptide chains. The term protein broadly defines molecules composed of one or more polypeptide chains. Proteins with one polypeptide chain are monomeric proteins. Proteins composed of more than one polypeptide chain are multimeric proteins. Multimeric proteins may contain only one kind of polypeptide, in which case they are homomultimeric, or they may be composed of several different kinds of polypeptide chains, in which instance they are heteromultimeric. Greek letters and subscripts are used to denote the polypeptide composition of multimeric proteins. Thus, an a2-type protein is a dimer of identical polypeptide subunits, or a homodimer. Hemoglobin (Table 4.2) consists of 4 polypeptides of 2 different kinds; it is an a2b2 heteromultimer. Polypeptide chains of proteins typically range in length from about 100 amino acids to around 2000, the number found in each of the 2 polypeptide chains of myosin, the contractile protein of muscle. However, exceptions abound, including human cardiac muscle titin, which has 26 926 amino acid residues and a molecular weight of 2 993 497. The average molecular weight of polypeptide chains in eukaryotic cells is about 31 700, corresponding to about 270 amino acid residues. Table 4.2 is a representative list of proteins according to size. The molecular weights (Mr) of proteins can be estimated by a number of physicochemical methods such as polyacrylamide gel electrophoresis or ultracentrifugation (see Chapter 5). Precise determinations of protein molecular masses can be obtained by simple calculations based on knowledge of their amino acid sequence, which is often available in genome databases. No simple generalizations correlate the size of proteins with their functions. For instance, the same function may be fulfilled in different cells by proteins of different molecular weight. The Escherichia coli enzyme responsible for glutamine synthesis (a protein known as glutamine synthetase) has a molecular weight of 600 000, whereas the analogous enzyme in brain tissue has a molecular weight of 380 000.

TABLE 4.2

Size of Protein Molecules*

Protein

Mr

Number of Residues per Chain

Insulin (bovine)

5 733

21 (A)

Subunit Organization

Cytochrome c (equine)

12 500

104

a1

Ribonuclease A (bovine pancreas)

12 640

124

a1

Lysozyme (egg white)

13 930

129

a1

Myoglobin (horse)

16 980

153

a1

Chymotrypsin (bovine pancreas)

22 600

13 (a)

abg

ab

30 (B)

132 (b) 97 (g) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 4

Hemoglobin (human)

64 500

Amino Acids

141 (a)

95

a 2b2

146 (b) Serum albumin (human)

68 500

550

a1

Hexokinase (yeast)

96 000

200

a4

g-Globulin (horse)

149 900

214 (a)

a 2b2

446 (b) Glutamate dehydrogenase (liver)

332 694

500

a6

Myosin (rabbit)

470 000

2 000 (heavy, h)

h2a1a´2b2

190 (a) 149 (a´) 160 (b) Ribulose bisphosphate carboxylase (spinach)

560 000

475 (a)

a 8b8

123 (b) Glutamine synthetase (E. coli)

600 000

468

a12

Insulin Cytochrome c

Ribonuclease

Lysozyme

Myoglobin

Hemoglobin

Immunoglobulin Glutamine synthetase *Illustrations of selected proteins listed in Table 4.2 are drawn to constant scale. Adapted from Goodsell, D. S., and Olson, A. J., 1993. Soluble proteins: Size, shape and function. Trends in Biochemical Sciences 18:65–68.

SUMMARY 4.1 What Are the Structures and Properties of Amino Acids? The central tetrahedral alpha (a) carbon (Ca) atom of typical amino acids is linked covalently to both the amino group and the carboxyl group. Also bonded to this a-carbon are a hydrogen and a variable side chain. It is the side chain, the so-called R group, that gives each amino acid its identity. In neutral solution (pH 7), the carboxyl group exists as i COO- and the amino group as i NH3+. The amino and carboxyl groups of amino acids can react in a head-to-tail fashion,

eliminating a water molecule and forming a covalent amide linkage, which, in the case of peptides and proteins, is typically referred to as a peptide bond. Amino acids are also chiral molecules. With 4 different groups attached to it, the a-carbon is said to be asymmetric. The 2  possible configurations for the a-carbon constitute nonidentical mirror-image isomers, or enantiomers. The structures of the 20 common amino acids are grouped into the following categories: (1) non-polar, or hydrophobic, amino acids; (2) neutral (uncharged) but

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96

Part 1 Building Blocks of the Cell

polar amino acids; (3) acidic amino acids (which have a net negative charge at pH 7.0); and (4) basic amino acids (which have a net positive charge at neutral pH). Derivatives of amino acids are obtained by their chemical modification and retain many of the amino acid structural characteristics in their final form. Many amino acids are used synthetically in the production of potential drugs, inhibitors of enzymes, and antagonists of receptors and small peptides. 4.2 What Are the Acid–Base Properties of Amino Acids? The common amino acids are all weak polyprotic acids. The ionizable groups are not strongly dissociating ones, and the degree of dissociation thus depends on the pH of the medium. All the amino acids contain at least 2 dissociable hydrogens. The side chains of several of the amino acids also contain dissociable groups. Thus, aspartic and glutamic acids contain an additional carboxyl function, and lysine possesses an aliphatic amino function. Histidine contains an ionizable imidazolium proton, and arginine carries a guanidinium function. The pI of an amino acid is the pH at which it will have an overall net charge of zero. In general, the pIs for amino acids will lie between the pH values at which the amino acids have net charges of +1 and -1 respectively. 4.3 What Are the Post-translational Modifications of Amino Acids? Most functional groups of individual amino acids can undergo modification. These amino acid side chain post-translational modifications include but are not limited to carboxylation, hydroxylation, acetylation, methylation, sulfation, formylation, deamidation, glycosylation, and phosphorylation.

4.4 What Are the Optical and Stereochemical Properties of Amino Acids? Except for glycine, all amino acids isolated from proteins are said to be asymmetric, or chiral (from the Greek cheir, meaning “hand”), and the 2 possible configurations for the a-carbon constitute non-superimposable mirror-image isomers, or enantiomers. Enantiomeric molecules display a special property called optical activity—the ability to rotate the plane of polarization of plane-polarized light. The magnitude and direction of the optical rotation depend on the nature of the amino acid side chain. 4.5 What Are the Spectroscopic Properties of Amino Acids? Many details of the structure and chemistry of the amino acids have been elucidated or at least confirmed by spectroscopic measurements. None of the amino acids absorbs light in the visible region of the electromagnetic spectrum. Several of the amino acids, however, do absorb ultraviolet radiation, and all absorb in the infrared region. The absorption of UV light by the aromatic amino acids permits the determination of protein concentration. Proton NMR spectra of amino acids are highly sensitive to their environment, and the chemical shifts of individual NMR signals can detect the pH-dependent ionizations of amino acids. 4.6 What Is the Fundamental Structural Pattern in Proteins? Proteins are linear polymers joined by peptide bonds. The defining characteristic of a protein is the amino acid sequence. The partial double-bonded character of the peptide bond has profound influences on protein conformation. Proteins are also classified according to the length of their polypeptide chains (how many amino acid residues they contain) and the number and kinds of polypeptide chains.

PROBLEMS Login to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. Without consulting chapter figures, draw Fischer projection formulas for glycine, aspartate, leucine, isoleucine, methionine, and threonine. 2. Without reference to the text, give the 1-letter and 3-letter abbreviations for asparagine, arginine, cysteine, lysine, proline, tyrosine, and tryptophan. 3. Write equations for the ionic dissociations of alanine, glutamate, histidine, lysine, and phenylalanine. 4. How is the pKa of the a-NH3+ group affected by the presence on an amino acid of the a-COO- ? 5. (Integrates with Chapter 2) Draw an appropriate titration curve for aspartic acid, labelling the axes and indicating the equivalence points and the pKa values. 6. (Integrates with Chapter 2) Calculate the concentrations of all ionic species in a 0.25 M solution of histidine at pH 2, pH 6.4, and pH 9.3. 7. (Integrates with Chapter 2) Calculate the pH at which the g-carboxyl group of glutamic acid is two-thirds dissociated. 8. (Integrates with Chapter 2) Calculate the pH at which the e-amino group of lysine is 20% dissociated. 9. (Integrates with Chapter 2) Calculate the pH of a 0.3 M solution of (a) leucine hydrochloride, (b) sodium leucinate, and (c) isoelectric leucine. 10. By drawing the appropriate titration curve for cysteine, determine its respective pI. 11. How many moles of NaOH are required to adjust 0.75 L of a 0.2 M solution of Arg–Glu–Trp–Ala from pH 2.4 to 12.5? Calculate the pI of the tetrapeptide.

12. Absolute configurations of the amino acids are referenced to D- and L-glyceraldehyde on the basis of chemical transformations that can convert the molecule of interest to either of these reference isomeric structures. In such reactions, the stereochemical consequences for the asymmetric centres must be understood for each reaction step. Propose a sequence of reactions that would demonstrate that L(-)-serine is stereochemically related to L(-)-glyceraldehyde. 13. Describe the stereochemical aspects of the structure of cystine, the structure that is a disulfide-linked pair of cysteines. 14. Draw a simple mechanism for the reaction of a cysteine sulfhydryl group with iodoacetamide. 15. A previously unknown protein has been isolated in your laboratory. Others in your lab have determined that the protein sequence contains 172 amino acids. They have also determined that this protein has no tryptophan and no phenylalanine. You have been asked to determine the possible tyrosine content of this protein. You know from your study of this chapter that there is a relatively easy way to do this. You prepare a pure 50 μM solution of the protein and you place it in a sample cell with a 1 cm path length. You measure the absorbance of this sample at 280 nm in a UV-visible spectrophotometer. The absorbance of the solution is 0.372. Are there tyrosines in this protein? How many? (Hint: You will need to use Beer’s Law, which is described on page 89 in this chapter. You will also find it useful to know that the units of molar absorptivity are M-1cm-1.) 16. The simple average molecular weight of the 20 common amino acids is 138, but most biochemists use 110 when estimating the number of amino acids in a protein of known molecular weight. Why do you suppose this is? (Hint: There are 2 contributing factors to the answer. One of them will be apparent from a brief consideration of the amino acid compositions of common proteins. See for example Figure 5.26 of this text.) NEL

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Chapter 4

17. The artificial sweeteners Equal and Nutrasweet contain aspartame, which has the structure

CH2 O CH

C

CH2 O NH

CH

C

97

only with aspartame, and you stored it in a thermos for several hours before drinking it. What might it taste like? 18. Individuals with phenylketonuria must avoid dietary phenylalanine because they are unable to convert phenylalanine to tyrosine. Look up this condition and find out what happens if phenylalanine accumulates in the body. Would you advise a person with phenylketonuria to consume foods sweetened with aspartame? Why or why not?

CO2– + H3N

Amino Acids

OCH3

Aspartame

What are the 2 amino acids that are components of aspartame? What kind of bond links these amino acids? What do you suppose might happen if a solution of aspartame was heated for several hours at a pH near neutrality? Suppose you wanted to make hot chocolate sweetened

19 (The concept of prochirality is discussed in Chapter 8, Section 8.3.) Citrate (see Figure 18.6) is a prochiral molecule. Describe the process by which you would distinguish between the (R-) and (S-) portions of this molecule and how an enzyme could discriminate between similar but distinct moieties. 20. Amino acids are frequently used as buffers. Describe the pH range of acceptable buffering behaviour for the amino acids alanine, histidine, aspartic acid, and lysine.

FURTHER READING General Amino Acid Chemistry Alberty, R.A., 1953. The Protein’s Chemistry, Biological Activity and Methods, Neurath, A.H., and Bailey, K., Eds. Vol I. New York: Academic Press. Atkins, J. F., and Gesteland, R., 2002. The 22nd amino acid. Science 296:1409–1410. Barker, R., 1971. Organic Chemistry of Biological Compounds, Chap. 4. Englewood Cliffs, NJ: Prentice Hall. Barrett, G. C., Ed., 1985. Chemistry and Biochemistry of the Amino Acids. New York: Chapman and Hall. Beynon, R.J., and Easterby, J.S., 1996. Buffer Solutions. New York: IRL Press at Oxford University Press. Gianazza, E., 1995. Isoelectric focusing as a tool for the investigation of posttranslational processing and chemical modification of proteins. Journal of Chromatography A 705:67–87. Greenstein, J. P., and Winitz M., 1961. Chemistry of the Amino Acids. New York: John Wiley & Sons. Herod, D. W., and Menzel, E. R., 1982. Laser detection of latent fingerprints: Ninhydrin. Journal of Forensic Science 27:200–204. Knowles, W. S., 2001. Asymmetric hydrogenations. Nobel Lecture Transcript. Macleod, A. M., Cascieri, M. A., et al. 1995. Synthesis and biological evaluation of NK1 antagonists derived from L-tryptophan. Journal of Medicinal Chemistry 38:934–941. Meister, A., 1965. Biochemistry of the Amino Acids, 2nd ed., Vol. 1. New York: Academic Press. Segel I. H., 1976. Biochemical Calculations, 2nd ed. New York: John Wiley & Sons.

Iizuke, E., and Yang, J. T., 1964. Optical rotatory dispersion of L-amino acids in acid solution. Biochemistry 3:1519–1524. Kauffman, G. B., and Priebe, P. M., 1990. The Emil Fischer-William Ramsey friendship. Journal of Chemical Education 67:93–101. Noble, J., and Bailey, M.J.A., 2009. Methods in Enzymology, Vol 463. Elsevier Inc. Surprenant, H. L., Sarneski, J. E., et al., 1980. Carbon-13 NMR studies of amino acids: Chemical shifts, protonation shifts, microscopic protonation behavior. Journal of Magnetic Resonance 40:231–243. NMR Spectroscopy Bovey, F. A., and Tiers, G. V. D., 1959. Proton N.S.R. spectroscopy. V. Studies of amino acids and peptides in trifluoroacetic acid. Journal of the American Chemical Society 81:2870–2878. de Groot, H. J., 2000. Solid-state NMR spectroscopy applied to membrane proteins. Current Opinion in Structural Biology 10:593–600. Hinds, M. G., and Norton, R. S., 1997. NMR spectroscopy of peptides and proteins. Practical considerations. Molecular Biotechnology 7:315–331. James, T. L., Dötsch, V., and Schmitz, U., Eds., 2001. Nuclear Magnetic Resonance of Biological Macromolecules. San Diego: Academic Press. Krishna, N. R., and Berliner, L. J. Eds. 2003. Protein NMR for the Millennium. New York: Kluwer Academic/Plenum. Opella, S. J., Nevzorov, A., Mesleb, M. F., and Marassi, F. M., 2002. Structure determination of membrane proteins by NMR spectroscopy. Biochemistry and Cell Biology 80:597–604. Roberts, G. C. K., and Jardetzky, O., 1970. Nuclear magnetic resonance spectroscopy of amino acids, peptides and proteins. Advances in Protein Chemistry 24:447–545.

Srinivasan, G., James, C., and Krzycki, J., 2002. Pyrrolysine encoded by UAG in Archaea: Charging of a UAG-decoding specialized tRNA. Science 296:1459–1462.

Amino Acid Analysis

Post-translational Modifications of Proteins

Prata C., Bonnafous P., et al., 2001. Recent advances in amino acid analysis by capillary electrophoresis. Electrophoresis 22:4129–4138.

Teerlink, T., 1994. Derivatization of posttranslationally modified amino acids. Journal of Chromatography B 659:185–207.

Smith, A. J., 1997. Amino acid analysis. Methods in Enzymology 289:419–426.

Optical and Stereochemical Properties Cahn, R. S., 1964. An introduction to the sequence rule. Journal of Chemical Education 41:116–125.

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5

Proteins: Their Primary Structure and Biological Functions

! Although helices sometimes appear as

decorative or utilitarian motifs in manmade structures, they are a common structural theme in biological macromolecules— proteins, nucleic acids, and even polysaccharides.

. . . by small and simple things are great things brought to pass. ALMA 37.6

© Jan Halaska/Photo Researchers, Inc.

The Book of Mormon

KEY QUESTIONS 5.1

What Architectural Arrangements Characterize Protein Structure?

5.2

How Are Proteins Isolated and Purified from Cells?

5.3

How Are Amino Acid Mixtures Separated?

5.4

How Is the Amino Acid Analysis of Proteins Performed?

ESSENTIAL QUESTIONS

5.5

How Is the Primary Structure of a Protein Determined?

5.6

What Is the Nature of Amino Acid Sequences?

Proteins are polymers composed of hundreds or even thousands of amino acids linked in series by peptide bonds.

5.7

Can Polypeptides Be Synthesized in the Laboratory?

What structural forms do these polypeptide chains assume, how can the sequence of amino acids in a protein be determined, and what are the biological roles played by proteins?

5.8

Do Proteins Have Chemical Groups Other Than Amino Acids?

WHY IT MATTERS

5.9

What Are the Many Biological Functions of Proteins?

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

Before we begin our discussion of proteins and their properties, we pose the question, “Why do proteins matter?” Proteins are important because they are the ‘end point’ in the central dogma of molecular biology, and thus are vital for a cell’s survival. Quite simply, life would cease to exist without them. As we will reveal in upcoming paragraphs and subsequent chapters, proteins are essentially the link between genetic information and your existence. Proteins are a diverse and abundant class of biomolecules, constituting more than 50% of the dry weight of cells. Their diversity and abundance reflect the central role of proteins in virtually all aspects of cell structure and function. An extraordinary diversity of cellular activity is possible only because of the versatility inherent in proteins, each of which is specifically tailored to its biological role. The pattern by which each is tailored resides within the genetic information of cells, encoded in a specific sequence of nucleotide bases in DNA. Each such segment of encoded information defines a gene, and expression of the gene leads to synthesis of the specific protein encoded by it, endowing the cell with the functions unique to that particular protein. Proteins are the agents of biological function; they are also the expressions of genetic information.

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Chapter 5

Proteins: Their Primary Structure and Biological Functions

" Canada’s National Bird—The Canadian

Thinkstock

Goose and her offspring

5.1

What Architectural Arrangements Characterize Protein Structure?

Protein Structure Is Described in Terms of Four Levels of Organization The architecture of protein molecules is complex. Nevertheless, this complexity can be resolved by defining various levels of structural organization. Primary Structure The amino acid sequence is, by definition, the primary (1°) structure of a protein, such as that for bovine pancreatic RNase in Figure 5.1, for example. Secondary Structure Through hydrogen-bonding interactions between adjacent amino acid residues (discussed in detail in Chapter 6), the polypeptide chain can arrange itself into characteristic helical or pleated segments. These segments constitute structural conformities, so-called regular structures, which extend along 1 dimension, like the coils of a spring. Such architectural features of a protein are designated secondary (2°) structures (Figure 5.2). Secondary structures are just one of the higher levels of structure that represent the 3-dimensional arrangement of the polypeptide in space. Tertiary Structure When the polypeptide chains of protein molecules bend and fold in order to assume a more compact 3-dimensional shape, the tertiary (3°) level of structure is generated (Figure 5.3 on page 101). It is by virtue of their tertiary structure that proteins adopt a globular shape. A globular conformation gives the lowest surface-to-volume ratio, minimizing interaction of the protein with the surrounding environment. Quaternary Structure Many proteins consist of 2 or more interacting polypeptide chains of characteristic tertiary structure, each of which is commonly referred to as a subunit of the protein. Subunit organization constitutes another level in the hierarchy of protein structure, defined as the protein’s quaternary (4°) structure (Figure 5.4 on page 101). Questions of quaternary structure address the various kinds of subunits within a protein molecule, the number of each, and the ways in which they interact with one another.

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99

100

Part 1 Building Blocks of the Cell

FIGURE 5.1 Bovine pancreatic ribonuclease A contains 124 amino acid residues, none of which are tryptophan. Four intrachain disulfide bridges (S!S) form cross-links in this polypeptide between Cys26 and Cys84, Cys40 and Cys95, Cys58 and Cys110, and Cys65 and Cys72.

50 Ser Glu His Val Phe Thr Asn Val 100 Asp Pro 41 Gln Ala Asn Val Thr Thr Lys Lys His Lys Gln 40 124 Tyr Ile Cys Ala Ala HOOC Val Ser Ala Ile Arg Val Asp Cys Val 58 95 Asp Cys 120 Phe Asn Ala Pro Lys Val Ser Val His 119 Pro Cys Glu 60 Gly Thr Gln 110 Asn Pro Tyr Tyr 80 Leu Lys 90 Lys Ser Thr Met Glu Thr Ser Ile Gly Asn Thr Arg Ser Ser Asn Tyr Arg Asp Cys 84 Val Ser 26 Ser Cys 30 Ala Gln Lys 21 Asn Gln 20 Met Met Tyr 65 Cys Ser Ser Tyr Asn Ala Ser 72 Lys Cys Ala Asn 12 10 Asn Ser Thr Ser Ser Asp Met His Gln Arg Gly Thr 70 Glu Gln 7 Glu Thr Ala Ala Ala Lys Phe Ala

Leu

H2N Lys 1 FIGURE 5.2 The a-helix and the b-pleated strand are the 2 principal secondary structures found in protein. Simple representations of these structures are the flat, helical ribbon for the a-helix and the flat, wide arrow for b-structures.

!-Helix Only the N — C! — C backbone is represented. The vertical line is the helix axis.

"-Strand The N — C! — C backbone as well as the C" of R groups are represented here. Note that the amide planes are perpendicular to the page. C! C" C N O C

N C C ! C

C! N N C

C!

N N

C C

C! C N

C! C

C! C!

C

N

N

C N

C!

C! C!

N C

C" C N O C

C C!

N

C C"

H

N

C!

C!

C! C C"

H C" C

C!

C

N C N O C C!

“Shorthand” !-helix

C C"

“Shorthand” "-strand

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Chapter 5 (a)

Chymotrypsin tertiary structure

(b)

(c)

Proteins: Their Primary Structure and Biological Functions

101

Chymotrypsin ribbon

Chymotrypsin space-filling model

FIGURE 5.3 Folding of the polypeptide chain into a compact, roughly spherical conformation creates the tertiary level of protein structure. Shown here are (a) a tracing showing the position of all of the Ca carbon atoms, (b) a ribbon diagram that shows the 3-dimensional track of the polypeptide chain, and (c) a space-filling representation of the atoms as spheres. The protein is chymotrypsin.

Non-covalent Forces Drive Formation of the Higher Orders of Protein Structure

"-Chains

Heme

Whereas the primary structure of a protein is determined by the covalently linked amino acid residues in the polypeptide backbone, secondary and higher orders of structure are determined principally by non-covalent forces, such as hydrogen bonds, and ionic, van der Waals, and hydrophobic interactions. It is important to emphasize that all the information necessary for a protein molecule to achieve its intricate architecture is contained within its 1° structure, that is, within the amino acid sequence of its polypeptide chain(s). Chapter 6 presents a detailed discussion of the 2°, 3°, and 4° structure of protein molecules.

Proteins Fall into Three Basic Classes According to Shape and Solubility As a first approximation, proteins can be assigned to 1 of 3 global classes on the basis of shape and solubility: fibrous, globular, or membrane (Figure 5.5). Fibrous proteins tend to have relatively simple, regular linear structures. These proteins often serve structural roles in cells. Typically, they are insoluble in water or in dilute salt solutions. In contrast, globular proteins are roughly spherical in shape. The polypeptide chain is compactly folded so that hydrophobic amino acid side chains are in the interior of the molecule and the hydrophilic side chains are on the outside exposed to the solvent, water. Consequently, globular proteins are usually very soluble in aqueous solutions. Most soluble proteins of the cell, such as the cytosolic enzymes, are globular in shape. Membrane proteins are found in association with the various membrane systems of cells. For interaction with the non-polar phase within membranes, membrane proteins have hydrophobic amino acid side chains oriented outward. As such, membrane proteins are insoluble in aqueous solutions but can be solubilized in solutions of detergents. Membrane proteins characteristically have fewer hydrophilic amino acids than cytosolic proteins.

!-Chains FIGURE 5.4 Hemoglobin is a tetramer consisting of 2 a and 2 b polypeptide chains.

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102

Part 1 Building Blocks of the Cell (a)

(b)

(c)

Myoglobin, a globular protein

Bacteriorhodopsin, a membrane protein

Collagen, a fibrous protein FIGURE 5.5 (a) Proteins having structural roles in cells are typically fibrous and often water insoluble. (b) Myoglobin is a globular protein. (c) Membrane proteins fold so that hydrophobic amino acid side chains are exposed in their membrane-associated regions. Bacteriorhodopsin binds the light-absorbing pigment cis-retinal, shown here in blue.

A Protein’s Conformation Can Be Described as Its Overall Three-Dimensional Structure The overall 3-dimensional architecture of a protein is generally referred to as its conformation. This term is not to be confused with configuration, which denotes the geometric possibilities for a particular set of atoms (Figure 5.6). In going from one configuration to another, covalent bonds must be broken and rearranged. In contrast, the conformational possibilities of a molecule are achieved without breaking any covalent bonds. In proteins, rotations about each of the single bonds along the peptide backbone have the potential to alter the course of the polypeptide chain in 3-dimensional space. These rotational possibilities create many possible orientations for the protein chain, referred to as its conformational possibilities. Of the great number of theoretical conformations a given protein might adopt, only a very few are favoured energetically under physiological conditions. At this time, the rules that direct the folding of protein chains into energetically favourable conformations are still not entirely clear; accordingly, they are the subject of intensive contemporary research.

5.2

How Are Proteins Isolated and Purified from Cells?

Cells contain thousands of different proteins. As a result, a major problem for protein chemists is the ability to purify a chosen protein so that they can study its specific properties in the absence of other proteins. The first step in the path to achieving this is through the use of dialysis and ultrafiltration. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5 (a)

H

OH

C

HO

H

C

C N

CH2OH

CH2OH

D -Glyceraldehyde

L -Glyceraldehyde

Cl H

H

C H

H

C Cl

1,2-Dichloroethane

H

C

H

Cl H

H Cl

Cl

C

H

H

H

H

C

H

H

H

Amide planes

Cl Cl

H

C

O

(b) Cl

103

(c)

CHO

CHO

Proteins: Their Primary Structure and Biological Functions

C

H

N Side chain

H H

C O C

Amino acids

FIGURE 5.6 Configuration and conformation are not synonymous. (a) Rearrangements between configurational alternatives of a molecule can be achieved only by breaking and remaking bonds, as in the transformation between the D- and L-configurations of glyceraldehyde. (b) The intrinsic free rotation around single covalent bonds creates a great variety of 3-dimensional conformations, even for relatively simple molecules, such as 1,2-dichloroethane. (c) Imagine the conformational possibilities for a protein in which 2 of every 3 bonds along its backbone are freely rotating single bonds.

Dialysis and Ultrafiltration If a solution of protein is separated from a bathing solution by a semipermeable membrane, small molecules and ions can pass through the semipermeable membrane to equilibrate between the protein solution and the bathing solution, called the dialysis bath or dialysate (Figure 5.7). This method is useful for removing small molecules from macromolecular solutions or for altering the composition of the protein-containing solution. Ultrafiltration is an improvement on the dialysis principle. Filters with pore sizes over the range of biomolecular dimensions are used to filter solutions to select for molecules in a particular size range. Because the pore sizes in these filters are microscopic, high pressures are often required to force the solution through the filter. This technique is useful for concentrating dilute solutions of macromolecules. The concentrated protein can then be diluted into the solution of choice. In addition to dialysis and ultrafiltration, other separation methods that exploit size differences, such as size exclusion chromatography and ultracentrifugation, can be used (see pages 105 through 110).

A Number of Protein Separation Methods Exploit Differences in Size and Charge Another prominent physical property of proteins is electrical charge. In this respect, the ionic properties of peptides and proteins are determined principally by their complement of amino acid side chains. Furthermore, the ionization of these groups is pH dependent. A variety of procedures have been designed to exploit the electrical charges on a protein as a means to separate proteins in a mixture. These procedures include ion

Semipermeable bag containing protein solution

Dialysate

Stir bar

Magnetic stirrer for mixing FIGURE 5.7 A dialysis experiment. The solution of macromolecules to be dialyzed is placed in a semipermeable membrane bag, and the bag is immersed in a bathing solution. A magnetic stirrer gently mixes the solution to facilitate equilibrium of diffusible solutes between the dialysate and the solution contained in the bag.

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Solubility, milligrams of protein per millilitre

104

Part 1 Building Blocks of the Cell

3

20 mM 2

1

10 mM

1 mM

5 mM

4M 0 4.8

5.0

5.2 pH

5.4

5.6

5.8

FIGURE 5.8 The solubility of most globular proteins is markedly influenced by pH and ionic strength. This figure shows the solubility of a typical protein as a function of pH and various salt concentrations.

exchange chromatography, electrophoresis, and solubility. Proteins tend to be least soluble at their isoelectric point (see Chapter 4, page 85), the pH value at which the sum of their positive and negative electrical charges is zero. At this pH, electrostatic repulsion between protein molecules is minimal and they are more likely to coalesce and precipitate out of solution. Ionic strength also profoundly influences protein solubility. Most globular proteins tend to become increasingly soluble as the ionic strength is raised. This phenomenon, the salting-in of proteins, is attributed to the diminishment of electrostatic attractions between protein molecules by the presence of abundant salt ions. Such electrostatic interactions between the protein molecules would otherwise lead to precipitation. However, as the salt concentration reaches high levels (greater than 1 M) the effect may reverse so that the protein is salted out of solution. In such cases, the numerous salt ions begin to compete with the protein for waters of solvation, and as they win out, the protein becomes insoluble. The solubility properties of a typical protein are shown in Figure 5.8. Although the side chains of non-polar amino acids in soluble proteins are usually buried in the interior of the protein away from contact with the aqueous solvent, a portion of them may be exposed at the protein’s surface, giving it a partially hydrophobic character. Hydrophobic interaction chromatography is a protein purification technique that exploits this hydrophobicity (see Section 5.3).

A Typical Protein Purification Scheme Uses a Series of Separation Methods Most purification procedures for a particular protein are developed in an empirical manner, the overriding principle being purification of the protein to a homogeneous state with acceptable yield. Table 5.1 presents a summary of a purification scheme for a desired enzyme. Note that the specific activity of the enzyme in the immunoaffinity purified fraction (fraction 5) has been increased 152/0.108, or 1407 times the specific activity in the crude extract (fraction 1). Thus, the concentration of this protein has been enriched more than 1400-fold by the purification procedure.

A DEEPER LOOK

Estimation of Protein Concentrations in Solutions of Biological Origin Biochemists are often interested in knowing the protein concentration in various preparations of biological origin. Such quantitative analysis is not straightforward. Cell extracts are complex mixtures that typically contain protein molecules of many different molecular weights, so the results of protein estimations cannot be expressed on a molar basis. Also, aside from the rather unreactive repeating peptide backbone, little common chemical identity is seen among the many proteins found in cells that might be readily exploited for exact chemical analysis. Most of their chemical properties vary with their amino acid composition, for example, nitrogen or sulfur content or the presence of aromatic, hydroxyl, or other functional groups. Several methods rely on the reduction of Cu2+ ions to Cu+ by readily oxidizable protein components, such as cysteine or the phenols and indoles of tyrosine and tryptophan. For example, bicinchoninic acid (BCA) forms a purple complex with Cu+ in alkaline solution, and the amount of this product can be easily measured spectrophotometrically to provide an estimate of protein concentration. Other assays are based on dye binding by proteins. The Bradford assay is a rapid and reliable technique that uses a dye called Coomassie Brilliant Blue G-250, which undergoes a change in its colour upon

non-covalent binding to proteins. The binding is quantitative and less sensitive to variations in the protein’s amino acid composition. The colour change is easily measured by a spectrophotometer. A  similar, very sensitive method capable of quantifying nanogram amounts of protein is based on the shift in colour of colloidal gold upon binding to proteins.

–OOC

Cu+

+

N

N

COO–

N

COO–

Cu+

BCA –OOC

N

BCA–Cu+ complex

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Chapter 5

TABLE 5.1

Proteins: Their Primary Structure and Biological Functions

105

Example of a Protein Purification Scheme: Purification of an Enzyme from a Cell Extract Volume (mL)

Total Protein (mg)

Total Activity*

Specific Activity**

Percent Recovery†

1. Crude extract

3800

22 800

2460

0.108

100

2. Salt precipitate

165

2 800

1190

0.425

48

3. Ion exchange chromatography

65

100

720

7.2

29

4. Molecular sieve chromatography

40

14.5

555

38.3

23

6

1.8

275

Fraction

5. Immunoaffinity

chromatography††

152

*The

11

relative enzymatic activity of each fraction is cited as arbitrarily defined units. specific activity is the total activity of the fraction divided by the total protein in the fraction. This value gives an indication of the increase in purity attained during the course of the purification as the samples become enriched for the enzyme. †The percent recovery of total activity is a measure of the yield of the desired enzyme. ††The last step in the procedure is an affinity method in which antibodies specific for the enzyme are covalently coupled to a chromatography matrix and packed into a glass tube to make a chromatographic column through which fraction 4 is passed. The enzyme is bound by this immunoaffinity matrix while other proteins pass freely out. The enzyme is then recovered by passing a strong salt solution through the column, which dissociates the enzyme–antibody complex. **The

5.3

How Are Amino Acid Mixtures Separated?

Amino Acids Can Be Separated by Chromatography A wide variety of methods are available for the separation and analysis of amino acids (and other biological molecules and macromolecules). All these methods take advantage of the relative differences in the physical and chemical characteristics of amino acids, particularly ionization behaviour and solubility characteristics. Separations of amino acids are usually based on partition properties (the tendency to associate with one solvent or phase over another) and separations based on electrical charge. In all the partition methods discussed here, the molecules of interest are allowed (or forced) to flow through a medium consisting of 2 phases: solid–liquid, liquid–liquid, or gas–liquid. The molecules partition, or distribute themselves, between the 2 phases in a manner based on their particular properties and their consequent preference for associating with one or the other phase. In 1903, a separation technique based on repeated partitioning between phases was developed by Mikhail Tswett for the separation of plant pigments (carotenes and chlorophylls). Due to the colourful nature of the pigments thus separated, Tswett called his technique chromatography. This term is now applied to a wide variety of separation methods, regardless of whether the products are coloured. The success of all chromatography techniques depends on the repeated microscopic partitioning of a solute mixture between the available phases. The more frequently this partitioning can be made to occur within a given time span or over a given volume, the more efficient is the resulting separation. Chromatographic methods have advanced rapidly in recent years, due in part to the development of sophisticated new solid-phase materials. Methods important for amino acid separations include ion exchange chromatography, gas chromatography (GC), and high-performance liquid chromatography (HPLC). A typical HPLC chromatogram using pre-column modification of amino acids to form phenylthiohydantoin (PTH) derivatives is shown in Figure 5.9. HPLC is the chromatographic technique of choice for most modern biochemists. The very high resolution, excellent sensitivity, and high speed of this technique usually outweigh the disadvantage of relatively low capacity.

Summary of the Types of Chromatographic Techniques Ion Exchange Chromatography Can Be Used to Separate Molecules on the Basis of Charge Charged molecules can be separated using ion exchange chromatography, a process in which the charged molecules of interest (ions) are exchanged for another ion (usually a salt ion) on a charged solid support. In a typical procedure, solutes in a liquid phase, usually water, are passed through a column filled with a porous solid phase composed of synthetic resin particles containing charged groups. Resins containing positively charged groups attract negatively charged solutes and are referred to as anion exchange resins. Resins with negatively charged groups are cation exchangers. Figure 5.10 shows NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Part 1 Building Blocks of the Cell

FIGURE 5.9 Gradient separation of common PTH-amino acids, which absorb UV light. Absorbance was monitored at 269 nm. PTH peaks are identified by single-letter notation for amino acid residues and by other abbreviations. D, Asp; CMC, carboxymethyl Cys; E, Glu; N, Asn; S, Ser; Q, Gln; H, His; T, Thr; G, Gly; R, Arg; MO2, Met sulfoxide; A, Ala; Y, Tyr; M, Met; V, Val; P, Pro; W, Trp; K, Lys; F, Phe; I, Ile; L, Leu. See Figure 5.19a for PTH derivatization. (Adapted from Persson, B., and Eaker, D., 1990. An optimized procedure for the separation of amino acid phenylthiohydantoins by reversed phase HPLC. Journal of Biochemical and Biophysical Methods 21:341–350.)

D

A Y

Q

M

W

V

G Absorbance

106

E

MO2

N S

T

CMC

F

P

R

I L

K

H

Elution time

FIGURE 5.10 Cation (a) and anion (b) exchange resins commonly used for biochemical separations.

Structure

(a) Cation Exchange Media

O Strongly acidic, polystyrene resin (Dowex-50)

O–

S O

O Weakly acidic, carboxymethyl (CM) cellulose

O

C

CH2

O– O

Weakly acidic, chelating, polystyrene resin (Chelex-100)

CH2

CH2C

O–

CH2C

O–

N

O

(b) Anion Exchange Media

Structure

CH3 Strongly basic, polystyrene resin (Dowex-1)

CH2

N

+

CH3

CH3 CH2CH3 Weakly basic, diethylaminoethyl (DEAE) cellulose

OCH2CH2

N

+

H

CH2CH3

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5

Proteins: Their Primary Structure and Biological Functions

Sample containing several proteins

107

FIGURE 5.11 The separation of amino acids on a cation exchange column.

Chromatography column containing cation exchange resin beads

The elution process separates proteins into discrete bands

Eluant emerging from the column is collected

Amino acid concentration

Some fractions do not contain proteins

Elution time

several typical anion and cation exchange resins. Weakly acidic or basic groups on ion exchange resins exhibit charges that are dependent on the pH of the bathing solution. Changing the pH will alter the ionic interaction between the resin groups and the bound ions. In all cases, the bare charges on the resin particles must be counterbalanced by oppositely charged ions in solution (counter-ions); salt ions (e.g., Na+ or Cl−) usually serve this purpose. The separation of a mixture of several amino acids on a column of cation exchange resin is illustrated in Figure 5.11. Increasing the salt concentration in the solution passing through the column leads to competition between the cationic amino acid bound to the column and the cations in the salt for binding to the column. Bound cationic amino acids that interact weakly with the charged groups on the resin wash out first, and those interacting strongly are washed out only at high salt concentrations. Size Exclusion Chromatography Size exclusion chromatography is also known as gel filtration chromatography or molecular sieve chromatography. In this method, fine, porous beads are packed into a chromatography column. The beads are composed of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or Bio-Gel P). The pore sizes of these beads approximate the dimensions of macromolecules. The total bed volume (Figure 5.12) of the packed chromatography column, Vt, is equal to the volume outside the porous beads (Vo) plus the volume inside the beads (Vi) plus the NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

108

Part 1 Building Blocks of the Cell

FIGURE 5.12 (a) A gel filtration chromatography column. Larger molecules are excluded from the gel beads and emerge from the column sooner than smaller molecules, whose migration is retarded because they can enter the beads. (b) An elution profile.

(a) Small molecule Large molecule Porous gel beads Elution column

Protein concentration

(b)

Elution profile of a large macromolecule (excluded from pores) (Ve ≅ Vo) A smaller macromolecule

Vo

Volume (mL)

Ve

Vt

volume actually occupied by the bead material (Vg): Vt = Vo + Vi + Vg. (Vg is typically less than 1% of Vt and can be conveniently ignored in most applications.) As a solution of molecules is passed through the column, the molecules passively distribute between Vo and Vi, depending on their ability to enter the pores (i.e., their size). If a molecule is too large to enter at all, it is totally excluded from Vi and emerges first from the column at an elution volume, Ve, equal to Vo (Figure 5.12). If a particular molecule can enter the pores in the gel, its distribution is given by the distribution coefficient, KD: KD = (Ve - Vo)>Vi, where Ve is the molecule’s characteristic elution volume (Figure 5.12). The chromatography run is complete when a volume of solvent equal to Vt has passed through the column. Electrophoresis Electrophoretic techniques are based on the movement of ions in an electrical field. An ion of charge q experiences a force F, given by F = Eq"d, where E is the voltage (or electrical potential) and d is the distance between the electrodes. In a vacuum, F would cause the molecule to accelerate. In solution, the molecule experiences frictional drag, Ff, due to the solvent: Ff = 6prhn, where r is the radius of the charged molecule, h is the viscosity of the solution, and v is the velocity at which the charged molecule is moving. So, the velocity of the charged molecule is proportional to its charge q and the voltage E, but inversely proportional to the viscosity of the medium h and d, the distance between the electrodes. Generally, electrophoresis is carried out not in free solution but in a porous support matrix such as polyacrylamide or agarose, which retards the movement of molecules according to their dimensions relative to the size of the pores in the matrix. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS is sodium dodecyl sulfate (sodium lauryl sulfate) (Figure 5.13). The hydrophobic tail of dodecyl sulfate interacts strongly with polypeptide chains. The number of SDS molecules bound by a polypeptide is proportional to the length (number of amino acid residues) of the polypeptide. Each dodecyl sulfate contributes 2 negative charges. Collectively, these charges overwhelm any NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5

Proteins: Their Primary Structure and Biological Functions

S O– Na+

O

CH2 CH2 CH2 CH2 CH2 CH3 CH2 CH2 CH2 CH2 CH2 CH2

intrinsic charge that the protein might have. SDS is also a detergent that disrupts protein folding (protein 3° structure). SDS-PAGE is usually run in the presence of sulfhydrylreducing agents such as b-mercaptoethanol so that any disulfide links between polypeptide chains are broken. The electrophoretic mobility of proteins upon SDS-PAGE is inversely proportional to the logarithm of the protein’s molecular weight (Figure 5.14). SDS-PAGE is often used to determine the molecular weight of a protein. Isoelectric Focusing Isoelectric focusing is an electrophoretic technique for separating proteins according to their isoelectric points (pIs). A solution of ampholytes (amphoteric electrolytes) is first electrophoresed through a gel, usually contained in a small tube. The  migration of these substances in an electric field establishes a pH gradient in the tube. Then a protein mixture is applied to the gel, and electrophoresis is resumed. As the protein molecules move down the gel, they experience the pH gradient and migrate to a position corresponding to their respective pIs. At its pI, a protein has no net charge and thus moves no farther.

Log molecular weight

FIGURE 5.13 The structure of sodium dodecyl sulfate (SDS).

O Na+ –O

109

Relative electrophoretic mobility FIGURE 5.14 A plot of the relative electrophoretic mobility of proteins in SDS-PAGE versus the log of the molecular weights of the individual polypeptides.

Two-Dimensional Gel Electrophoresis This separation technique uses isoelectric focusing in 1 dimension and SDS-PAGE in the second dimension to resolve protein mixtures. The proteins in a mixture are first separated according to pI by isoelectric focusing in a polyacrylamide gel in a tube. The gel is then removed and laid along the top of an SDS-PAGE slab, and the proteins are electrophoresed into the SDS polyacrylamide gel, where they are separated according to size (Figure 5.15). The gel slab can then be stained FIGURE 5.15 A 2-dimensional electrophoresis separation. A mixture of macromolecules is first separated according to charge by isoelectric focusing in a gel tube. The gel containing separated molecules is then placed on top of an SDS-PAGE slab, and the molecules are electrophoresed into the SDS-PAGE gel, where they are separated according to size.

Isoelectric focusing gel

10

pH

pH 10

pH 4 High MW

4 Direction of electrophoresis

Low MW SDS-poly- Protein spot acrylamide slab NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

110

Part 1 Building Blocks of the Cell

to reveal the locations of the individual proteins. Using this powerful technique, researchers have the potential to visualize and construct catalogues of virtually all the proteins present in particular cell types. The ExPASy server (http://us.expasy.org/) provides access to a 2-dimensional polyacrylamide gel electrophoresis database named SWISS-2DPAGE. This database contains information on proteins, identified as spots on 2-dimensional electrophoresis gels, from many different cell and tissue types.

A protein interacts with a metabolite. The metabolite is thus a ligand that binds specifically to this protein

+ Protein

Metabolite

The metabolite can be immobilized by covalently coupling it to an insoluble matrix such as an agarose polymer. Cell extracts containing many individual proteins may be passed through the matrix.

Specific protein binds to ligand. All other unbound material is washed out of the matrix.

Adding an excess of free metabolite that will compete for the bound protein dissociates the protein from the chromatographic matrix. The protein passes out of the column complexed with free metabolite.

Hydrophobic Interaction Chromatography Hydrophobic interaction chromatography (HIC) exploits the hydrophobic nature of proteins in purifying them. Proteins are passed over a chromatographic column packed with a support matrix to which hydrophobic groups are covalently linked. Phenyl Sepharose, an agarose support matrix to which phenyl groups are affixed, is a prime example of such material. In the presence of high salt concentrations, proteins bind to the phenyl groups by virtue of hydrophobic interactions. Proteins in a mixture can be differentially eluted from the phenyl groups by lowering the salt concentration or by adding solvents such as polyethylene glycol to the elution fluid. High-Performance Liquid Chromatography The principles exploited in high-performance (or high-pressure) liquid chromatography (HPLC) are the same as those used in the common chromatographic methods such as ion exchange chromatography or size exclusion chromatography. Very high resolution separations can be achieved quickly and with high sensitivity in HPLC using automated instrumentation. Reverse-phase HPLC is a widely used chromatographic procedure for the separation of non-polar solutes. In reverse-phase HPLC, a solution of non-polar solutes is chromatographed on a column having a nonpolar liquid immobilized on an inert matrix; this non-polar liquid serves as the stationary phase. A more polar liquid that serves as the mobile phase is passed over the matrix, and solute molecules are eluted in proportion to their solubility in this more polar liquid. Affinity Chromatography Affinity purification strategies for proteins exploit the biological function of the target protein. In most instances, proteins carry out their biological activity through binding or complex formation with specific small biomolecules, or ligands, as in the case of an enzyme binding its substrate. If this small molecule can be immobilized through covalent attachment to an insoluble matrix, such as a chromatographic medium like cellulose or polyacrylamide, then the protein of interest, in displaying affinity for its ligand, becomes bound and immobilized itself. It can then be removed from contaminating proteins in the mixture by simple means such as filtration and washing the matrix. Finally, the protein is dissociated or eluted from the matrix by the addition of high concentrations of the free ligand in solution. Figure 5.16 depicts the protocol for such an affinity chromatography scheme. Because this method of purification relies on the biological specificity of the protein of interest, it is a very efficient procedure and proteins can be purified several thousand-fold in a single step. Ultracentrifugation Centrifugation methods separate macromolecules on the basis of their characteristic densities. Particles tend to “fall” through a solution if the density of the solution is less than the density of the particle. The velocity of the particle through the medium is proportional to the difference in density between the particle and the solution. The tendency of any particle to move through a solution under centrifugal force is given by the sedimentation coefficient, S: S = ( rp - rm)V>f, where rp is the density of the particle or macromolecule, rm is the density of the medium or solution, V is the volume of the particle, and f is the frictional coefficient, given by

Purifications of proteins as much as 1000-fold or more are routinely achieved in a single affinity chromatographic step like this. FIGURE 5.16 Diagram illustrating affinity chromatography.

f = Ff >v,

where v is the velocity of the particle and Ff is the frictional drag. Non-spherical molecules have larger frictional coefficients and thus smaller sedimentation coefficients. The smaller the particle and the more its shape deviates from spherical, the more slowly that particle will sediment in a centrifuge. Centrifugation can be used as either a preparative technique for separating and purifying macromolecules and cellular components, or an analytical technique to characterize the hydrodynamic properties of macromolecules such as proteins and nucleic acids. NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5

5.4

Proteins: Their Primary Structure and Biological Functions

How Is the Amino Acid Analysis of Proteins Performed?

Acid Hydrolysis Liberates the Amino Acids of a Protein Peptide bonds of proteins are hydrolyzed by either strong acid or strong base. Acid hydrolysis is the method of choice for analysis of the amino acid composition of proteins and polypeptides because it proceeds without racemization and with less destruction of certain amino acids (Ser, Thr, Arg, and Cys). Typically, samples of a protein are hydrolyzed with 6 N HCl at 110°C. Tryptophan is destroyed by acid and must be estimated by other means to determine its contribution to the total amino acid composition. The OH-containing amino acids serine and threonine are destroyed slowly. In contrast, peptide bonds involving hydrophobic residues such as valine and isoleucine are only slowly hydrolyzed in acid. Another complication arises because the b- and g-amide linkages in asparagine (Asn) and glutamine (Gln) are acid labile. The amino nitrogen is released as free ammonium, and all the Asn and Gln residues of the protein are converted to aspartic acid (Asp) and glutamic acid (Glu) respectively. The amount of ammonium released during acid hydrolysis gives an estimate of the total number of Asn and Gln residues in the original protein, but not the amounts of either.

Chromatographic Methods Are Used to Separate the Amino Acids The complex amino acid mixture in the hydrolysate obtained after digestion of a protein in 6 N HCl can be separated into the component amino acids using either ion exchange chromatography or reversed-phase high-pressure liquid chromatography (HPLC) (see Section 5.3). The amount of each amino acid can then be determined. These methods of separation and analysis are fully automated in instruments called amino acid analyzers. Analysis of the amino acid composition of a 30 kD protein by these methods requires less than 1 hour and only 6 mg (0.2 nmol) of the protein.

The Amino Acid Compositions of Different Proteins Are Different Amino acids almost never occur in equimolar ratios in proteins, indicating that proteins are not composed of repeating arrays of amino acids. There are a few exceptions to this rule. Collagen, for example, contains large proportions of glycine and proline, and much of its structure is composed of (Gly-x-Pro) repeating units, where x is any amino acid. Other proteins show unusual abundances of various amino acids. For example, histones are rich in positively charged amino acids such as arginine and lysine. Histones are a class of proteins found associated with the anionic phosphate groups of eukaryotic DNA. Amino acid analysis itself does not directly give the number of residues of each amino acid in a polypeptide, but if the molecular weight and the exact amount of the protein analyzed are known (or the number of amino acid residues per molecule is known), the molar ratios of amino acids in the protein can be calculated. Amino acid analysis provides no information on the order or sequence of amino acid residues in the polypeptide chain.

5.5

How Is the Primary Structure of a Protein Determined?

The Sequence of Amino Acids in a Protein Is Distinctive The unique characteristic of each protein is the distinctive sequence of amino acid residues in its polypeptide chain(s). Indeed, it is the amino acid sequence of proteins that is encoded by the nucleotide sequence of DNA. This amino acid sequence, then, is a form of genetic information. Because polypeptide chains are unbranched, a polypeptide chain NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

111

112

Part 1 Building Blocks of the Cell N

N

Gly

Phe

Ile

Val

Val

Asn

Glu

Gln

5 Gln

His

Cys

Leu

Cys

S

S

Cys

S

Ala

Gly

S

Ser

Ser

10 Val

His

Cys

Leu

Ser

Val

Leu

Glu

Tyr

Ala

15 Gln

Leu

Leu

Tyr

Glu

Leu

Asn

Val

Tyr

Cys

20 Cys

S

S

has only 2 ends, an amino-terminal, or N-terminal, end and a carboxy-terminal, or C-terminal, end. By convention, the amino acid sequence is read from the N-terminal end of the polypeptide chain through to the C-terminal end. As an example, every molecule of ribonuclease A from bovine pancreas has the same amino acid sequence, beginning with N-terminal lysine at position 1 and ending with C-terminal valine at position 124 (Figure 5.1). Given the possibility of any of the 20 amino acids at each position, the number of unique amino acid sequences is astronomically large. The astounding sequence variation possible within polypeptide chains provides a key insight into the incredible functional diversity of protein molecules in biological systems discussed later in this chapter.

Sanger Was the First to Determine the Sequence of a Protein In 1953, Frederick Sanger of Cambridge University in England reported the amino acid sequences of the 2 polypeptide chains composing the protein insulin (Figure 5.17). Not only was this a remarkable achievement in analytical chemistry, but it helped demystify speculation about the chemical nature of proteins. Sanger’s results clearly established that all the molecules of a given protein have a fixed amino acid composition, a defined amino acid sequence, and therefore an invariant molecular weight. In short, proteins are well defined chemically. Today, the amino acid sequences of hundreds of thousands of proteins are known. Although many sequences have been determined from application of the principles first established by Sanger, most are now deduced from knowledge of the nucleotide sequence of the gene that encodes the protein. In addition, in recent years, the application of mass spectrometry to the sequence analysis of proteins has largely superseded the protocols based on chemical and enzymatic degradation of polypeptides that Sanger pioneered.

Gly

Asn

Glu

C A chain

Arg Gly Phe 25 Phe Tyr Thr Pro Lys 30 Ala C B chain

FIGURE 5.17 The hormone insulin consists of 2 polypeptide chains, A and B, held together by 2 disulfide cross-bridges (S!S). The A chain has 21 amino acid residues and an intrachain disulfide; the B polypeptide contains 30 amino acids. The sequence shown is for bovine insulin.

Both Chemical and Enzymatic Methodologies Are Used in Protein Sequencing The chemical strategy for determining the amino acid sequence of a protein involves 6 basic steps: 1. If the protein contains more than 1 polypeptide chain, the chains are separated and purified. 2. Intrachain S!S (disulfide) cross-bridges between cysteine residues in the polypeptide chain are cleaved. (If these disulfides are interchain linkages, then step 2 precedes step 1.) 3. The N-terminal and C-terminal residues are identified. 4. Each polypeptide chain is cleaved into smaller fragments, and the amino acid composition and sequence of each fragment are determined. 5. Step 4 is repeated, using a different cleavage procedure to generate a different and therefore overlapping set of peptide fragments. 6. The overall amino acid sequence of the protein is reconstructed from the sequences in overlapping fragments. Each of these steps is discussed in greater detail in the following sections.

Step 1. Separation of Polypeptide Chains If the protein of interest is a heteromultimer (composed of more than 1 type of polypeptide chain), then the protein must be dissociated into its component polypeptide chains, which then must be separated from one another and sequenced individually. Because subunits in multimeric proteins typically associate through non-covalent NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5

Proteins: Their Primary Structure and Biological Functions

113

A DEEPER LOOK

The Virtually Limitless Number of Different Amino Acid Sequences Given 20 different amino acids, a polypeptide chain of n residues can have any one of 20n possible sequence arrangements. To portray this, consider the number of tripeptides possible if there were only 3 different amino acids, A, B, and C (tripeptide = 3 = n; 3n = 33 = 27):

AAA AAB AAC ABA ACA ABC ACB ABB ACC

BBB BBA BBC BAB BCB BAA BCC BAC BCA

CCC CCA CCB CBC CAC CBA CAB CBB CAA

For a polypeptide chain of 100 residues in length, a modest size, the number of possible sequences is 20100, or because 20 = 101.3, 10130 unique possibilities. These numbers are more than astronomical! Because an average protein molecule of 100 residues would have a mass of 12 000 daltons (D; assuming the average molecular mass of an amino acid residue = 120), 10130 such molecules would have a mass of 1.2 * 10134 D. The mass of the observable universe is estimated to be 1080 proton masses (about 1080 D). Thus, the universe lacks enough material to make just 1 molecule of each possible polypeptide sequence for a protein only 100 residues in length.

interactions, most multimeric proteins can be dissociated by exposure to pH extremes, 8 M urea, 6 M guanidinium hydrochloride, or high salt concentrations. (All these treatments disrupt polar interactions such as hydrogen bonds both within the protein molecule and between the protein and the aqueous solvent.) Once dissociated, the individual polypeptides can be isolated from one another on the basis of differences in size and/or charge. Occasionally, heteromultimers are linked together by interchain S—S bridges. In such instances, these cross-links must be cleaved before dissociation and isolation of the individual chains. The methods described under step 2 are applicable for this purpose.

Step 2. Cleavage of Disulfide Bridges A number of methods exist for cleaving disulfides. An important consideration is to carry out these cleavages so that the original or even new S—S links do not form. Oxidation of a disulfide by performic acid results in the formation of 2 equivalents of cysteic acid (Figure 5.18a). Because these cysteic acid side chains are ionized SO3- groups, electrostatic repulsion (as well as altered chemistry) prevents S—S recombination. Alternatively, sulfhydryl compounds such as 2-mercaptoethanol or dithiothreitol (DTT) readily reduce S—S bridges to regenerate 2 cysteine—SH side chains, as in a reversal of the reaction shown in Figure 5.19b on page 115. However, these SH groups recombine to re-form either the original disulfide link or, if other free Cys—SHs are available, new disulfide links. To prevent this, S—S reduction must be followed by treatment with alkylating agents such as iodoacetate or 3-bromopropylamine, which modify the SH groups and block disulfide bridge formation (Figure 5.18b).

Step 3. N- and C-Terminal Analysis A. N-Terminal Analysis The amino acid residing at the N-terminal end of a protein can be identified in a number of ways; one method, Edman degradation, has become the procedure of choice. This method is preferable because it allows the sequential identification of a series of residues beginning at the N-terminus. The reaction with phenylisothiocyanate, or Edman reagent, involves nucleophilic attack by the amino acid a-amino nitrogen, followed by cyclization, to yield a phenylthiohydantoin (PTH) derivative of the amino acid (Figure 5.19a on page 115). PTH-amino acids can be identified and quantified easily, as shown in Section 5.3. An important amino acid side-chain reaction is formation of disulfide bonds via reaction between 2 cysteines. In proteins, cysteine residues form disulfide

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

114

Part 1 Building Blocks of the Cell (a)

...

Oxidative cleavage R O N

CH

C

H

O

N

CH

H

CH2 S S

...

N

R′

O

CH

C

H (b)

C

N

...

...

H

N

H

C

O

O

CH

H

C

N

CH

C

O N

CH

H

CH2

...

H

N

R′

O

CH

C

H

N

...

H Cysteic acid residues

SO3–

Performic acid

...

C

SO3–

H

CH2 O N

O

H

O

Disulfide bond

R

CH2 O N

CH

C

H

N

...

H

SH modification (1)

...

H

O

N

C

C

H

CH2

... +

ICH2COOH Iodoacetic acid

HI

+ ...

SH

H

O

N

C

C

H

CH2 S

...

CH2

COO–

S-carboxymethyl derivative (2)

...

H

O

N

C

C

H

CH2 SH

... +

Br CH2 CH2 CH2 NH2 3-Bromopropylamine

HBr

+ ...

H

O

N

C

C

H

CH2 S

...

CH2 CH2 CH2

NH2

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins. (a) Oxidative cleavage by reaction with performic acid. (b) Disulfide bridges can be broken by reduction with sulfhydryl agents such as b-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced !SH groups to reestablish disulfide bonds is a likelihood, S!S reduction must be followed by !SH modification: (1) alkylation with iodoacetate (ICH2COOH) or (2) modification with 3-bromopropylamine [Br!(CH2)3!NH2].

linkages that stabilize protein structure (Figure 5.19b). Chromatographic methods can be used to identify this PTH derivative. Importantly, in this procedure, the rest of the polypeptide chain remains intact and can be subjected to further rounds of Edman degradation to identify successive amino acid residues in the chain. Often, the carboxyl terminus of the polypeptide under analysis is coupled to an insoluble matrix, allowing the polypeptide to be easily recovered by filtration or centrifugation following each round of Edman reaction. Thus, the Edman reaction not only identifies the N-terminal residue of proteins but through successive reaction cycles can reveal further information about sequence. Automated instruments (so-called Edman sequenators) have been designed to carry out repeated rounds of the Edman procedure. In practical terms, as many as 50 cycles of reaction can be accomplished on 50 pmol (about 0.1 mg) of a polypeptide 100–200 residues long, revealing the sequential order of the first 50 amino acid residues in the protein. The efficiency with larger proteins is less: a typical 2000–amino acid protein provides only 10–20 cycles of reaction. B. C-Terminal Analysis For the identification of the C-terminal residue of polypeptides, an enzymatic approach is commonly used. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use: A, B, C, and Y. Carboxypeptidase A (from bovine NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5

S

(a)

H N

C

S

R

H

N

R′

CH C

O C

S

O

H

O

N

C

H

H2C

N

CH2 C

C

O H Cys residues in two peptide chains

R′

CH

H N

Thiazoline derivative

+ H3N R′

N

C

C C H

O

H

S N

TFA

CH

C

O

H

S

N H

C

O

C N

R

C Weak aqueous acid

N C

H O PTH-amino acid

CH C

O

O

H C

N

S

+

S H

C

R

H C

H2C

N Mild alkali

CH C

H

S N

NH2

(b)

115

Proteins: Their Primary Structure and Biological Functions

2 H+

+

2 e–

CH2 C

C

O H Disulfide

FIGURE 5.19 Some reactions of amino acids. (a) Edman reagent, phenylisothiocyanate, reacts with the a-amino group of an amino acid or peptide to produce a phenylthiohydantoin (PTH) derivative. (b) Cysteines react to form disulfides.

pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys is the C-terminal residue. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved, and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators.

Steps 4 and 5. Fragmentation of the Polypeptide Chain The aim at this step is to produce fragments useful for sequence analysis. The cleavage methods employed are usually enzymatic, but proteins can also be fragmented by specific or nonspecific chemical means (such as partial acid hydrolysis). Proteolytic enzymes offer an advantage in that many hydrolyze only specific peptide bonds, and this specificity immediately gives information about the peptide products. As a first approximation, fragments produced upon cleavage should be small enough to yield their sequences through end-group analysis and Edman degradation, yet not so small that an overabundance of products must be resolved before analysis. A. Trypsin The digestive enzyme trypsin is the most commonly used reagent for specific proteolysis. Trypsin will only hydrolyze peptide bonds in which the carbonyl function is contributed by an arginine or a lysine residue. That is, trypsin cleaves on the C-side of Arg or Lys, generating a set of peptide fragments having Arg or Lys at their C-termini. The number of smaller peptides resulting from trypsin action is equal to the total number of Arg and Lys residues in the protein plus one—the protein’s C-terminal peptide fragment (Figure 5.20). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

H R

116

Part 1 Building Blocks of the Cell

FIGURE 5.20 (a) Trypsin is a proteolytic enzyme, or protease, that specifically cleaves only those peptide bonds in which arginine or lysine contributes the carbonyl function. (b) The products of the reaction are a mixture of peptide fragments with C-terminal Arg or Lys residues and a single peptide derived from the polypeptide’s C-terminal end.

NH2

(a)

C

+ NH2

+ NH3

HN

CH2 CH2

CH2 OH

CH2 CH3 O

...

N H

CH Ala

C

N H

CH2

O

CH Arg

C

N H

COO–

CH2

CH2

O

CH Ser

C

N H

Trypsin

CH2

O

CH Lys

C

CH2 O N H

CH C Asp

...

Trypsin

(b) N—Asp—Ala—Gly—Arg—His—Cys—Lys—Trp—Lys—Ser—Glu—Asn—Leu—Ile—Arg—Thr—Tyr—C

Trypsin Asp—Ala—Gly—Arg His—Cys—Lys Trp—Lys Ser—Glu—Asn—Leu—Ile—Arg Thr—Tyr

B. Chymotrypsin Chymotrypsin shows a strong preference for hydrolyzing peptide bonds formed by the carboxyl groups of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. However, over time, chymotrypsin also hydrolyzes amide bonds involving amino acids other than Phe, Tyr, or Trp. For instance, peptide bonds having leucine-donated carboxyls are also susceptible. Thus, the specificity of chymotrypsin is only relative. Because chymotrypsin produces a very different set of products than trypsin, treatment of separate samples of a protein with these 2 enzymes generates fragments whose sequences overlap. Resolution of the order of amino acid residues in the fragments yields the amino acid sequence in the original protein. C. Other Endopeptidases A number of other endopeptidases (proteases that cleave peptide bonds within the interior of a polypeptide chain) are also used in sequence investigations. These include clostripain, which acts only at Arg residues; endopeptidase Lys-C, which cleaves only at Lys residues; and staphylococcal protease, which acts at the acidic residues Asp and Glu. Other, relatively non-specific endopeptidases are handy for digesting large tryptic or chymotryptic fragments. Pepsin, papain, subtilisin, thermolysin, and elastase are some examples. Papain is the active ingredient in meat tenderizer, soft contact lens cleaner, and some laundry detergents. D. Cyanogen Bromide Several highly specific chemical methods of proteolysis are available, the most widely used being cyanogen bromide (CNBr) cleavage. CNBr acts upon methionine residues (Figure 5.21). The nucleophilic sulfur atom of Met reacts with CNBr, yielding a sulfonium ion that undergoes a rapid intramolecular rearrangement to form a cyclic iminolactone. Water readily hydrolyzes this iminolactone, cleaving the polypeptide and generating peptide fragments having C-terminal homoserine lactone residues at the former Met positions. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5 CH3

Brδ–

S

Cδ+

CH2

N

CH3 + S Br–

CH2 O

...

N

C

H

H

C

N

...

Methyl thiocyanate C

H3C

N

S

+

CH2

...

H

N

C

H

H

C

N

C

N

(C-terminal peptide) H+3N Peptide CH2

CH2

2

CH2 O

1

Proteins: Their Primary Structure and Biological Functions

...

...

H

CH2

O

N

C

C

H

H

+ N H

...

3

...

CH2

O

N

C

C

H

H

O

H2O

OVERALL REACTION: CH3 S CH2 CH2 O

...

C

N

C

CH2

BrCN N

...

70% HCOOH

...

N

CH2

O

C

C

O H H Peptide H+3N Peptide with C-terminal (C-terminal peptide) homoserine lactone

H H H Polypeptide

FIGURE 5.21 Cyanogen bromide (CNBr) is a highly selective reagent for cleavage of peptides only at methionine residues. (1) Nucleophilic attack of the Met S atom on the !C≡N carbon atom, with displacement of Br. (2) Nucleophilic attack by the Met carbonyl oxygen atom on the R group. The cyclic derivative is unstable in aqueous solution. (3) Hydrolysis cleaves the Met peptide bond. C-terminal homoserine lactone residues occur where Met residues once were.

E. Other Chemical Methods of Fragmentation A number of other chemical methods give specific fragmentation of polypeptides, including cleavage at asparagine–glycine bonds by hydroxylamine (NH2OH) at pH 9, and selective hydrolysis at aspartyl–prolyl bonds under mildly acidic conditions. Table 5.2 summarizes the various procedures described here for polypeptide cleavage. These methods are only a partial list of the arsenal of reactions available to protein chemists. Cleavage products generated by these procedures must be

TABLE 5.2 Specificity of Representative Polypeptide Cleavage Procedures Used in Sequence Analysis* Peptide Bond on Carboxyl (C) or Amino (N) Side of Susceptible Residue

Susceptible Residue(s)

Trypsin

C

Arg or Lys

Chymotrypsin

C

Phe, Trp, or Tyr; Leu

Method Proteolytic

enzymes*

Clostripain

C

Arg

Staphylococcal protease

C

Asp or Glu

Cyanogen bromide

C

Met

NH2OH

Asn-Gly bonds

pH 2.5, 40°C

Asp-Pro bonds

Chemical methods

*Some proteolytic enzymes, including trypsin and chymotrypsin, will not cleave peptide bonds where proline is the amino acid contributing the N-atom.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

117

118

Part 1 Building Blocks of the Cell

FIGURE 5.22 Summary of the sequence analysis of catrocollastatin-C, a 23.6 kD protein found in the venom of the western diamondback rattlesnake Crotalus atrox. Sequences shown are given in the 1-letter amino acid code. The overall amino acid sequence (216 amino acid residues long) for catrocollastatin-C as deduced from the overlapping sequences of peptide fragments is shown on the lines headed CAT-C. The other lines report the various sequences used to obtain the overlaps. These sequences were obtained from (a) N-term: Edman degradation of the intact protein in an automated Edman sequenator; (b) M: proteolytic fragments generated by CNBr cleavage, followed by Edman sequencing of the individual fragments (numbers denote fragments M1 through M5); (c) K: proteolytic fragments from endopeptidase Lys-C cleavage, followed by Edman sequencing (only fragments K3 through K6 are shown); (d) E: proteolytic fragments from Staphylococcus protease digestion of catrocollastatin sequenced in the Edman sequenator (only E13 through E15 are shown). (Adapted from Shimokawa, K., et al., 1997. Sequence and biological activity of catrocollastatin-C: A disintegrin-like/ cysteine-rich two-domain protein from Crotalus atrox venom. Archives of Biochemistry and Biophysics 343:35–43.)

1 10 20 30 40 50 60 CAT-C LGTDIISPPVCGNELLEVGEECDCGTPENCQNECCDAATCKLKSGSQCGHGDCCEQCKFS N-Term LGTDIISPPVCGNELLEVGEECDCGTPENCQNECCDAAT LGTDIISPPVCGNELLEVGEECDCGTPENCQNECCDAATCKLKSGSQCGHGDCCEQCK M1 GSQCGHGDCCEQCK K3 SGSQCGHGDCCEQCK FS K4

CAT-C M2 M3 K4 K5 K6

70 80 90 100 110 120 KSGTECRASMSECDPAEHCTGQSSECPADVFHKNGQPCLDNYGY CYNGNCPIMYHQCYDL SECDPAEHCTGQSSECPADVFHKNGQPCLDNYGYCY YHQCYDL K SGTECRASMSECDPAEHCTGQSSECPADVF NGQPCLDNYGYCYNGNCPIMYHQCYDL

CAT-C M3 K6 E13 E15

130 140 150 160 170 180 FGADVYEAEDSCFERNQKGNYYGYCRKENGNKIPCCAPEDVKCGRLYCKDNSPGQNNPCKM FGADVYEAEDSCF –RNQKGNYYGYCRKENGNKIPCCAPEDVKCGRLYCKDN–PGQN– PCK FGA –SCFERNQKGN DVKCGRLYCKDNSPGQNNPCKM

CAT-C M4 M5 E15

190 200 210 FYSNEDEHKGMVLPGTKCADGKVCSNGHCVDVATAY FYSNEDEHKGM VLPGTKCADGKVCSNGHCVDVATAY FYSNEDEHKGMVLPGTKCADGKVC

isolated and individually sequenced to accumulate the information necessary to reconstruct the protein’s complete amino acid sequence. Peptide sequencing today is most commonly done by Edman degradation of relatively large peptides or by mass spectrometry (see following discussion).

Step 6. Reconstruction of the Overall Amino Acid Sequence The sequences obtained for the sets of fragments derived from 2 or more cleavage procedures are now compared, with the objective being to find overlaps that establish continuity of the overall amino acid sequence of the polypeptide chain. The strategy is illustrated by the example shown in Figure 5.22. Peptides generated from specific fragmentation of the polypeptide can be aligned to reveal the overall amino acid sequence. Such comparisons are also useful in eliminating errors and validating the accuracy of the sequences determined for the individual fragments.

The Amino Acid Sequence of a Protein Can Be Determined by Mass Spectrometry Mass spectrometers exploit the difference in the mass-to-charge (m/z) ratio of ionized atoms or molecules to separate them from each other. The m/z ratio of a molecule is also a highly characteristic property that can be used to acquire chemical and structural information. Furthermore, molecules can be fragmented in distinctive ways in mass spectrometers, and the fragments that arise also provide specific structural information about the molecule. The basic operation of a mass spectrometer is to (1) evaporate and ionize molecules in a vacuum, creating gas-phase ions; (2) separate the ions in space and/or time based on their m/z ratios; and (3) measure the amount of ions with specific m/z ratios. Because proteins (as well as nucleic acids and carbohydrates) decompose upon heating, rather than evaporating, methods to ionize such molecules for mass spectrometry (MS) analysis require innovative approaches. The 2 most prominent MS modes for protein analysis are summarized in Table 5.3. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5

Proteins: Their Primary Structure and Biological Functions

TABLE 5.3 The Two Most Common Methods of Mass Spectrometry for Protein Analysis Electrospray Ionization (ESI-MS) A solution of macromolecules is sprayed in the form of fine droplets from a glass capillary under the influence of a strong electrical field. The droplets pick up positive charges as they exit the capillary; evaporation of the solvent leaves multiply charged molecules. The typical 20 kD protein molecule will pick up 10–30 positive charges. The MS spectrum of this protein reveals all the differently charged species as a series of sharp peaks whose consecutive m/z values differ by the charge and mass of a single proton (see Figure 5.24). Note that decreasing m/z values signify increasing number of charges per molecule, z. Tandem mass spectrometers downstream from the ESI source (ESI-MS/MS) can analyze complex protein mixtures (such as tryptic digests of proteins or chromatographically separated proteins emerging from a liquid chromatography column), selecting a single m/z species for collision-induced dissociation and acquisition of amino acid sequence information. Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS) The protein sample is mixed with a chemical matrix that includes a light-absorbing substance excitable by a laser. A laser pulse is used to excite the chemical matrix, creating a microplasma that transfers the energy to protein molecules in the sample, ionizing them and ejecting them into the gas phase. Among the products are protein molecules that have picked up a single proton. These positively charged species can be selected by the MS for mass analysis. MALDI-TOF MS is very sensitive and very accurate; as little as attomole (10−18 moles) quantities of a particular molecule can be detected at accuracies better than 0.001 atomic mass units (0.001 D). MALDI-TOF MS is best suited for very accurate mass measurements.

Figure 5.23 illustrates the basic features of electrospray mass spectrometry (ESI-MS). In this technique, the high voltage at the electrode causes proteins to pick up protons from the solvent, such that, on average, individual protein molecules acquire about 1 positive charge (proton) per kilodalton, leading to the spectrum of m/z ratios for a single protein Countercurrent

Glass capillary

++ + + + + + + + + ++ +

+ +

Sample solution

+

+ + +

Mass spectrometer

(c) (a) High voltage

Vacuum (b) interface FIGURE 5.23 The 3 principal steps in electrospray ionization mass spectrometry (ESI-MS). (a) Small, highly charged droplets are formed by electrostatic dispersion of a protein solution through a glass capillary subjected to a high electric field; (b) protein ions are desorbed from the droplets into the gas phase (assisted by evaporation of the droplets in a stream of hot N2 gas); and (c) the protein ions are separated in a mass spectrometer and identified according to their m/z ratios. (Adapted from Figure 1 in Mann, M., and Wilm, M., 1995. Electro-spray mass spectrometry for protein characterization. Trends in Biochemical Sciences 20:219–224.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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47342

100

50+ 100

50

40+

75

0 47000 Intensity (%)

120

48000

Molecular weight

30+

50

25

0 800

1000

1200

1400

1600

m/z FIGURE 5.24 Electrospray ionization mass spectrum of the protein aerolysin K. The attachment of many protons per protein molecule (from less than 30 to more than 50 here) leads to a series of m/z peaks for this single protein. The equation describing each m/z peak is m/z = [M + n(mass of proton)]/n(charge on proton), where M = mass of the protein and n = number of positive charges per protein molecule. Thus, if the number of charges per protein molecule is known and m/z is known, M can be calculated. The inset shows a computer analysis of the data from this series of peaks that generates a single peak at the correct molecular mass of the protein. (Adapted from Figure 2 in Mann, M., and Wilm, M., 1995. Electrospray mass spectrometry for protein characterization. Trends in Biochemical Sciences 20:219–224.)

species (Figure 5.24). Computer analysis can convert these data into a single spectrum that has a peak at the correct protein mass (Figure 5.24, inset). Sequencing by Tandem Mass Spectrometry Tandem MS (or MS/MS) allows sequencing of proteins by hooking 2 mass spectrometers in tandem. The first mass spectrometer is used as a filter to sort the oligopeptide fragments in a protein digest based on differences in their m/z ratios. Each of these oligopeptides can then be selected by the mass spectrometer for further analysis. A selected ionized oligopeptide is directed toward the second mass spectrometer; on the way, this oligopeptide is fragmented by collision with helium or argon gas molecules (a process called collision-induced dissociation, or c.i.d.), and the fragments are analyzed by the second mass spectrometer (Figure 5.25). Fragmentation occurs primarily at the peptide bonds linking successive amino acids in the oligopeptide. Thus, the products include a series of fragments that represent a nested set of peptides differing in size by 1  amino acid residue. The various members of this set of fragments differ in mass by 56  atomic mass units [the mass of the peptide backbone atoms (NH!CH!CO)] plus the mass of the R group at each position, which ranges from 1 atomic mass unit (Gly) to 130 (Trp). MS sequencing has the advantages of very high sensitivity, fast sample processing, and the ability to work with mixtures of proteins. Sub-picomoles (less than NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5

Proteins: Their Primary Structure and Biological Functions

10−12 moles) of peptide can be analyzed with these spectrometers. In practice, tandem MS is limited to short sequences (no longer than 15 or so amino acid residues). Nevertheless, capillary HPLC-separated peptide mixtures from trypsin digests of proteins can be directly loaded into the tandem MS spectrometer. Furthermore, separation of a complex mixture of proteins from a whole-cell extract by 2-dimensional gel electrophoresis (see Section 5.3), followed by trypsin digestion of a specific protein spot on the gel and injection of the digest into the HPLC/tandem MS, gives sequence information that can be used to identify specific proteins. Often, by comparing the mass of tryptic peptides from a protein digest with a database of all possible masses for tryptic peptides (based on all known protein and DNA sequences), one can identify a protein of interest without actually sequencing it. Peptide Mass Fingerprinting Peptide mass fingerprinting is used to uniquely identify a protein based on the masses of its proteolytic fragments, usually produced by trypsin digestion. MALDI-TOF MS instruments are ideal for this purpose because they yield highly accurate mass data. The measured masses of the proteolytic fragments can be compared to databases (see following discussion) of peptide masses of known sequence. Such information is easily generated from genomic databases: Nucleotide sequence information can be translated into amino acid sequence information, from which very accurate peptide mass

Electrospray Ionization Tandem Mass Spectrometer

(a)

Electrospray ionization source

MS-1

Collision cell

(b)

Detector

Collision cell

P1 P2

MS-1

MS-2

He gas

P3 P4

MS-2

P5

F1 F2 F3 F4

F5

IS Electrospray ionization

Det

(c)

...

N H

R1

O

C H

C

N H

R2

O

C H

C

N H

R3

O

C H

C

...

Fragmentation at peptide bonds FIGURE 5.25 Tandem mass spectrometry. (a) Configuration used in tandem MS. (b) Schematic description of tandem MS: Tandem MS involves electrospray ionization of a protein digest (IS in this figure), followed by selection of a single peptide ion mass for collision with inert gas molecules (He) and mass analysis of the fragment ions resulting from the collisions. (c) Fragmentation usually occurs at peptide bonds, as indicated. (Adapted from Yates, J. R., 1996. Protein structure analysis by mass spectrometry. Methods in Enzymology 271:351–376; and Gillece-Castro, B. L., and Stults, J. T., 1996. Peptide characterization by mass spectrometry. Methods in Enzymology 271:427–447.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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compilations are readily calculated. For example, the SWISS-PROT database lists 1197 proteins with a tryptic fragment of m"z = 1335.63 (; 0.2 D), 16 proteins with tryptic fragments of m"z = 1335.63 and m"z = 1405.60, but only a single protein [human tissue plasminogen activator (tPA)] with tryptic fragments of m"z = 1335.63, m"z = 1405.60, and m"z = 1272.60.1 Although the identities of many proteins revealed by genomic analysis remain unknown, peptide mass fingerprinting can assign a particular protein exclusively to a specific gene in a genomic database.

Sequence Databases Contain the Amino Acid Sequences of Millions of Different Proteins The first protein sequence databases were compiled by protein chemists using chemical sequencing methods. Today, the vast preponderance of protein sequence information has been derived from translating the nucleotide sequences of genes into codons and, thus, amino acid sequences (see Chapter 27). Sequencing the order of nucleotides in cloned genes is a more rapid, efficient, and informative process than determining the amino acid sequences of proteins by chemical methods. Several electronic databases containing continuously updated sequence information are accessible by personal computer. Prominent among these is the SWISS-PROT protein sequence database on the ExPASy (Expert Protein Analysis System) Molecular Biology server at http://us.expasy.org/ and the PIR (Protein Identification Resource Protein Sequence Database) at http://pir.georgetown.edu/, as well as protein information from genomic sequences available in databases such as GenBank, accessible via the National Center for Biotechnology Information (NCBI) website at www.ncbi.nlm.nih.gov/. The protein sequence databases contain several hundred thousand entries, whereas the genomic databases list nearly 100 million nucleotide sequences covering over 100 gigabases (100 billion bases) from over 165 000 organisms. The Protein Data Bank (PDB; www.rcsb.org/pdb/) is a protein database that provides 3-dimensional structure information on more than 50 000 proteins and nucleic acids.

5.6

What Is the Nature of Amino Acid Sequences?

Figure 5.26 illustrates the relative frequencies of the amino acids in proteins. It is very unusual for a globular protein to have an amino acid composition that deviates substantially from these values. Apparently, these abundances reflect a distribution of amino acid polarities that is optimal for protein stability in an aqueous milieu. Membrane proteins tend to have relatively more hydrophobic and fewer ionic amino acids, a condition consistent with their location. Fibrous proteins may show compositions that are atypical with respect to these norms, indicating an underlying relationship between the composition and the structure of these proteins. Proteins have unique amino acid sequences, and it is this uniqueness of sequence that ultimately gives each protein its own particular personality. Because the number of possible amino acid sequences in a protein is astronomically large, the probability that 2 proteins will, by chance, have similar amino acid sequences is negligible. Consequently, sequence similarities between proteins imply evolutionary relatedness.

Homologous Proteins from Different Organisms Have Homologous Amino Acid Sequences Proteins sharing a significant degree of sequence similarity and structural resemblance are said to be homologous. Proteins that perform the same function in different organisms are also referred to as homologous. For example, the oxygen transport protein hemoglobin serves a similar role and has a similar structure in all vertebrates. The study of the amino 1 The tPA amino acid sequences corresponding to these masses are m/z = 1335.63: HEALSPFYSER; m/z = 1405.60: ATCYEDQGISYR; and m/z = 1272.60:

DSKPWCYVFK.

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Chapter 5

Proteins: Their Primary Structure and Biological Functions

Amino Acid Composition Key: 10

8

Aliphatic

Aromatic (Phe, Trp, Tyr)

Acidic

Amide

Small hydroxy (Ser and Thr)

Sulfur

Basic

%

6

4

2

0 Leu Ala Ser Gly Val Glu Lys Ile Thr Asp Arg Pro Asn Phe Gln Tyr Met His Cys Trp

FIGURE 5.26 Amino acid composition: frequencies of the various amino acids in proteins for all the proteins in the SWISS-PROT protein knowledgebase. These data are derived from the amino acid composition of more than 100 000 different proteins (representing more than 40 000 000 amino acid residues). The range is from leucine at 9.55% to tryptophan at 1.18% of all residues.

acid sequences of homologous proteins from different organisms provides very strong evidence for their evolutionary origin within a common ancestor. Homologous proteins characteristically have polypeptide chains that are nearly identical in length, and their sequences share identity in direct correlation to the relatedness of the species from which they are derived. Homologous proteins can be further subdivided into orthologous and paralogous proteins. Orthologous proteins are proteins from different species that have homologous amino acid sequences (and often a similar function). Orthologous proteins arose from a common ancestral gene during evolution. Paralogous proteins are proteins found within a single species that have homologous amino acid sequences; paralogous proteins arose through gene duplication. For example, the a- and b-globin chains of hemoglobin are paralogs. How is homology revealed?

Computer Programs Can Align Sequences and Discover Homology between Proteins Protein and nucleic acid sequence databases (see page 122) provide enormous resources for sequence comparisons. If 2 proteins share homology, it can be revealed through alignment of their sequences using powerful computer programs. In such studies, a given amino acid sequence is used to query the databases for proteins with similar sequences. BLAST (Basic Local Alignment Search Tool) is one commonly used program for rapid searching of sequence databases. The BLAST program detects local as well as global alignments where sequences are in close agreement. Even regions of similarity shared between otherwise unrelated proteins can be detected. Discovery of sequence similarities between proteins can be an important clue to the function of uncharacterized proteins. Similarities are also useful in assigning related proteins to protein families. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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S. acidocaldarius F P IAKGGTAAIPGPFGSGKTVT L Q SLAKWSAAK – – – VVIYVGCGERGNEMTD E. coli CPFAKGGKVGLFGGAGVGKTVNMMELI R N IAIEHSGYSVFAGVGERTREGND FIGURE 5.27 Alignment of the amino acid sequences of 2 protein homologues using gaps. Shown are parts of the amino acid sequences of the catalytic subunits from the major ATP-synthesizing enzyme (ATP synthase) in a representative archaea (Sulfolobus acidocaldarius) and a bacterium (Escherichia coli). These protein segments encompass the nucleotide-binding site of these enzymes. Identical residues in the 2 sequences are shown in red. Introduction of a 3-residue-long gap in the archaeal sequence optimizes alignment of the 2 sequences.

The process of sequence alignment is an operation akin to sliding 1 sequence along another in a search for regions where the 2 sequences show a good match. Positive scores are assigned everywhere the amino acid in 1 sequence is similar to or identical with the amino acid in the other; the greater the overall score, the better the match between the 2 protein sequences. Sometimes 2 sequences match well at several places along their lengths, but, in 1 of the proteins, the matching segments are interrupted by a sequence that is dissimilar. When such an interruption is found by the computer program, it inserts a gap in the uninterrupted sequence to bring the matching segments of the 2 sequences into better alignment (Figure 5.27 above). Because any 2 sequences would show similarity if a sufficient number of gaps were introduced, a gap penalty is imposed for each gap. Gap penalties are negative numbers that lower the overall similarity score. Gaps arise naturally during evolution through insertion and deletion mutations, so-called indels, which add or remove residues in the gene and, consequently, the protein. The optimal sequence alignment between 2 proteins is one that maximizes sequence alignments while minimizing gaps. Methods for alignment and comparison of protein sequences depend upon some quantitative measure of how similar any 2 sequences are. One way to measure similarity is to use a matrix that assigns scores for all possible substitutions of 1 amino acid for another. BLOSUM62 is the substitution matrix most often used with BLAST. This matrix assigns a probability score for each position in an alignment based on the frequency with which that substitution occurs in the consensus sequences of related proteins. BLOSUM is an acronym for Blocks Substitution Matrix, a matrix that scores each position on the basis of observed frequencies of different amino acid substitutions within blocks of local alignments in related proteins. In the BLOSUM62 matrix, the most commonly used matrix, the scores are derived using sequences sharing no more than 62% identity (Figure 5.28). BLOSUM FIGURE 5.28 The BLOSUM62 substitution matrix provides scores for all possible exchanges of 1 amino acid with another. (From Henikoff, S., and Henikoff, J. G., 1992. Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences, USA 89:10915–10919.)

A 4 R –1 5 N –2 0 6 D –2 –2 1 6 C 0 –3 –3 –3 9 Q –1 1 0 0 –3 5 E –1 0 0 2 –4 2 5 G 0 –2 0 –1 –3 –2 –2 6 H –2 0 1 –1 –3 0 0 –2 8 I –1 –3 –3 –3 –1 –3 –3 –4 –3 4 L –1 –2 –3 –4 –1 –2 –3 –4 –3 2 4 K –1 2 0 –1 –3 1 1 –2 –1 –3 –2 5 M –1 –1 –2 –3 –1 0 –2 –3 –2 1 2 –1 5 F –2 –3 –3 –3 –2 –3 –3 –3 –1 0 0 –3 0 6 P –1 –2 –2 –1 –3 –1 –1 –2 –2 –3 –3 –1 –2 –4 7 S 1 –1 1 0 –1 0 0 0 –1 –2 –2 0 –1 –2 –1 4 T 0 –1 0 –1 –1 –1 –1 –2 –2 –1 –1 –1 –1 –2 –1 1 5 W –3 –3 –4 –4 –2 –2 –3 –2 –2 –3 –2 –3 –1 1 –4 –3 –2 11 Y –2 –2 –2 –3 –2 –1 –2 –3 2 –1 –1 –2 –1 3 –3 –2 –2 2 7 V 0 –3 –3 –3 –1 –2 –2 –3 –3 3 1 –2 1 –1 –2 –2 0 –3 –1 4 A R N D C Q E G H I L K M F P S T W Y V NEL

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Chapter 5

Proteins: Their Primary Structure and Biological Functions

substitution scores range from −4 (lowest probability of substitution) to 11 (highest probability of substitution). For example, to look up the value corresponding to the substitution of an asparagine (N) by a tryptophan (W), or vice versa, find the intersection of the “N” column with the “W” row in Figure 5.28. The value –4 means that the substitution of N for W, or vice versa, is not very likely. On the other hand, the substitution of V for I, (BLOSUM score: 3) or vice versa, is very likely. Amino acids whose side chains have unique qualities (such as C, H, P, or W) have high BLOSUM62 scores because replacing them with any other amino acid may change the protein significantly. Amino acids that are similar (such as R and K, or D and E, or A, V, L, and I) have low scores, since one can replace the other with less likelihood of serious change to the protein structure. Cytochrome c The electron transport protein cytochrome c, found in the mitochondria of all eukaryotic organisms, provides a well-studied example of orthology. Amino acid sequencing of cytochrome c from more than 40 different species has revealed that there are 28 positions in the polypeptide chain where the same amino acid residues are always found (Figure 5.29). These invariant residues serve roles crucial to the biological function of this protein, and thus substitutions of other amino acids at these positions cannot be tolerated. The number of amino acid differences between 2 cytochrome c sequences is proportional to the phylogenetic difference between the species from which they are derived. Cytochrome c in humans and in chimpanzees is identical; human and another mammalian (sheep) cytochrome c differ at 10 residues. The human cytochrome c sequence has 14 variant residues from a reptile sequence (rattlesnake), 18 from a fish (carp), 29 from a mollusc (snail), 31 from an insect (moth), and more than 40 from yeast or higher plants (cauliflower). The Phylogenetic Tree for Cytochrome c Figure 5.30 displays a phylogenetic tree (a diagram illustrating the evolutionary relationships among a group of organisms) constructed from the sequences of cytochrome c. The tips of the branches are occupied by contemporary species whose sequences have been determined. The tree has been deduced by computer analysis of these sequences to find the minimum number of mutational changes connecting the branches. Other computer methods can be used to infer potential ancestral sequences represented by nodes, or branch points, in the tree. Such analysis ultimately suggests a primordial cytochrome c sequence lying at the base of the tree. Evolutionary trees constructed in this manner, that is, solely on the basis of amino acid differences occurring in the primary sequence of one selected protein, show remarkable agreement with phylogenetic relationships derived from more classic approaches and have given rise to the field of molecular evolution.

Related Proteins Share a Common Evolutionary Origin Amino acid sequence analysis reveals that proteins with related functions often show a high degree of sequence similarity. Such findings suggest a common ancestry for these proteins. Oxygen-Binding Heme Proteins Myoglobin and the a- and b-globin chains of hemoglobin constitute a set of paralogous proteins. Myoglobin, the oxygen-binding heme protein of muscle, consists of a single polypeptide chain of 153 residues. Hemoglobin, the oxygen transport protein of erythrocytes, is a tetramer composed of 2 !-chains (141 residues each) and 2 "-chains (146 residues each). These globin paralogs—myoglobin, a-globin, and b-globin—share a strong degree of sequence homology (Figure 5.31 on page 127). Human myoglobin and the human a-globin chain show 38 amino acid identities, whereas human a-globin and human b-globin have 64 residues in common. The relatedness suggests an evolutionary sequence of events in which chance mutations led to amino acid substitutions and divergence in primary structure. The ancestral myoglobin gene diverged first, after duplication of a primordial globin gene had given rise to its progenitor and an ancestral hemoglobin gene (Figure 5.32 on page 127). Subsequently, the ancestral hemoglobin gene duplicated to generate the progenitors of the present-day a-globin and b-globin genes. The ability to bind O2 via a heme prosthetic group is retained by all 3 of these polypeptides.

1

Gly

6

Gly

10

Phe

125

Heme 17 18

Cys His

29 30

Gly Pro

32

Leu

34

Gly

38

Arg

41

Gly

45

Gly

48

Tyr

52

Asn

59

Trp

68

Leu

70 71 72 73 74

Asn Pro Lys Lys Tyr

76

Pro

78 79 80

Thr Lys Met

82

Phe

84

Gly

91

100

Arg

FIGURE 5.29 The sequence of cytochrome c from more than 40 different species reveals that 28 residues are invariant. These invariant residues are scattered irregularly along the polypeptide chain, except for a cluster between residues 70 and 80. All cytochrome c polypeptide chains have a cysteine residue at position 17, and all but 1 have another Cys at position 14. These Cys residues serve to link the heme prosthetic group of cytochrome c to the protein.

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Part 1 Building Blocks of the Cell

Human, chimpanzee

Horse Pig, bovine, sheep

Rabbit

2

12.5

Pekin duck

3

6

6.5

4

Bonito

11

2

7.5

Snapping turtle

Tuna

Carp

Fruit fly

Hornworm moth

4

2

6

Silkworm moth

Pigeon

2.5 3

Gray whale

Puget Sound dogfish

13

Gray kangaroo

2.5 Bullfrog

Baker's yeast

6

Dog

14.5

Chicken, turkey

King penguin

3

Debaryomyces kloeckri Candida krusei

Monkey

6

Pacific lamprey

5 Screwworm fly

Neurospora crassa

11

Mungbean

7.5 12

25

5

Wheat

Sesame 2

2 7.5

Castor

4

15 4

12

6 Sunflower

25

Ancestral cytochrome c Human cytochrome c

1

Pro Ala Gly Asp

?

10

Lys Lys Gly Ala Lys Ile Phe

Gly Asp Val Glu Lys Gly Lys

Lys Ile Phe

30

Lys Thr Ile

?

Cys Ala

Met Lys Cys Ser

40

His Lys Thr Gly Pro Asn Leu His Gly Leu

Phe Gly

Arg Lys Thr Gly Gln Ala Pro

80

Pro Gly Thr Lys Met

?

Phe

60

?

Gly Leu

Pro Gly Thr Lys Met Ile Phe Val Gly

Ala Thr Ala Ala Thr Asn Glu

Ile

?

Gly Gln Ala

Arg Lys

? Trp ? Ile Trp Gly

Gly Gly

Gly Gly Lys

Phe Gly

? Ile

?

Gln Cys His Thr Val Glu Lys

His Lys Val Gly Pro Asn Leu His Gly Leu

Ala Asn Lys Asn Lys Gly Ala Asn Lys Asn Lys Gly

?

20

Gln Cys His Thr Val Glu

?

50

Gly Tyr

Ser

Tyr Thr Asp

Gly Tyr

Ser

Tyr Thr Ala

Glu Asn Thr Leu Phe Glu Tyr Leu Glu Asn Pro Lys Glu Asp Thr Leu Met Gln Tyr Leu Glu Asn Pro Lys

Lys Tyr Ile Lys Tyr Pro

70

Lys Lys Lys Lys

?

?

90

Asp Arg

Lys Glu Glu Arg

Ala Asp Leu Ile Ala

Tyr Leu Lys

100 ?

Ala Asp Leu Ile Ala Tyr Leu Lys Lys

FIGURE 5.30 This phylogenetic tree depicts the evolutionary relationships among organisms as determined by the similarity of their cytochrome c amino acid sequences. The numbers along the branches give the amino acid changes between a species and a hypothetical progenitor. Note that extant species are located only at the tips of branches. Below, the sequence of human cytochrome c is compared with an inferred ancestral sequence represented by the base of the tree. Uncertainties are denoted by question marks. (Adapted from Creighton, T. E., 1983. Proteins: Structure and Molecular Properties. San Francisco: W. H. Freeman.) NEL

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Chapter 5 1 Myoglobin Gly Hemoglobin

! Val

Proteins: Their Primary Structure and Biological Functions

127

10 Leu Ser Asp Gly Glu Trp Gln Leu Val Leu Asn Val Trp

20 Gly Lys Val Glu Ala Asp Ile Pro Gly His Gly Gln Glu Val

Leu Ser Pro Ala Asp Lys

" Val His Leu Thr Pro Glu Glu Lys

30 Leu Ile Arg Leu Phe Lys Gly His Pro Glu Leu Glu Arg Met Phe Leu Ser Phe Pro Thr Leu Gly Arg Leu Leu Val Val Tyr Pro Trp

Thr Asn Val Lys

Ala

Ala Trp

Gly Lys Val Gly

Ala His Ala Gly Gln Tyr Gly Ala

Glu Ala

Ser Ala

Ala

Leu Trp

Gly Lys Val Asn

Val Asp Glu Val Gly Gly

Glu Ala

Ser Glu Asp Glu Met Lys Ala His Gly Thr Pro Asp Ala Val Met Gly

60 Ser Glu Ser Ala Asn Pro

Val Thr

40 50 Thr Leu Glu Lys Phe Asp Lys Phe Lys His Leu Lys Thr Lys Thr Tyr Phe Pro His Phe Asp Leu Ser Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp Leu Ser

70

80

Asp Leu Lys Lys His Gly Ala Thr Val Leu Thr Ala Gln Val Lys Gly His Gly Lys Lys Val Ala Asp Ala Lys Val Lys Ala His Gly Lys Lys Val Leu Gly Ala 100 Gln Ser His Ala Thr Lys His Lys Ile Pro Asp Leu His Ala His Lys Leu Arg Val Asp Glu Leu His Cys Asp Lys Leu His Val Asp

Val Lys Tyr Leu Glu Phe Ile Ser Pro Val Asn Phe Lys Leu Leu Ser Pro Glu Asn Phe Arg Leu Leu Gly

130 Asp Phe Gly Ala Asp Ala Gln Gly Ala Met Asn Lys Glu Phe Thr Pro Ala Val His Ala Ser Leu Asp Lys Glu Phe Thr Pro Pro Val Gln Ala Ala Tyr Gln Lys

!-chain of horse methemoglobin

Leu Gly Gly Ile Leu Lys Leu Thr Asn Ala Val Ala Phe Ser Asp Gly Leu Ala

90

Lys Lys Gly His His Glu Ala Glu Ile Lys Pro Leu His Val Asp Asp Met Pro Asn Ala Leu Ser Ala Leu His Leu Asp Asn Leu Lys Gly Thr Phe Ala Thr Leu

Ala Ser Ser

110 Glu Cys Ile Ile Gln Val Leu Gln Ser Lys His Cys Leu Leu His Thr Leu Ala Ala His Asn Val Leu Val Asn Val Leu Ala His His

Gly Ala Lys

120 His Pro Leu Pro Phe Gly

140 150 Ala Leu Glu Leu Phe Arg Lys Asp Met Ala Ser Asn Tyr Lys Glu Leu Gly Phe Gln Gly Phe Leu Ala Ser Val Ser Thr Val Leu Thr Ser Lys Tyr Arg Val Val Ala Gly Val Ala Asn Ala Leu Ala His Lys Tyr His

"-chain of horse methemoglobin

Sperm whale myoglobin

FIGURE 5.31 The amino acid sequences of the globin chains of human hemoglobin and myoglobin show a strong degree of homology. The a- and b-globin chains share 64 residues of their approximately 140 residues in common. Myoglobin and the a-globin chain have 38 amino acid sequence identities. This homology is further reflected in these proteins’ tertiary structure.

Myoglobin

"

Ancestral "-globin

!

Ancestral !-globin

FIGURE 5.32 This evolutionary tree is inferred from the homology between the amino acid sequences of the a-globin, b-globin, and myoglobin chains. Duplication of an ancestral globin gene allowed the divergence of the myoglobin and ancestral hemoglobin genes. Another gene duplication event subsequently gave rise to ancestral a and b forms, as indicated. Gene duplication is an important evolutionary force in creating diversity.

Ancestral hemoglobin

Ancestral globin NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Serine Proteases Whereas the globins provide an example of gene duplication giving rise to a set of proteins in which the biological function has been highly conserved, other sets of proteins united by strong sequence homology show more divergent biological functions. Trypsin, chymotrypsin (see Chapter 13), and elastase are members of a class of proteolytic enzymes called serine proteases because of the central role played by specific serine residues in their catalytic activity. Thrombin, an essential enzyme in blood clotting, is also a serine protease. These enzymes show sufficient sequence homology to conclude that they arose via duplication of a progenitor serine protease gene, even though their substrate preferences are now different.

Apparently Different Proteins May Share a Common Ancestry A more remarkable example of evolutionary relatedness is inferred from sequence homology between hen egg white lysozyme and human milk !-lactalbumin, proteins of different biological activity and origin. Lysozyme (129 residues) and a-lactalbumin (123 residues) are identical at 48 positions. Lysozyme hydrolyzes the polysaccharide wall of bacterial cells, whereas a-lactalbumin regulates milk sugar (lactose) synthesis in the mammary gland. Although both proteins act in reactions involving carbohydrates, their functions show little similarity otherwise. Nevertheless, their tertiary structures are strikingly similar (Figure 5.33). It is conceivable that many proteins are related in this way, but time and the course of evolutionary change erased most evidence of their common ancestry. In contrast to this case, the proteins G-actin and hexokinase (Figure 5.34) share essentially no sequence homology, yet they have strikingly similar 3-dimensional structures, even though their biological roles and physical properties are very different. Actin forms a filamentous polymer that is a principal component of the contractile apparatus in muscle; hexokinase is a cytosolic enzyme that catalyzes the first reaction in glucose catabolism.

A Mutant Protein Is a Protein with a Slightly Different Amino Acid Sequence Given a large population of individuals, a considerable number of sequence variants can be found for a protein. These variants are a consequence of mutations in a gene (base substitutions in DNA) that have arisen naturally within the population. Gene mutations lead to mutant forms of the protein in which the amino acid sequence is altered at 1 or more positions. Many of these mutant forms are “neutral” in that the functional properties of the protein are unaffected by the amino acid substitution. Others may be non-functional FIGURE 5.33 The tertiary structures of hen egg white lysozyme and human a-lactalbumin are very similar.

N N

123 C Human milk !-lactalbumin

C 129 Hen egg white lysozyme NEL

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Chapter 5

Proteins: Their Primary Structure and Biological Functions

(b)

(a)

FIGURE 5.34 The tertiary structures of (a) hexokinase and (b) actin; ADP is bound to both proteins (purple).

(if loss of function is not lethal to the individual), and still others may display a range of aberrations between these 2 extremes. The severity of the effects on function depends on the nature of the amino acid substitution and its role in the protein. These conclusions are exemplified by the hundreds of human hemoglobin variants that have been discovered to date. Some of these are listed in Table 5.4. A variety of effects on the hemoglobin molecule are seen in these mutants, including alterations in oxygen affinity, heme affinity, stability, solubility, and subunit interactions between the a-globin and b-globin polypeptide chains. Some variants show no apparent changes, whereas others, such as HbS, sickle-cell hemoglobin (see Chapter 14), result in serious illness. This diversity of response indicates that some amino acid changes are relatively unimportant, whereas others drastically alter 1 or more functions of a protein.

TABLE 5.4 Some Pathological Sequence Variants of Human Hemoglobin Abnormal Hemoglobin*

Normal Residue and Position

Substitution

Torino

phenylalanine 43

valine

MBoston

histidine 58

tyrosine

Chesapeake

arginine 92

leucine

GGeorgia

proline 95

leucine

Tarrant

aspartate 126

asparagine

Suresnes

arginine 141

histidine

glutamate 6

valine

a-chain

b-chain S Riverdale–Bronx

glycine 24

arginine

Genova

leucine 28

proline

Zurich

histidine 63

arginine

MMilwaukee

valine 67

glutamate

MHyde Park

histidine 92

tyrosine

Yoshizuka

asparagine 108

aspartate

Hiroshima

histidine 146

aspartate

*Hemoglobin

variants are often given the geographical name of their origin. Adapted from Dickerson, R. E., and Geis, I., 1983. Hemoglobin: Structure, Function, Evolution and Pathology. Menlo Park, CA: Benjamin/Cummings. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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A DEEPER LOOK

Bovine Spongiform Encephalopathy (BSE) and Its Effect on the Canadian Economy

Thinkstock

A brief introduction to prions can be found near the end of Chapter 1. We return to prions in Chapter 5 because of their role in transmissible neurodegenerative diseases such as bovine spongiform encephalopathy (BSE, a.k.a. ‘Mad Cow Disease’), Creutzfeldt–Jakob Disease, and scrapie. As previously mentioned, the misfolding of the distinct cellular prion protein PrPc leads to its accumulation and subsequent spongiform change found in brain. The devastating socio-economic impact as a result of the discovery of BSE in the Canadian cattle industry was one that garnered international attention when it was first reported back in May 2003. Within the first year of the initial discovery, losses in the Canadian livestock sector totalled more than $6 billion due to the banned and sanctioned imports on livestock imposed by the majority of its trading partners, including the United States. Despite the fact that the number of BSE cases have dwindled since then, the aftermath of the crisis is still ongoing even after almost 10 years following the initial detection of the first domestic case of BSE in Canada. A Canadian Cow

5.7

Can Polypeptides Be Synthesized in the Laboratory?

Chemical synthesis of peptides and polypeptides of defined sequence can be carried out in the laboratory. Formation of peptide bonds linking amino acids together is not a chemically complex process, but making a specific peptide can be challenging because various functional groups present on side chains of amino acids may also react under the conditions used to form peptide bonds. Furthermore, if correct sequences are to be synthesized, the a-COOH group of residue x must be linked to the a-NH2 group of neighbouring residue y in a way that prevents reaction of the amino group of x with the carboxyl group of y. In essence, any functional groups to be protected from reaction must be blocked while the desired coupling reactions proceed. Also, the blocking groups must be removable later under conditions in which the newly formed peptide bonds are stable. An ingenious synthetic strategy to circumvent these technical problems is orthogonal synthesis. An orthogonal system is defined as a set of distinctly different blocking groups: one for side-chain protection, another for a-amino protection, and a third for a-carboxyl protection or anchoring to a solid support (see following discussion). Ideally, any of the 3 classes of protecting groups can be removed in any order and in the presence of the other 2 because the reaction chemistries of the 3 classes are sufficiently different from one another. In peptide synthesis, all reactions must proceed with high yield if peptide recoveries are to be acceptable. Peptide formation between amino and carboxyl groups is not spontaneous under normal conditions (see Chapter 4), so one or the other of these groups must be activated to facilitate the reaction. Despite these difficulties, biologically active peptides and polypeptides have been recreated by synthetic organic chemistry. Milestones include the pioneering synthesis of the nonapeptide posterior pituitary hormones oxytocin and vasopressin by Vincent du Vigneaud in 1953 and, in later years, larger proteins such as insulin (21 A-chain and 30 B-chain residues), ribonuclease A (124 residues), and HIV protease (99 residues).

Solid-Phase Methods Are Very Useful in Peptide Synthesis Bruce Merrifield and his collaborators pioneered a clever solution to the problem of recovering intermediate products in the course of a synthesis. The carboxyl-terminal residues of synthesized peptide chains are covalently anchored to an insoluble resin (polystyrene particles) that can be removed from reaction mixtures simply by filtration. After each new residue is added successively at the free amino-terminus, the elongated product is recovered by filtration and readied for the next synthetic step. Because the growing peptide chain is coupled to an insoluble resin bead, the method is called solid-phase synthesis. The procedure is detailed in Figure 5.35. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5

131

Proteins: Their Primary Structure and Biological Functions Aminoacylresin particle R1

6

7

5

H3C 8

H CH2

9

4

O O

C

CH3 N

R2

+

NHCHCOOH

NHCHC

Fmoc

N

Fmoc blocking group

O

H3C

C

H3C

DIPCDI (diisopropyl) carbodiimide

1

CHC O

CH3 NH

2

NH

O CH3

H3 C

Incoming blocked amino acid

2

H 2N

CH3 N

R2

C

1 3

H3C

C

CH3

O

NH

Activated amino acid

H3C

CH3

Diisopropylurea CH3 CH3

C

Amino-blocked dipeptidylresin particle

CH3

R2 Fmoc

NHCHCNHCHC O

t Butyl group H 3C

CH3

H3C

N

+

C

H2N

N H3 C

R

O

C

C

OH

H2N

H

O

C

C

CH3

O

CH3 N

3 Base

Fmoc removal

C

O

NH

H H3C

DIPCDI (diisopropyl) carbodiimide

CH3

R2

Activated amino acid

Dipeptide-resin particle

R1

H2NCHCNHCHC O

H 3C R3 Fmoc

R

R1

NHCHCOOH

CH3 R3

N

+

C

4

H3 C

CH3

DIPCDI

CH3 N

NHCHC

O

O

N Incoming blocked amino acid

H 3C

O

H3C

C

NH

NH H3C

C

CH3

O

NH

Activated amino acid

FIGURE 5.35 Solid-phase synthesis of a peptide. The 9-fluorenylmethoxycarbonyl (Fmoc) group is an excellent orthogonal blocking group for the a-amino group of amino acids during organic synthesis because it is readily removed under basic conditions that do not affect the linkage between the insoluble resin and the a-carboxyl group of the growing peptide chain. (inset) N,N¿-diisopropylcarbodiimide (DIPCDI) is one agent of choice for activating carboxyl Amino-blocked Fmoc groups to condense with amino groups to form peptide bonds. (1) The carboxyl group of the first tripeptidylresin particle amino acid (the carboxyl-terminal amino acid of the peptide to be synthesized) is chemically attached to an insoluble resin particle (the amin oacyl-resin particle). (2) The second amino acid, with its amino group blocked by an Fmoc group and its carboxyl group activated with DIPCDI, is reacted with the aminoacyl-resin particle to form a peptide linkage, with elimination of DIPCDI as diisopropylurea. (3) Then, basic treatment (with piperidine) removes the N-terminal Fmoc blocking group, exposing the N-terminus of the dipeptide for another cycle of amino acid addition. (4) Any reactive side chains on amino acids are blocked by addition of acid-labile tertiary butyl (tBu) Tripeptidyl-resin groups as an orthogonal protective function. (5) After each step, the peptide product is recovered particle by collection of the insoluble resin beads by filtration or centrifugation. Following cyclic additions of amino acids, the completed peptide chain is hydrolyzed from linkage to the insoluble resin by treatment with HF; HF also removes any tBu protecting groups from side chains on the peptide.

CH3

H3C

R1

R2

R3

NHCHC NHCHCN HCHC O

O

5 Base

R3

O Fmoc removal R1

R2

H2NCHC NHCHC NHCHC O

O

O

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CH3

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Part 1 Building Blocks of the Cell

This cyclic process is automated and computer controlled so that the reactions take place in a small cup with reagents being pumped in and removed as programmed.

5.8

Do Proteins Have Chemical Groups Other Than Amino Acids?

Many proteins consist of only amino acids and contain no other chemical groups. The enzyme ribonuclease and the contractile protein actin are 2 such examples. Such proteins are called simple proteins. However, many other proteins contain various chemical constituents as an integral part of their structure. Some of these constituents arise through covalent modification of amino acid side chains in proteins after the protein has been synthesized. Such alterations are called post-translational modifications. For example, the reaction of 2 cysteine residues in a protein to form a disulfide linkage (Figure 5.19b) is a post-translational modification. Many of the prominent post-translational modifications, such as those listed in Table 5.5, can act as “on–off switches” that regulate the function or cellular location of the protein. Approximately 500 post-translational modifications are listed in the RESID database accessible through The National Cancer Institute at Frederick Advanced Biomedical Computing Center: www.ncifcrf.gov/RESID/ A common form of post-translational modification not to be found in such a database or in Table 5.5 is the removal of amino acids from the protein by proteolytic cleavage. Many proteins localized in specific subcellular compartments have N-terminal signal sequences that stipulate their proper destination. Such signal sequences typically are clipped off during their journey. Other proteins, such as some hormones or potentially destructive proteases, are synthesized in an inactive form and converted into an active form through proteolytic removal of some of their amino acids. The general term for proteins containing non-protein constituents is conjugated proteins (Table 5.6). Because association of the protein with the conjugated group does not occur until the protein has been synthesized, these associations are post-translational as

TABLE 5.5 Some Prominent Post-translational Modifications Found in Proteins Amino Acid Side Chain Modified

Name

Non-protein Part

Phosphorylation

!PO32−

Acetylation

!CH2COO−

Methylation

!CH3

Acylation

Palmitic acid

C

G protein–coupled receptors

Prenylation

Prenyl group

C

Ras p21

ADP-ribosylation

ADP-ribose

H, R

G proteins, eukaryotic elongation factors

Adenylylation

AMP

Y

Glutamine synthetase

S, T, Y

Examples Hormone receptors, regulatory enzymes

K

Histones

K, R

Histones

TABLE 5.6 Some Common Conjugated Proteins Name

Non-protein Part

Association

Examples

Lipoproteins

Lipids

Non-covalent

Blood lipoprotein complexes (HDL, LDL)

Nucleoproteins

RNA, DNA

Non-covalent

Ribosomes, chromosomes

Glycoproteins

Carbohydrate groups

Covalent

Immunoglobulins, LDL receptor

Metalloproteins and metal-activated proteins

Ca2+, K+, Fe2+, Zn2+, Co2+, others

Covalent to non-covalent

Metabolic enzymes, kinases, phosphatases, among others

Hemoproteins

Heme group

Covalent or non-covalent

Hemoglobin, cytochromes

Flavoproteins

FMN, FAD

Covalent or non-covalent

Electron transfer enzymes NEL

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133

well, although such terminology is usually not applied to these proteins (with the possible exception of glycoproteins). As Table 5.6 indicates, conjugated proteins are typically classified according to the chemistry of the non-protein part. If the non-protein part participates in the protein’s function, it is referred to as a prosthetic group. Conjugation of proteins with these different non-protein constituents dramatically enhances the repertoire of functionalities available to proteins.

5.9

What Are the Many Biological Functions of Proteins?

Proteins are the agents of biological function. Virtually every cellular activity is dependent on 1 or more particular proteins. Thus, a convenient way to classify the enormous number of proteins is to group them according to the biological roles they serve. Figure 5.36 summarizes the classification of proteins found in the human proteome according to their function. Proteins fill essentially every biological role, with the exception of information storage. The ability to bind other molecules (ligands) is common to many proteins. Binding proteins typically interact non-covalently with their specific ligands. Transport proteins are one class of binding proteins. Transport proteins include membrane proteins that

Proteome: The complete catalogue of proteins encoded by a genome. In cell-specific terms, a proteome is the complete set of proteins found in a particular cell type at a particular time.

Cell adhesion (577, 1.9%) Miscellaneous (1318, 4.3%)

Chaperone (159, 0.5%) Cytoskeletal structural protein (876, 2.8%)

Viral protein (100, 0.3%)

Extracellular matrix (437, 1.4%) Immunoglobulin (264, 0.9%)

Transfer/carrier protein (203, 0.7%)

Ion channel (406, 1.3%)

Transcription factor (1850, 6.0%)

None

Motor (376, 1.2%) Structural protein of muscle (296, 1.0%) Protooncogene (902, 2.9%) Select calcium-binding protein (34, 0.1%) Intracellular transporter (350, 1.1%)

id ac c ng i le i uc nd N bi

Nucleic acid enzyme (2308, 7.5%)

Transporter (533, 1.7%)

Signalling molecule (376, 1.2%)

Signal transduction

Receptor (1543, 5.0%)

Kinase (868, 2.8%) Select regulatory molecule (988, 3.2%) Transferase (610, 2.0%)

En m

zy

Synthase and synthetase (313, 1.0%)

e

Oxidoreductase (656, 2.1%) Lyase (117, 0.4%) Ligase (56, 0.2%) Isomerase (163, 0.5%) Hydrolase (1227, 4.0%)

Molecular function unknown (12809, 41.7%)

FIGURE 5.36 Proteins of the human genome grouped according to their molecular function. The numbers and percentages within each functional category are enclosed in parentheses. Note that the function of more than 40% of the proteins encoded by the human genome remains unknown. Considering those of known function, enzymes (including kinases and nucleic acid enzymes) account for about 20% of the total number of proteins, and nucleic acid–binding proteins of various kinds, about 14%, among which almost half are gene-regulatory proteins (transcription factors). Transport proteins collectively constitute about 5% of the total and structural proteins, another 5%. (Adapted from Figure 15 in Venter, J. C., et al., 2001. The sequence of the human genome. Science 291:1304–1351.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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transport substances across membranes, as well as soluble proteins that deliver specific nutrients or waste products throughout the body. Scaffold proteins are a class of binding proteins that uses protein–protein interactions to recruit other proteins into multimeric assemblies whose purpose is to mediate and coordinate the flow of information in cells. Catalytic proteins (enzymes) mediate almost every metabolic reaction. Regulatory proteins that bind to specific nucleotide sequences within DNA control gene expression. Hormones are another kind of regulatory protein in that they convey information about the environment and deliver this information to cells when they bind to specific receptors. Switch proteins such as G proteins can switch between 2 conformational states—an “on” state and an “off ” state—and act, via this conformational switching, as regulatory proteins. Structural proteins give form to cells and subcellular structures. The great diversity in function that characterizes biological systems is based on attributes that proteins possess. All Proteins Function through Specific Recognition and Binding of Some Target Molecule Although the classification of proteins according to function has advantages, many proteins are not assigned readily to 1 of the traditional groupings. Further, classification can be somewhat arbitrary because many proteins fit more than 1 category. However, for all categories, the protein always functions through specific recognition and binding of some other molecule, although for structural proteins, it is usually self-recognition and assembly into stable multimeric arrays. Protein behaviour provides the cardinal example of molecular recognition through structural complementarity, a fundamental principle of biochemistry that was presented in Chapter 1. Protein Binding The interaction of a protein with its target can be described usually in simple quantitative terms. Let’s explore the simplest situation in which a protein has a single binding site for the molecule it binds (its ligand; Chapter 1). If we treat the interaction between the protein (P) and the ligand (L) as a dissociation reaction: PL ∆ P + L. The equilibrium constant for the reaction, written as Keq = [P][L]>[PL], is a dissociation constant because it describes the dissociation of the ligand from the protein. Biochemists typically use dissociation constants (KD) to describe binding phenomena. Because brackets ([ ]) denote molar concentrations, dissociation constants have the units of M. Typically, the ligand concentration is much greater than the protein concentration. Under such conditions, a plot of the moles of ligand bound per mole of protein (defined as [PL]>([P] + [PL])) versus [L] yields a hyperbolic curve known as a saturation curve or binding isotherm (Figure 5.37).

FIGURE 5.37 Saturation curve or binding isotherm.

0.5

#=

[PL] [P]+[PL]

1.0

[L] = K D

0

0

Ligand concentration([L]) NEL

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135

If we define the fractional saturation of P with L, [PL]"([P] + [PL]), as v, a little algebra yields n = [L]>(KD + [L]). Thus, when v = 0.5, [L] = KD. That is, the concentration of L where half the protein has L bound is equal to the value of KD. The smaller this number is, the better the ligand binds to the protein; that is, a small KD means that the protein is half-saturated with L at a low concentration of L. In other words, if KD is small, the protein binds the ligand avidly. Typical KD values fall in a range from 10−3 M to 10−12 M. The Ligand-Binding Site Ligand binding occurs through non-covalent interactions between the protein and ligand. The lack of covalent interactions means that binding is readily reversible. Proteins display specificity in ligand binding because they possess a specific site, the binding site, within their structure that is complementary to the structure of the ligand, its charge distribution, and any H-bond donors or acceptors it might have. Structural complementarity within the binding site is achieved because part of the 3-dimensional structure of the protein provides an ensemble of amino acid side chains (and polypeptide backbone atoms) that establish an interactive cavity complementary to the ligand molecule. When a ligand binds to the protein, the protein usually undergoes a conformational change. This new protein conformation provides an even better fit with the ligand than before. Such changes are called ligand-induced conformational changes, and the result is an even more stable interaction between the protein and its ligand. Thus, in a general sense, most proteins are binding proteins because ligand binding is a hallmark of protein function. Catalytic proteins (enzymes) bind substrates; regulatory proteins bind hormones or other proteins or regulatory sequences in genes; structural proteins bind to and interact with each other; and the many types of transport proteins bind ligands, facilitating their movement from one place to another. Many proteins accomplish their function through the binding of other protein molecules, a phenomenon called protein–protein interaction. Some proteins engage in protein–protein interactions with proteins that are similar or identical to themselves so that an oligomeric structure is formed, as in hemoglobin. Other proteins engage in protein–protein interactions with proteins that are very different from themselves, as in the anchoring proteins or the scaffolding proteins of signalling pathways.

SUMMARY The primary structure (the amino acid sequence) of a protein is encoded in DNA in the form of a nucleotide sequence. Expression of this genetic information is realized when the polypeptide chain is synthesized and assumes its functional, 3-dimensional architecture. Proteins are the agents of biological function. 5.1 What Architectural Arrangements Characterize Protein Structure? Proteins are generally grouped into 3 fundamental structural classes (soluble, fibrous, and membrane) based on their shape and solubility. In more detail, protein structure is described in terms of a hierarchy of organization: Primary (1°) structure: the protein’s amino acid sequence Secondary (2°) structure: regular elements of structure (helices, sheets) within the protein and created by hydrogen bonds Tertiary (3°) structure: the folding of the polypeptide chain in 3-dimensional space Quaternary (4°) structure: the subunit organization of multimeric proteins The 3 higher levels of protein structure form and are maintained exclusively through non-covalent interactions.

5.2 How Are Proteins Isolated and Purified from Cells? Cells contain thousands of different proteins. A protein of choice can be isolated and purified from such complex mixtures by exploiting 2 prominent physical properties: size and electrical charge. A more direct approach is to employ affinity purification strategies that take advantage of the biological function or specific recognition properties of a protein. A typical protein purification strategy will use a series of separation methods to obtain a pure preparation of the desired protein. 5.3 How Are Amino Acid Mixtures Separated? Separation can be achieved on the basis of the relative differences in the physical and chemical characteristics of amino acids, particularly ionization behaviour and solubility characteristics. The methods important for amino acids include separations based on partition properties and separations based on electrical charge. HPLC is the chromatographic technique of choice for most modern biochemists. The very high resolution, excellent sensitivity, and high speed of this technique usually outweigh the disadvantage of relatively low capacity.

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Part 1 Building Blocks of the Cell

5.4 How Is the Amino Acid Analysis of Proteins Performed? Acid treatment of a protein hydrolyzes all the peptide bonds, yielding a mixture of amino acids. Chromatographic analysis of this hydrolysate reveals the amino acid composition of the protein. Proteins vary in their amino acid composition, but most proteins contain at least 1 of each of the 20 common amino acids. To a very rough approximation, proteins contain about 30% charged amino acids and about 30% hydrophobic amino acids (when aromatic amino acids are included in this number), the remaining being polar, uncharged amino acids. 5.5 How Is the Primary Structure of a Protein Determined? The primary structure (amino acid sequence) of a protein can be determined by a variety of chemical and enzymatic methods. Alternatively, mass spectroscopic methods can be used. In the chemical and enzymatic protocols, a pure polypeptide chain whose disulfide linkages have been broken is the starting material. Methods that identify the N-terminal and C-terminal residues of the chain are used to determine which amino acids are at the ends, and then the protein is cleaved into defined sets of smaller fragments using enzymes such as trypsin or chymotrypsin, or chemical cleavage by agents such as cyanogen bromide. The sequences of these products can be obtained by Edman degradation. Edman degradation is a powerful method for stepwise release and sequential identification of amino acids from the N-terminus of the polypeptide. The amino acid sequence of the entire protein can be reconstructed once the sequences of overlapping sets of peptide fragments are known. In mass spectrometry, an ionized protein chain is broken into an array of overlapping fragments. Small differences in the masses of the individual amino acids lead to small differences in the masses of the fragments, and the ability of mass spectrometry to measure mass-to-charge ratios very accurately allows computer devolution of the data into an amino acid sequence. The amino acid sequences of about a million different proteins are known. The vast majority of these amino acid sequences were deduced from nucleotide sequences available in genomic databases. 5.6 What Is the Nature of Amino Acid Sequences? Proteins have unique amino acid sequences, and similarity in sequence between proteins implies evolutionary relatedness. Homologous proteins share sequence similarity and show structural resemblance. These relationships can be used to trace evolutionary histories of proteins and the organisms that contain them, and the study of such relationships has given rise to the field of molecular evolution. Related proteins, such as the oxygen-binding proteins of myoglobin and hemoglobin or the serine proteases, share a common evolutionary origin. Sequence variation within a protein arises from mutations that result in amino acid

substitution, and the operation of natural selection on these sequence variants is the basis of evolutionary change. Occasionally, a sequence variant with a novel biological function may appear, upon which selection can operate. 5.7 Can Polypeptides Be Synthesized in the Laboratory? It is possible, although difficult, to synthesize proteins in the laboratory. The major obstacles involve joining desired amino acids to a growing chain using chemical methods that avoid side reactions and the creation of undesired products, such as the modification of side chains or the addition of more than 1 residue at a time. Solid-state techniques along with orthogonal protection methods circumvent many of these problems, and polypeptide chains having more than 100 amino acid residues have been artificially created. 5.8 Do Proteins Have Chemical Groups Other Than Amino Acids? Although many proteins are composed of just amino acids, other proteins undergo post-translational modifications to certain amino acid side chains. These modifications often regulate the function of those proteins. In addition, many proteins are conjugated with various other chemical components, including carbohydrates, lipids, nucleic acids, metal and other inorganic ions, and a host of novel structures such as heme or flavin. Association with these non-protein substances dramatically extends the physical and chemical properties that proteins possess, in turn creating a much greater repertoire of functional possibilities. 5.9 What Are the Many Biological Functions of Proteins? Proteins are the agents of biological function. Their ability to bind various ligands is intimately related to their function and thus forms the basis of most classification schemes. Transport proteins bind molecules destined for transport across membranes or around the body. Enzymes bind the reactants unique to the reactions they catalyze. Regulatory proteins are of 2 general sorts: those that bind small molecules that are physiological or environmental cues, such as hormone receptors, or those that bind to DNA and regulate gene expression, such as transcription activators. These are just a few prominent examples. Indeed, the great diversity in function that characterizes biological systems is based on the attributes that proteins possess. Proteins usually interact non-covalently with their ligands, and often the interaction can be defined in simple, quantitative terms by a protein–ligand dissociation constant. Proteins display specificity in ligand binding because the structure of the protein’s ligand-binding site is complementary to the structure of the ligand. Some proteins act through binding other proteins. Such protein–protein interactions lie at the heart of many biological functions.

PROBLEMS Login to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. The element molybdenum (atomic weight 95.95) constitutes 0.08% of the weight of nitrate reductase. If the molecular weight of nitrate reductase is 240 000, what is its likely quaternary structure? 2. Identify the amino acid composition of the following peptide: +

NH3

H

O

C

C

NH

H

O

C

C

CH2

CH2

CH2

OH

NH

H

O

C

C

CH2

NH

H

O

C

CO

CH2

3. How many fragments will be generated from the following polypeptide if it is first treated with trypsin, followed by cyanogen bromide, and lastly by chymotrypsin?

MTSRAVLYFLKHINDRM 4. Amino acid analysis of an oligopeptide 7 residues long gave

Asp

Leu

Lys

Met

Phe

Tyr

The following facts were observed: a. Trypsin treatment had no apparent effect. b. The phenylthiohydantoin released by Edman degradation was

S CH3

OH NEL

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Chapter 5 O C C

C

CH2

N

2 Ala H

S

1 Arg

1 Asp

H3+N

C

Leu

Lys

Met

Tyr

Trp

NH4+

N C

C

CH2

2 Arg

OH

Glu

Met

Pro

Lys

Tyr Ser

C

Phe

C

H

N H

9. Amino acid analysis of an octapeptide gave the following results:

1 Ala

1 Arg

1 Asp

1 Gly

3 Ile

NH4+

1 Val

The following facts were observed: a. Trypsin treatment yielded a pentapeptide and a tripeptide. b. Chemical reduction of the free a-COOH and subsequent acid hydrolysis yielded 2-aminopropanol. c. Partial acid hydrolysis of the tryptic pentapeptide yielded, among other products, 2 dipeptides, each of which contained C-terminal isoleucine. One of these dipeptides migrated as an anionic species upon electrophoresis at neutral pH. d. The tryptic tripeptide was degraded in an Edman sequenator, yielding first A, then B: O

H C

N

What is the amino acid sequence of this decapeptide?

1 Lys

What is the amino acid sequence of this octapeptide?

CH2OH H

1 Phe

b. CNBr treatment yielded a pentapeptide and a tripeptide containing phenylalanine. c. Chymotrypsin treatment yielded a tetrapeptide containing 1 C-terminal indole amino acid and 2 dipeptides. d. Trypsin treatment yielded a tetrapeptide, a dipeptide, and free Lys and Phe. e. Clostripain yielded a pentapeptide, a dipeptide, and free Phe.

H C

1 Tyr

H

S

Arg

The following facts were observed: a. Carboxypeptidase A or B treatment of the decapeptide had no effect. b. Trypsin treatment yielded 2 tetrapeptides and free Lys. c. Clostripain treatment yielded a tetrapeptide and a hexapeptide. d. Cyanogen bromide treatment yielded an octapeptide and a dipeptide of sequence NP (using the 1-letter codes). e. Chymotrypsin treatment yielded 2 tripeptides and 1 tetrapeptide. The N-terminal chymotryptic peptide had a net charge of –1 at neutral pH, and a net charge of –3 at pH 12. f. One cycle of Edman degradation gave the PTH derivative

S

1 Trp

C

6. Amino acid analysis of a decapeptide revealed the presence of the following products:

N

1 Met

N

What is the amino acid sequence of this heptapeptide?

C

1 Gly

C

c. Brief chymotrypsin treatment yielded several products, including a dipeptide and a tetrapeptide. The amino acid composition of the tetrapeptide was Glx, Leu, Lys, and Met. d. Cyanogen bromide treatment yielded a tetrapeptide that had a net positive charge at pH 7 and a tripeptide that had a zero net charge at pH 7.

Asp

COOH

H

O

NH4+

C

The following facts were observed: a. Edman degradation gave

H

O

N

8. Amino acid analysis of an octapeptide revealed the following composition:

N

S

C

What is the amino acid sequence of this octapeptide?

H C

CH3

b. Chymotrypsin treatment of the octapeptide yielded 2 tetrapeptides, each containing an alanine residue. c. Trypsin treatment of 1 of the tetrapeptides yielded 2 dipeptides. d. Cyanogen bromide treatment of another sample of the same tetrapeptide yielded a tripeptide and free Tyr. e. End-group analysis of the other tetrapeptide gave Asp.

(NH4+ is released by acid hydrolysis of N and/or Q amides.) The following facts were observed: a. Trypsin had no effect. b. The phenylthiohydantoin released by Edman degradation was

NH4+

1 Val

CH

H

5. Amino acid analysis of another heptapeptide gave

Glu

2 Tyr

H3C CH3 O

What is the amino acid sequence of this heptapeptide?

Asp

1 Met

The following facts were observed: a. Partial acid hydrolysis of the octapeptide yielded a dipeptide of the structure

c. Brief chymotrypsin treatment yielded several products, including a dipeptide and a tetrapeptide. The amino acid composition of the tetrapeptide was Leu, Lys, and Met. d. Cyanogen bromide treatment yielded a dipeptide, a tetrapeptide, and free Lys.

O

137

7. Analysis of the blood of a catatonic football fan revealed large concentrations of a psychotoxic octapeptide. Amino acid analysis of this octapeptide gave the following results:

H

N

Proteins: Their Primary Structure and Biological Functions

A.

N C S

O

H CH3

C

C

N

CH3 H

H C

B.

N C S

H CH2

C

C

N

CH3 H

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CH3

138

Part 1 Building Blocks of the Cell

What is an amino acid sequence of the octapeptide? Four sequences are possible, but only one suits the authors. Why? 10. An octapeptide consisting of 2 Gly, 1 Lys, 1 Met, 1 Pro, 1 Arg, 1 Trp, and 1 Tyr was subjected to sequence studies. The following was found: a. Edman degradation yielded O

H C

N C

C

What is the amino acid sequence of this nonapeptide?

H

12. Describe the synthesis of the dipeptide Lys-Ala by Merrifield’s solidphase chemical method of peptide synthesis. What pitfalls might be encountered if you attempted to add a leucine residue to Lys-Ala to make a tripeptide?

N

S

H

b. Upon treatment with carboxypeptidases A, B, and C, only carboxypeptidase C had any effect. c. Trypsin treatment gave 2 tripeptides and 1 dipeptide. d. Chymotrypsin treatment gave 2 tripeptides and 1 dipeptide. Acid hydrolysis of the dipeptide yielded only Gly. e. Cyanogen bromide treatment yielded 2 tetrapeptides. f. Clostripain treatment gave a pentapeptide and a tripeptide. What is the amino acid sequence of this octapeptide? 11. Amino acid analysis of an oligopeptide containing 9 residues revealed the presence of the following amino acids:

Arg

Cys

Gly

Leu

Met

Pro

Tyr

Val

The following was found: a. Carboxypeptidase A treatment yielded no free amino acid. b. Edman analysis of the intact oligopeptide released O

H C

N C S

C N

C

13. Electrospray ionization mass spectrometry (ESI-MS) of the polypeptide chain of myoglobin yielded a series of m/z peaks (similar to those shown in Figure 5.24 for aerolysin K). Two successive peaks had m/z values of 1304.7 and 1413.2 respectively. Calculate the mass of the myoglobin polypeptide chain from these data. 14. Phosphoproteins are formed when a phosphate group is esterified to an —OH group of a Ser, Thr, or Tyr side chain. At typical cellular pH values, this phosphate group bears 2 negative charges —OP23 - . Compare this side-chain modification to the 20 side chains of the common amino acids found in proteins, and comment on the novel properties that it introduces into side-chain possibilities. 15. A quantitative study of the interaction of a protein with its ligand yielded the following results: Ligand concentration 1 (mM) v (moles of ligand bound per mole of protein)

2

3

4

5

6

9

12

0.28 0.45 0.56 0.60 0.71 0.75 0.79 0.83

Plot a graph of [L] versus v. Determine KD, the dissociation constant for the interaction between the protein and its ligand, from the graph.

H CH2

d. CNBr treatment of the 8-residue fragment left after combined trypsin and chymotrypsin action yielded a 6-residue fragment containing Cys, Gly, Pro, Tyr, and Val; and a dipeptide. e. Treatment of the 6-residue fragment with b-mercaptoethanol yielded 2 tripeptides. Brief Edman analysis of the tripeptide mixture yielded only PTH-Cys. (The sequence of each tripeptide, as read from the N-terminal end, is alphabetical if the 1-letter designation for amino acids is used.)

CH3

CH3 H

c. Neither trypsin nor chymotrypsin treatment of the nonapeptide released smaller fragments. However, combined trypsin and chymotrypsin treatment liberated free Arg.

Biochemistry on the Web 16. The human insulin receptor substrate-1 (IRS-1) is designated protein P35568 in the protein knowledge base on the ExPASy website (http:// us.expasy.org/). Go to the PeptideMass tool on this website and use it to see the results of trypsin digestion of IRS-1. How many amino acids does IRS-1 have? What is the average molecular mass of IRS-1? What is the amino acid sequence of the tryptic peptide of IRS-1 that has a mass of 1741.9629?

FURTHER READING General References on Protein Structure and Function Creighton, T. E., 1983. Proteins: Structure and Molecular Properties. San Francisco: W. H. Freeman and Co. Creighton, T. E., Ed., 1997. Protein Function—A Practical Approach, 2nd ed. Oxford: CRI. Press at Oxford University Press. Fersht, A., 1999. Structure and Mechanism in Protein Science. New York: W. H. Freeman and Co. Goodsell, D. S., and Olson, A. J., 1993. Soluble proteins: Size, shape and function. Trends in Biochemical Sciences 18:65–68.

Dennison, C., 1999. A Guide to Protein Isolation. Norwell, MA: Kluwer Academic Publish. Separation Methods Heiser, T., 1990. Amino acid chromatography: The “best” technique for student labs. Journal of Chemical Education 67:964–966. Mabbott, G., 1990. Qualitative amino acid analysis of small peptides by GC/MS. Journal of Chemical Education 67:441–445. Moore, S., Spackman, D., et al., 1958. Chromatography of amino acids on sulfonated polystyrene resins. Analytical Chemistry 30:1185–1190.

Lesk, A. M., 2001. Introduction to Protein Architecture: The Structural Biology of Proteins. Oxford: Oxford University Press.

Amino Acid Sequence Analysis

Petsko, G. A., and Ringe, D., 2004. Protein Structure and Function. Sunderland, MA: Sinauer Associates.

Dahoff, M. O., 1972–1978. The Atlas of Protein Sequence and Structure, Vols. 1–5. Washington, DC: National Medical Research Foundation.

Protein Purification

Hsieh, Y. L., Wang, H.-Q., et al., 1996. Automated analytical system for the examination of protein primary structure. Analytical Chemistry 68:455–462. [An analytical system is described in which a protein is

Ahmed, H., 2005. Principles and Reactions of Protein Extraction. Boca Raton, FL: CRC Press.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 5

purified by affinity chromatography, digested with trypsin, and its peptides separated by HPLC and analyzed by tandem MS in order to determine its amino acid sequence.] Karger, B. L., and Hancock, W. S., Eds. 1996. High resolution separation and analysis of biological macromolecules. Part B: Applications. Methods in Enzymology 271. New York: Academic Press. [Sections on liquid chromatography, electrophoresis, capillary electrophoresis, mass spectrometry, and interfaces between chromatographic and electrophoretic separations of proteins followed by mass spectrometry of the separated proteins.] von Heijne, G., 1987. Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit? San Diego, CA: Academic Press. Mass Spectrometry Bienvenut, W. V., 2005. Introduction: Proteins analysis using mass spectrometry. In: Acceleration and Improvement of Protein Identification by Mass Spectrometry, pp. 1–138. Norwell, MA: Springer. Burlingame, A. L., Ed., 2005. Biological mass spectrometry. Methods in Enzymology 402. New York: Academic Press. Hamdan, M., and Gighetti, P. G., 2005. Proteomics Today. Hoboken, NJ: John Wiley & Sons. Hernandez, H., and Robinson, C. V., 2001. Dynamic protein complexes: Insights from mass spectrometry. Journal of Biological Chemistry 276:46685–46688. [Advances in mass spectrometry open a new view onto the dynamics of protein function, such as protein–protein interactions and the interaction between proteins and their ligands.]

Proteins: Their Primary Structure and Biological Functions

139

Liebler, D. C., 2002. Introduction to Proteomics. Towata, NJ: Humana Press. [An excellent primer on proteomics, protein purification methods, sequencing of peptides and proteins by mass spectrometry, and identification of proteins in a complex mixture.] Mann, M., and Wilm, M., 1995. Electrospray mass spectrometry for protein characterization. Trends in Biochemical Sciences 20:219–224. [A review of the basic application of mass spectrometric methods to the analysis of protein sequence and structure.] Quadroni, M., Staudenmann, W., et al., 1996. Analysis of global responses by protein and peptide fingerprinting of proteins isolated by twodimensional electrophoresis. Application to sulfate-starvation response of Escherichia coli. European Journal of Biochemistry 239:773–781. [This paper describes the use of tandem MS in the analysis of proteins in cell extracts.] Vestling, M. M., 2003. Using mass spectrometry for proteins. Journal of Chemical Education 80:122–124. [A report on the 2002 Nobel Prize in Chemistry honouring the scientists who pioneered the application of mass spectrometry to protein analysis.] Solid-Phase Synthesis of Proteins Aparicio, F., 2000. Orthogonal protecting groups for N-amino and C-terminal carboxyl functions in solid-phase peptide synthesis. Biopolymers 55:123–139. Fields, G. B. Ed., 1997. Solid-phase peptide synthesis. Methods in Enzymology 289. San Diego, CA: Academic Press. Merrifield, B., 1986. Solid phase synthesis. Science 232:341–347.

Hunt, D. F., Shabanowitz, J., et al., 1987. Tandem quadrupole Fouriertransform mass spectrometry of oligopeptides and small proteins. Proceedings of the National Academy of Sciences, U.S.A. 84:620–623.

Wilken, J., and Kent, S. B. H., 1998. Chemical protein synthesis. Current Opinion in Biotechnology 9:412–426.

Johnstone, R. A. W., and Rose, M. E., 1996. Mass Spectrometry for Chemists and Biochemists, 2nd ed. Cambridge, England: Cambridge University Press.

Dudas, S., Yang, J., et al., 2010. Molecular, biochemical and genetic characteristics of BSE in Canada. PLoS ONE 5:1–8.

Kamp, R. M., Cakvete, J. J., and Choli-Papadopoulou, T., Eds., 2004. Methods in Proteome and Protein Analysis. New York: Springer. Karger, B. L., and Hancock, W. S., Eds. 1996. High resolution separation and analysis of biological macromolecules. Part A: Fundamentals. Methods in Enzymology 270. New York: Academic Press. [Separate sections discussing liquid chromatography, columns and instrumentation, electrophoresis, capillary electrophoresis, and mass spectrometry.]

Prions and BSE

Mitra, D., Amaratunga, C., et al., 2009. The psychosocial and socioeconomic consequences of bovine spongiform encephalopathy (BSE): A community impact study. Journal of Toxicology and Environmental Health, Part A 72:1106–1112. Mitura, V., and Di Pietro, L., 2004. Canada’s beef cattle sector and the impact of BSE on farm family income 2000–2003. Agriculture and Rural Working Paper Series, Working paper 69:1–35. Watts, J.C., Balachandran, A., et al., 2006. The expanding universe of prion disease. PLoS Pathogens 2:152–163.

Kinter, M., and Sherman, N. E., 2001. Protein Sequencing and Identification Using Tandem Mass Spectrometry. Hoboken, NJ: Wiley-Interscience.

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6

Proteins: Secondary, Tertiary, and Quaternary Structure

! Like the Greek sea god Proteus, who could

National Archaeological Museum, Athens, Greece/Bridgeman Art Library

assume different forms, proteins act through changes in conformation. Proteins (from the Greek proteios, meaning “primary”) are the primary agents of biological function. (“Proteus, Old Man of the Sea; Roman period mosaic, from Thessaloniki, 1st century CE. National Archaeological Museum, Athens/ Ancient Art and Architecture Collection)

Growing in size and complexity Living things, masses of atoms, DNA, protein Dancing a pattern ever more intricate. Out of the cradle onto the dry land Here it is standing Atoms with consciousness Matter with curiosity. Stands at the sea Wonders at wondering I A universe of atoms An atom in the universe. Richard P. Feynman (1918–1988) From “The Value of Science” in Edward Hutchings, Jr., Ed., 1958. Frontiers of Science: A Survey. New York: Basic Books.

KEY QUESTIONS 6.1

What Non-covalent Interactions Stabilize Protein Structure?

6.2

What Role Does the Amino Acid Sequence Play in Protein Structure?

6.3

What Are the Elements of Secondary Structure in Proteins, and How Are They Formed?

6.4

How Do Polypeptides Fold into Three-Dimensional Protein Structures?

6.5

How Do Protein Subunits Interact at the Quaternary Level of Protein Structure?

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

ESSENTIAL QUESTION Linus Pauling received the Nobel Prize in Chemistry in 1954. The award cited “his research into the nature of the chemical bond and its application to the elucidation of the structure of complex substances.” Pauling pioneered the study of secondary structure in proteins. How do the forces of chemical bonding determine the formation, stability, and myriad functions of proteins?

WHY IT MATTERS? Before we begin our discussion of proteins and their structures, we pose the question, “Why does protein structure matter?” All cells rely on particular proteins to carry out their biological functions. One can then appreciate the destructive outcome on cellular function if these proteins were not assembled and/or folded properly into the functioning state. As we will see, even a single amino acid mutation in a protein’s sequence can have catastrophic effects on its structure and hence function. Nearly all biological processes involve the specialized functions of 1 or more protein molecules. Proteins function to produce other proteins, control all aspects of cellular metabolism, regulate the movement of various molecular and ionic species across membranes, convert and store cellular energy, and carry out many other activities. Essentially all the information required to initiate, conduct, and regulate each of these functions must be contained in the structure of the protein itself. The previous chapter described the details of protein primary structure. However, proteins do not normally exist as fully extended polypeptide chains but rather as compact structures that biochemists refer to as “folded.” The ability of a particular protein to carry out its function in nature is normally determined by its overall 3-dimensional shape, or conformation. NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

141

" A mutation to a residue in the protein’s

core, or one that disrupts either a region of secondary structure or a disulphide bond, can also prevent the protein folding, possibly leading to aggregation. (Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Genetics 9, Understanding the molecular machinery of genetics through 3D structures Roman A. Laskowski & Janet M. Thornton 141–151 (February 2008).)

This chapter reveals and elaborates upon the exquisite beauty of protein structures. What will become apparent in this discussion is that the 3-dimensional structure of proteins and their biological function are linked by several overarching principles: 1. 2. 3. 4. 5. 6.

Function depends on structure. Structure depends on both amino acid sequence and weak, non-covalent forces. The number of protein folding patterns is very large but finite. The structures of globular proteins are marginally stable. Marginal stability facilitates motion. Motion enables function.

6.1

What Non-covalent Interactions Stabilize Protein Structure?

The amino acid sequence (primary structure) of any protein is dictated by covalent bonds, but the higher levels of structure—secondary, tertiary, and quaternary—are formed and stabilized by weak, non-covalent interactions (Figure 6.1). Hydrogen bonds, hydrophobic interactions, electrostatic bonds, and van der Waals forces are all non-covalent in nature, yet they are extremely important influences on protein conformation. The stabilization free energies afforded by each of these interactions may be highly dependent on the local environment within the protein, but certain generalizations can still be made. Main chain

Main chain H2O

NH O

Cl–

Na+

HN

O

C

+ – HC CH2CH2CH2CH2NH3 ....... O

HN

Lysine C

O

Na+

Cl–

C

C

O

CH2CH2 CH

Glutamate O

H2O

NH C

FIGURE 6.1 An electrostatic interaction between the !-amino group of a lysine and the g-carboxyl group of a glutamate. The protein is IRAK-4 kinase, an enzyme that phosphorylates other proteins (pdb id = 2NRY). The interaction shown is between Lys213 (left) and Glu233 (right). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

142

Part 1 Building Blocks of the Cell

Hydrogen Bonds Are Formed Whenever Possible Hydrogen bonds are generally made wherever possible within a given protein structure. In most protein structures that have been examined to date, component atoms of the peptide backbone tend to form hydrogen bonds with one another. Furthermore, side chains capable of forming H bonds are usually located on the protein surface and form such bonds either with the water solvent or with other surface residues. The strengths of hydrogen bonds depend to some extent on environment. The difference in energy between a side-chain hydrogen bonded to water and that same side-chain hydrogen bonded to another side chain is usually small. On the other hand, a hydrogen bond in the protein interior, away from bulk solvent, can provide substantial stabilization energy to the protein. Although each hydrogen bond may contribute an average of only a few kilojoules per mole in stabilization energy for the protein structure, the number of H bonds formed in the typical protein is very large. For example, in a-helices, the C “ O and N"H groups of every interior residue participate in H bonds. The importance of H bonds in protein structure cannot be overstated.

Hydrophobic Interactions Drive Protein Folding Hydrophobic “bonds,” or, more accurately, interactions, form because non-polar side chains of amino acids and other non-polar solutes prefer to cluster in a non-polar environment rather than to intercalate in a polar solvent such as water. The forming of hydrophobic “bonds” minimizes the interaction of non-polar residues with water and is therefore highly favourable. Such clustering is entropically driven, and it is in fact the principal impetus for protein folding. The side chains of the amino acids in the interior or core of the protein structure are almost exclusively hydrophobic. Polar amino acids are much less common in the interior of a protein, but the protein surface may consist of both polar and non-polar residues.

Ionic Interactions Usually Occur on the Protein Surface Ionic interactions arise either as electrostatic attractions between opposite charges or repulsions between like charges. Chapter 4 discusses the ionization behaviour of amino acids. Amino acid side chains can carry positive charges, as in the case of lysine, arginine, and histidine, or negative charges, as in aspartate and glutamate. In addition, the N-terminal and C-terminal residues of a protein or peptide chain usually exist in ionized states and carry positive or negative charges respectively. All of these may experience ionic interactions in a protein structure. Charged residues are normally located on the protein surface, where they may interact optimally with the water solvent. It is energetically unfavourable for an ionized residue to be located in the hydrophobic core of the protein. Ionic interactions between charged groups on a protein surface are often complicated by the presence of salts in the solution. For example, the ability of a positively charged lysine to attract a nearby negative glutamate may be weakened by dissolved salts such as NaCl (Figure 6.1). The Na+ and Cl- ions are highly mobile, compact units of charge, compared to the amino acid side chains, and thus compete effectively for charged sites on the protein. In this manner, ionic interactions among amino acid residues on protein surfaces may be damped out by high concentrations of salts. Nevertheless, these interactions are important for protein stability.

Van der Waals Interactions Are Ubiquitous Both attractive forces and repulsive forces are included in van der Waals interactions. The attractive forces are due primarily to instantaneous dipole-induced dipole interactions that arise because of fluctuations in the electron charge distributions of adjacent non-bonded atoms. Individual van der Waals interactions are weak ones (with stabilization energies of 0.4–4.0 kJ/mol), but many such interactions occur in a typical protein, and by sheer force of numbers, they can represent a significant contribution to the stability of a protein. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

Peter Privalov and George Makhatadze have shown that, for pancreatic ribonuclease A, hen egg white lysozyme, horse heart cytochrome c, and sperm whale myoglobin, van der Waals interactions between tightly packed groups in the interior of the protein are a major contribution to protein stability.

6.2

What Role Does the Amino Acid Sequence Play in Protein Structure?

It can be inferred from the first section of this chapter that many different forces work together in a delicate balance to determine the overall 3-dimensional structure of a protein. These forces operate both within the protein structure itself and between the protein and the water solvent. How, then, does nature dictate the manner of protein folding to generate the 3-dimensional structure that optimizes and balances these many forces? All the information necessary for folding the peptide chain into its “native” structure is contained in the amino acid sequence of the peptide. Just how proteins recognize and interpret the information that is stored in the amino acid sequence is not yet well understood. Certain loci along the peptide chain may act as nucleation points, which initiate folding processes that eventually lead to the correct structures. Regardless of how this process operates, it must take the protein correctly to the final native structure. Along the way, local energy-minimum states different from the native state itself must be avoided. A long-range goal of many researchers in the protein structure field is the prediction of 3-dimensional conformation from the amino acid sequence. As the details of secondary and tertiary structure are described in this chapter, the complexity and immensity of such a prediction will be more fully appreciated. This area is one of the greatest uncharted frontiers remaining in molecular biology.

6.3

What Are the Elements of Secondary Structure in Proteins, and How Are They Formed?

Any discussion of protein folding and structure must begin with the peptide bond, the fundamental structural unit in all proteins. As we saw in Chapter 4, the resonance structures experienced by a peptide bond constrain 6 atoms—the oxygen, carbon, nitrogen, and hydrogen atoms of the peptide group, as well as the adjacent a-carbons—to lie in a plane. The resonance stabilization energy of this planar structure is approximately 88 kJ/mol, and substantial energy is required to twist the structure about the C"N bond. A twist of u degrees involves a twist energy of 88 sin2u kJ/mol.

All Protein Structure Is Based on the Amide Plane The planarity of the peptide bond means that there are only two degrees of freedom per residue for the peptide chain. Rotation is allowed about the bond linking the a-carbon and the carbon of the peptide bond and also about the bond linking the nitrogen of the peptide bond and the adjacent a-carbon. As shown in Figure 6.2, each a-carbon is the joining point for 2 planes defined by peptide bonds. The angle about the Ca—N bond is denoted by the Greek letter f (phi), and that about the Ca—Co is denoted by c (psi). For either of these bond angles, a value of 0° corresponds to an orientation with the amide plane bisecting the H—Ca—R (side chain) angle and a cis conformation of the main chain around the rotating bond in question (Figure 6.3). The entire path of the peptide backbone in a protein is known if the f and c rotation angles are all specified. Some values of f and c are not allowed due to steric interference between non-bonded atoms. As shown in Figure 6.3, values of f = 180° and c = 0° are not allowed because of the forbidden overlap of the N—H hydrogens. Similarly, f = 0° and c = 180° are forbidden because of unfavourable overlap between the carbonyl oxygens. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

FIGURE 6.2 The amide or peptide bond planes are joined by the tetrahedral bonds of the a-carbon. The rotation parameters are f and c. The conformation shown corresponds to f = 180° and c = 180°. Note that positive values of f and c correspond to clockwise rotation as viewed from Ca. Starting from 0°, a rotation of 180° in the clockwise direction (+180°) is equivalent to a rotation of 180° in the counterclockwise direction (-180°). (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.)

C Amide plane N

O

H

C H

#-Carbon

C H

R

N

Side group

C O C

Amide plane ! = 180°, " =180°

G. N. Ramachandran and his co-workers in Madras, India, demonstrated that it was convenient to plot f values against c values to show the distribution of allowed values in a protein or in a family of proteins. A typical Ramachandran plot is shown in Figure 6.4. Note the clustering of f and c values in a few regions of the plot. Most combinations of f and c are sterically forbidden, and the corresponding regions of the Ramachandran plot are sparsely populated. The combinations that are sterically allowed represent the subclasses of structure described in the remainder of this section.

Cα

Ca

Non-bonded contact radius

ON

Ca N

H

C

Ca H

Non-bonded contact radius

H

Ca

O

H

Ca

C C

Ca

H

R

N

O

C

H

Ca

O

R

N

C

N

H

O

Ca

H

H

O

Cα

Ca

! = –60°, " = 180°

Cα ! = 0°, " = 180°

O

Ca

C

R

H

N

H

N

R C

O

H

Ca

O

N

H

C

N O

Ca

! = 180°, " = 0°

A further ! rotation of 120° removes the bulky carbonyl group as far as possible from the side chain

! = 0°, " = 0°

FIGURE 6.3 Many of the possible conformations about an a-carbon between 2 peptide planes are forbidden because of steric crowding. Several noteworthy examples are shown here. Note: The formal IUPAC-IUB Commission on Biochemical Nomenclature convention for the definition of the torsion angles f and c in a polypeptide chain (Biochemistry 9:3471–3479, 1970) is different from that used here, where the Ca atom serves as the point of reference for both rotations, but the result is the same. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

Parallel $-sheet Collagen triple helix

Antiparallel $-sheet

Proteins: Secondary, Tertiary, and Quaternary Structure

Left-handed #-helix

180 II

+5

C

–4

90

FIGURE 6.4 A Ramachandran diagram showing the sterically reasonable values of the angles f and c. The shaded regions indicate particularly favourable values of these angles. Dots in purple indicate actual angles measured for 1000 residues (excluding glycine, for which a wider range of angles is permitted) in 8 proteins. The lines running across the diagram (numbered +5 through 2 and -5 through -3) signify the number of amino acid residues per turn of the helix; “+” means right-handed helices; “-” means left-handed helices. (After Richardson, J. S., 1981. The anatomy and taxonomy of protein structure. Advances in Protein Chemistry 34:167–339.)

+4

–5

–3

2

! (deg)

#L

0 3

n=2

α π

–90

+3 +5 –4

–180 –180

Right-handed #-helix

+4

–5

–90

0 " (deg)

90

145

180

Closed ring

The Alpha-Helix Is a Key Secondary Structure As noted in Chapter 5, the term secondary structure describes local conformations of the polypeptide that are stabilized by hydrogen bonds. In nearly all proteins, the hydrogen bonds that make up secondary structures involve the amide proton of one peptide group and the carbonyl oxygen of another, as shown in Figure 6.5. These structures tend to form in cooperative fashion and involve substantial portions of the peptide chain. When a number of hydrogen bonds form between portions of the peptide chain in this manner, 2 basic types of structures can result: a-helices and b-pleated sheets. The earliest studies of protein secondary structure were those of William Astbury at the University of Leeds. Astbury carried out X-ray diffraction studies on wool and observed differences between unstretched wool fibres and stretched wool fibres. He proposed that the protein structure in unstretched fibres was a helix (which he called the alpha form). He also proposed that stretching caused the helical structures to uncoil, yielding an extended structure (which he called the beta form). Astbury was the first to propose that hydrogen bonds between peptide groups contributed to stabilizing these structures. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

C C N O

C

O

C N R

C C R

O

N

C C

O

N

FIGURE 6.5 A hydrogen bond between the backbone C “ O of Ala191 and the backbone N"H of Ser147 in the acetylcholine-binding protein of a snail, Lymnaea stagnalis (pdb id = 1I9B).

C

C

In 1951, Linus Pauling, Robert Corey, and their colleagues at the California Institute of Technology summarized a large volume of crystallographic data in a set of dimensions for polypeptide chains. (A summary of data similar to what they reported is shown in Figure 4.14.) With these data in hand, Pauling, Corey, and their colleagues proposed a new model for a helical structure in proteins, which they called the a-helix. The report from Caltech was of particular interest to Max Perutz in Cambridge, England, a crystallographer who was also interested in protein structure. By taking into account a critical but previously ignored feature of the X-ray data, Perutz realized that the a-helix existed in keratin, a protein from hair, and also in several other proteins. Since then, the a-helix has proved to be a fundamentally important peptide structure. Several representations of the a-helix are shown in Figure 6.6. One turn of the helix represents 3.6 amino acid residues. (A single turn of the a-helix involves 13 atoms from the O to the H of the H bond. For this reason, the a-helix is sometimes referred to as the 3.613 helix.) This is in fact the feature that most confused crystallographers before the Pauling and Corey a-helix model. Crystallographers were so accustomed

A DEEPER LOOK

Knowing What the Right Hand and Left Hand Are Doing Certain conventions related to peptide bond angles and the “handedness” of biological structures are useful in any discussion of protein structure. To determine the f and c angles between peptide planes, viewers should imagine themselves at the Ca looking outward and should imagine starting from the f = 0°, c= 0° conformation. From this perspective, positive values of f correspond to clockwise rotations about the Ca—N bond of the plane that includes the adjacent N—H group. Similarly, positive values of c correspond to clockwise

rotations about the Ca—C bond of the plane that includes the adjacent C “ O group. Biological structures are often said to exhibit “right-hand” or “left-hand” twists. For all such structures, the sense of the twist can be ascertained by holding the structure in front of you and looking along the polymer backbone. If the twist is clockwise as one proceeds outward and through the structure, it is said to be right-handed. If the twist is counterclockwise, it is said to be left-handed.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6 (a)

(b)

Proteins: Secondary, Tertiary, and Quaternary Structure

(c)

(d)

α-Carbon Side group

Hydrogen bonds stabilize the helix structure.

The helix can be viewed as a stacked array of peptide planes hinged at the α-carbons and approximately parallel to the helix.

FIGURE 6.6 Four different graphic representations of the a-helix. (a) A stick representation with H bonds as dotted lines, as originally conceptualized in Pauling’s 1960 The Nature of the Chemical Bond. (b) Showing the arrangement of peptide planes in the helix. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.) (c) A space-filling computer graphic presentation. (d) A “ribbon structure” with an inlaid stick figure, showing how the ribbon indicates the path of the polypeptide backbone.

to finding 2-fold, 3-fold, 6-fold, and similar integral axes in simpler molecules that the notion of a non-integral number of units per turn was never taken seriously before Pauling and Corey’s work. Each amino acid residue extends 1.5 Å (0.15 nm) along the helix axis. With 3.6 residues per turn, this amounts to 3.6 * 1.5 Å or 5.4 Å (0.54 nm), of travel along the helix axis per turn. This is referred to as the translation distance or the pitch of the helix. If one ignores side chains, the helix is about 6 Å in diameter. The side chains, extending outward from the core structure of the helix, are removed from steric interference with the polypeptide backbone. As can be seen in Figure 6.6, each peptide carbonyl is hydrogen bonded to the peptide N—H group 4 residues farther up the chain. Note that all the H bonds lie parallel to the helix axis and all the carbonyl groups are pointing in one direction along the helix axis, while the N—H groups are pointing in the opposite direction. Recall that the entire path of the peptide backbone can be known if the f and c twist angles are specified for each residue. The a-helix is formed if the values of f are approximately -60° and the values of f are in the range of -45° to -50°. Figure 6.7 shows the structures of 2 proteins that contain a-helical segments. The number of residues involved in a given a-helix varies from helix to helix and from protein to protein. On average, there are about 10 residues per helix. Myoglobin, one of the first proteins in which a-helices were observed, has 8 stretches of a-helix that form a box to contain the heme prosthetic group (see Figure 5.5).

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Part 1 Building Blocks of the Cell

FIGURE 6.7 The 3-dimensional structures of 2 proteins that contain substantial amounts of a-helix in their structures. The helices are represented by the regularly coiled sections of the ribbon drawings. Myohemerythrin is the oxygen-carrying protein in certain invertebrates, including Sipuncula, a phylum of marine worm. a-Hemoglobin subunit: pdb id = 1HGA; myohemerythrin pdb id = 1A7D. (Jane Richardson.)

(a)

–0.42

O

–

$-Hemoglobin subunit

Dipole moment

+0.42

–0.20 H +

+0.20

C (b)

N

FIGURE 6.8 The arrangement of N"H and C “ O groups (each with an individual dipole moment) along the helix axis creates a large net dipole for the helix. Numbers indicate fractional charges on respective atoms.

Myohemerythrin

As shown in Figure 6.6, all the hydrogen bonds point in the same direction along the a-helix axis. Each peptide bond possesses a dipole moment that arises from the polarities of the N—H and C “ O groups, and because these groups are all aligned along the helix axis, the helix itself has a substantial dipole moment, with a partial positive charge at the N-terminus and a partial negative charge at the C-terminus (Figure 6.8). Negatively charged ligands (e.g., phosphates) frequently bind to proteins near the N-terminus of an a-helix. By contrast, positively charged ligands are only rarely found to bind near the C-terminus of an a-helix. In a typical a-helix of 12 (or n) residues, there are 8 (or n - 4) hydrogen bonds. As shown in Figure 6.9, the first 4 amide hydrogens and the last 4 carbonyl oxygens cannot participate in helix H bonds. Also, non-polar residues situated near the helix termini can be exposed to solvent. Proteins frequently compensate for these problems by helix capping— providing H-bond partners for the otherwise bare N—H and C “ O groups and folding other parts of the protein to foster hydrophobic contacts with exposed non-polar residues at the helix termini. Careful studies of the polyamino acids, polymers in which all the amino acids are identical, have shown that certain amino acids tend to occur in a-helices, whereas others are less likely to be found in them. Polyleucine and polyalanine, for example, readily form a-helical structures. In contrast, polyaspartic acid and polyglutamic acid, which are highly negatively charged at pH 7.0, form only random structures because of strong charge repulsion between the R groups along the peptide chain. At pH 1.5–2.5, however, where the side chains are protonated and thus uncharged, these latter species spontaneously form a-helical structures. In similar fashion, polylysine is a random coil at pH values below about 11, where repulsion of positive charges prevents helix formation. At pH 12, where polylysine is a neutral peptide chain, it readily forms an a-helix. The tendencies of various amino acids to stabilize or destabilize a-helices are different in typical proteins than in polyamino acids. The occurrence of the common amino acids in helices is summarized in Table 6.1. Notably, proline and hydroxyproline act as helix breakers due to their unique structure, which fixes the value of the Ca—N—C bond angle. Helices can be formed from either D- or L-amino acids, but a given helix must be composed entirely of amino acids of one configuration. a-Helices cannot be formed from a mixed copolymer of D- and L-amino acids. An a-helix composed of D-amino acids is left-handed.

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Chapter 6

TABLE 6.1

O

Helix-Forming and Helix-Breaking Behaviour of the Amino Acids

Amino Acid

Helix Behaviour*

A

Ala

H

C

Cys

variable

D

Asp

variable

E

Glu

H

F

Phe

H

G

Gly

I

(B)

H

His

H

(I)

I

Ile

H

(C)

K

Lys

variable

L

Leu

H

M

Met

H

N

Asn

C

P

Pro

B

Q

Gln

H

(I)

R

Arg

H

(I)

S

Ser

C

(B)

T

Thr

variable

V

Val

variable

W

Trp

H

(C)

Y

Tyr

H

(C)

149

Proteins: Secondary, Tertiary, and Quaternary Structure

N

C#8

(I)

H

C#9

C#7 C#5 C#6

3.6 residues C#4

(I)

C#3

C#2 C#1

The B-Pleated Sheet Is a Core Structure in Proteins Another type of structure commonly observed in proteins also forms because of local, cooperative formation of hydrogen bonds. That is the pleated sheet, or b-structure, often called the B-pleated sheet. This structure was also first postulated by Pauling and Corey in 1951 and has now been observed in many natural proteins. A b-pleated sheet can be visualized by laying thin, pleated strips of paper side by side to make a “pleated sheet” of paper (Figure 6.10). Each strip of paper can then be pictured as a single peptide strand in which the peptide backbone makes a zigzag pattern along the strip, with the a-carbons lying at the folds of the pleats. The pleated sheet can exist in both parallel and antiparallel forms. In the parallel B-pleated sheet, adjacent chains run in the same direction. In the antiparallel B-pleated sheet, adjacent strands run in opposite directions. Each single strand of the b-sheet structure can be pictured as a 2-fold helix, that is, a helix with 2 residues per turn. The arrangement of successive amide planes has a pleated appearance due to the tetrahedral nature of the Ca atom. It is important to note that the hydrogen bonds in this structure are essentially interstrand rather than intrastrand. Optimum formation of H bonds in the parallel pleated sheet results in a slightly less extended conformation than in the antiparallel sheet. The H bonds thus formed in the parallel b-sheet are bent significantly. The distance between residues is 0.347 nm for the antiparallel pleated sheet, but only 0.325 nm for the parallel pleated sheet. Note that the side chains in the pleated sheet are oriented perpendicular or normal to the plane of the sheet, extending out from the plane on alternating sides. Parallel b-sheets tend to be more regular than antiparallel b-sheets. As can be seen in Figure 6.4, typical f, c values for a parallel b-sheet are f = -120°, c = 105°, and typical

FIGURE 6.9 Four N—H groups at the N-terminal end of an a-helix and 4 C “ O groups at the C-terminal end lack partners for H-bond formation. The formation of H bonds with other nearby donor and acceptor groups is referred to as helix capping. Capping may also involve appropriate hydrophobic interactions that accommodate non-polar side chains at the ends of helical segments.

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150

Part 1 Building Blocks of the Cell

FIGURE 6.10 A “pleated sheet” of paper with an antiparallel b-sheet drawn on it. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.)

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

In Bed with a Cold, Pauling Stumbles onto the a-Helix and a Nobel Prize* As high technology continues to transform the modern biochemical laboratory, it is interesting to reflect on Linus Pauling’s discovery of the a-helix. It involved only a piece of paper, a pencil, scissors, and a sick Linus Pauling, who had tired of reading detective novels. The story is told in the excellent book The Eighth Day of Creation, by Horace Freeland Judson: From the spring of 1948 through the spring of 1951…rivalry sputtered and blazed between Pauling’s lab and (Sir Lawrence) Bragg’s—over protein. The prize was to propose and verify in nature a general three-dimensional structure for the polypeptide chain. Pauling was working up from the simpler structures of components. In January 1948, he went to Oxford as a visiting professor for two terms, to lecture on the chemical bond and on molecular structure and biological specificity. “In Oxford, it was April, I believe, I caught cold. I went to bed, and read detective stories for a day, and got bored, and thought why don’t I have a crack at that problem of alpha keratin.” Confined, and still fingering the polypeptide chain in his mind, Pauling called for paper, pencil, and straightedge and attempted to reduce the problem to an almost Euclidean purity. “I took a sheet of paper—I still have this sheet of paper—and drew, rather roughly, the way that I thought a polypeptide chain would look if it were spread out into a plane.” The repetitious herringbone of the chain he could stretch across the paper as simply as this—

—putting in lengths and bond angles from memory…. He knew that the peptide bond, at the carbon-to-nitrogen link, was always rigid: (b) O C

αC

H

R

H N H

αC

H

R C O

N

O C

αC

H

R

H N H

αC

H

R C O

N

αC

H

R

And this meant that the chain could turn corners only at the alpha carbons…. “I creased the paper in parallel creases through the alpha carbon atoms, so that I could bend it and make the bonds to the alpha carbons, along the chain, have tetrahedral value. And then I looked to see if I could form hydrogen bonds from one part of the chain to the next.” He saw that if he folded the strip like a chain of paper dolls into a helix, and if he got the pitch of the screw right, hydrogen bonds could be shown to form, |N—H . . . O—, three or four knuckles apart along the backbone, holding the helix in shape. After several tries, changing the angle of the parallel creases in order to adjust the pitch of the helix, he found one where the hydrogen bonds would drop into place, connecting the turns, as straight lines of the right length. He had a model.

(a) O C

N H

*The discovery of the a-helix structure was only one of many achievements that led to Pauling’s Nobel Prize in Chemistry in 1954. The official citation for the prize was “for his research into the nature of the chemical bond and its application to the elucidation of the structure of complex substances.”

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Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

values for an antiparallel b-sheet are f = -135°, c = 140°. However, the range of f and c angles for the peptide bonds in parallel sheets is much smaller than that for antiparallel sheets. Parallel sheets are typically large structures; those composed of less than 5 strands are rare. Antiparallel sheets, however, may consist of as few as 2 strands. Parallel sheets characteristically distribute hydrophobic side chains on both sides of the sheet, whereas antiparallel sheets are usually arranged with all their hydrophobic residues on one side of the sheet. This requires an alternation of hydrophilic and hydrophobic residues in the primary structure of peptides involved in antiparallel b-sheets because every other side chain projects to the same side of the sheet (Figure 6.10). Antiparallel pleated sheets are the fundamental structure found in the fabric we know as silk, with the polypeptide chains forming the sheets running parallel to the silk fibres. The silk fibres thus formed have properties consistent with those of the b-sheets that form them. They are flexible but cannot be stretched or extended to any appreciable degree.

Helix–Sheet Composites in Spider Silk Although the intricate designs of spiderwebs are eye- (and fly-) catching, it might be argued that the composition of web silk itself is even more remarkable. Spider silk (a form of keratin) is synthesized in special glands in the spider’s abdomen. The silk strands produced by these glands are both strong and elastic. Dragline silk (that from which the spider hangs) has a tensile strength of 200 000 psi (pounds per square inch)—stronger than steel and similar to Kevlar, the synthetic material used in bulletproof vests! This same silk fibre is also flexible enough to withstand strong winds and other natural stresses. This combination of strength and flexibility derives from the composite nature of spider silk. As keratin protein is extruded from the spider’s glands, it endures shearing forces that break the H bonds stabilizing keratin a-helices (Figure 6.11). These regions then form microcrystalline arrays of b-sheets. These microcrystals are surrounded by the keratin strands, which adopt a highly disordered state composed of a-helices and random coil structures. The b-sheet microcrystals contribute strength, and the disordered array of

(a) Spider web

(b) Radial strand (c) Ordered $-sheets surrounded by disordered #-helices and $-bends.

(d) $-sheets impart strength and #-helices impart flexibility to the strand.

FIGURE 6.11 Spiderweb silks are composites of a-helices and b-sheets. The radial strands of webs must be strong and rigid; silks in radial strands contain a higher percentage of b-sheets. The circumferential strands (termed capture silk) must be flexible (to absorb the impact of flying insects); capture silk contains a higher percentage of a-helices. Spiders typically have several different silk gland spinnerets, which secrete different silks of differing composition. Spiders have inhabited the earth for about 470 million years. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

151

152

Part 1 Building Blocks of the Cell R2

R2

R3

R3

O C

α2

N

α3

C

α2

N

α3

O N

C

O

N

N

C

O

N

C

O α1

C

O α4

α1

α4

FIGURE 6.12 The structures of 2 kinds of b-turns (also called tight turns or b-bends). Four residues are required to form a b-turn. Left: type I; right: type II. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.)

helix and coil make the silk strand flexible. The resulting silk strand resembles modern human-engineered composite materials. Certain tennis racquets, for example, consist of fibreglass polymers impregnated with microcrystalline graphite. The fibreglass provides flexibility, and the graphite crystals contribute strength.

B-Turns Allow the Protein Strand to Change Direction Most proteins are globular structures. The polypeptide chain must therefore possess the capacity to bend, turn, and reorient itself to produce the required compact, globular structures. A simple structure observed in many proteins is the B-turn (also known as the tight turn or b-bend), in which the peptide chain forms a tight loop with the carbonyl oxygen of 1 residue hydrogen bonded with the amide proton of the residue 3 positions down the chain. This H bond makes the b-turn a relatively stable structure. As shown in Figure 6.12, the b-turn allows the protein to reverse the direction of its peptide chain. This figure shows the 2 major types of b-turns, but a number of less common types are also found in protein structures. Because it lacks a side chain, glycine is sterically the most adaptable of the amino acids, and it accommodates conveniently to other steric constraints in the b-turn. Proline, however, has a cyclic structure and a fixed f angle, so, to some extent, it forces the formation of a b-turn; in many cases this facilitates the turning of a polypeptide chain upon itself. Such bends promote formation of antiparallel b-pleated sheets. Type I turns (Figure 6.12) are more common than type II. Proline fits best in the 3 position of the type I turn. In the type II turn, proline is preferred at the 2 position, whereas the 3 position prefers glycine or small polar residues.

6.4

How Do Polypeptides Fold into Three-Dimensional Protein Structures?

The arrangement of all atoms of a single polypeptide chain in 3-dimensional space is referred to as its tertiary structure. As discussed in Section 6.2, all the information needed to fold the protein into its native tertiary structure is contained within the primary structure of the peptide chain itself. Sometimes proteins known as chaperones assist in the process of protein folding in the cell, but proteins in dilute solution can be unfolded and refolded without the assistance of such chaperones.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

The first determinations of the tertiary structure of a protein were by John Kendrew and Max Perutz. Kendrew’s structure of myoglobin and Perutz’s structure of hemoglobin, reported in the late 1950s, were each the result of more than 20 years of work. Ever since these first protein structures were elucidated, biochemists have sought to understand the principles by which proteins adopt their remarkable structures. Vigorous work in many laboratories has slowly brought important principles to light: • Secondary structures—helices and sheets—form whenever possible as a consequence of the formation of large numbers of hydrogen bonds. • a-Helices and b-sheets often associate and pack close together in the protein. No protein is stable as a single-layer structure, for reasons that become apparent later. There are a few common methods for such packing to occur. • Because the peptide segments between secondary structures in the protein tend to be short and direct, the peptide does not execute complicated twists and knots as it moves from one region of secondary structure to another. • Proteins generally fold so as to form the most stable structures possible. The stability of most proteins arises from (1) the formation of large numbers of intramolecular hydrogen bonds and (2) the reduction in the surface area accessible to solvent that occurs upon folding. Two factors lie at the heart of these 4 “principles.” First, proteins are typically a mixture of hydrophilic and hydrophobic amino acids. Why is this important? Imagine a protein that is composed only of polar and charged amino acids. In such a protein, every side chain could hydrogen bond to water. This would leave no reason for the protein to form a compact, folded structure. Now consider a protein composed of a mixture of hydrophilic and hydrophobic residues. In this case, the hydrophobic side chains cannot form H bonds with water, and their presence will disrupt the hydrogen-bonding structure of water itself. To minimize this, the hydrophobic groups will tend to cluster together. This hydrophobic effect induces formation of a compact structure—the folded protein. A potential problem with this rather simple folding model is that polar backbone N—H and C “ O groups on the hydrophobic residues accompany the hydrophobic side chains into the folded protein interior. This would be energetically costly to the protein, but the actual result is that the polar backbone groups form H bonds with one another, so that the polar backbone N—H and C “ O moieties are stabilized in a-helices and b-sheets in the protein interior.

Fibrous Proteins Usually Play a Structural Role In Chapter 5, we saw that proteins can be grouped into 3 large classes based on their structure and solubility: fibrous proteins, globular proteins, and membrane proteins. Fibrous proteins contain polypeptide chains organized approximately parallel along a single axis, producing long fibres or large sheets. Such proteins tend to be mechanically strong and resistant to solubilization in water and dilute salt solutions. Fibrous proteins often play a structural role in nature (see Chapter 5). A-Keratin The a-keratins are the predominant constituents of claws, fingernails, hair, and horns in mammals. As their name suggests, the structure of the a-keratins is dominated by a-helical segments of polypeptide. The amino acid sequence of a-keratin subunits is composed of central a-helix–rich rod domains about 311–314 residues in length, flanked by non-helical N- and C-terminal domains of varying size and composition (Figure 6.13a). The structure of the central rod domain of a typical a-keratin is shown in Figure 6.13b. Pairs of right-handed a-helices wrap around each other to form a left-twisted coiled coil. X-ray diffraction data (including the original studies by William Astbury) show that these structures resemble a-helices, but with a pitch of 0.51 nm rather than the expected 0.54 nm. This is consistent with a tilt of the helix relative to the long axis of the coiled coil (and the keratin fibre) in Figure 6.13.

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Part 1 Building Blocks of the Cell (a)

N-terminal domain

Keratin type I

H3+N

*

Keratin type II

H3+N

*

Rod domain

*

*

36

C-terminal domain

35

11 14

101

16 19 8

121

35

12

101

17 19 8

121

(b) #-Helix

*

20

*

*

COO–

20

COO–

(c) Periodicity of hydrophobic residues

Coiled coil of two #-helices

N Undistorted

Protofilament (pair of coiled coils)

Supercoiled

Left-handed coiled coil

Helices with a heptad repeat of hydrophobic residues Filament (four right-hand twisted protofibrils)

FIGURE 6.13 (a) Both type I and type II a-keratin molecules have sequences consisting of long, central rod domains with terminal cap domains. The numbers of amino acid residues in each domain are indicated. Asterisks denote domains of variable length. (b) The rod domains form coiled coils consisting of left-twisted right-handed a-helices. These coiled coils form protofilaments that then wind around each other in a right-handed twist. Keratin filaments consist of twisted protofibrils (each a bundle of 4 coiled coils). (c) Periodicity of hydrophobic residues. (Adapted from Steinert, P., and Parry, D., 1985. Intermediate filaments: Conformity and diversity of expression and structure. Annual Review of Cell Biology 1:41–65; and Cohlberg, J., 1993. Textbook error: The structure of alpha-keratin. Trends in Biochemical Sciences 18:360–362.)

The amino acid sequence of the central rod segments of a-keratin consists of quasirepeating 7-residue segments of the form (a-b-c-d-e-f-g)n. These units are not true repeats, but residues a and d are usually non-polar amino acids. In a-helices, with 3.6 residues per turn, these non-polar residues are arranged in an inclined row or stripe that twists around the helix axis (Figure 6.13c). These non-polar residues would make the helix highly unstable if they were exposed to solvent, but the association of hydrophobic stripes on 2 a-helices to form the 2-stranded coiled coil effectively buries the hydrophobic residues and forms a highly stable structure (Figure 6.13). The helices clearly sacrifice some stability in assuming this twisted conformation, but they gain stabilization energy from the packing of side chains between the helices. In other forms of keratin, covalent disulfide bonds form between cysteine residues of adjacent molecules, making the overall structure even more rigid, inextensible, and insoluble—important properties for structures such as claws, fingernails, hair, and horns. How and where these disulfides form determines the amount of curling in hair and wool fibres. When a hairstylist creates a permanent wave (simply called a “permanent”) in a hair salon, disulfides in the hair are first reduced and cleaved, then reorganized and reoxidized to change the degree of curl or wave. In contrast, a “set” that is created by wetting the hair, setting it with curlers, and then drying it represents merely a rearrangement of the hydrogen bonds between helices and between fibres. (On humid or rainy days, the hydrogen bonds in curled hair may rearrange, and the hair becomes “frizzy.”)

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Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

155

A DEEPER LOOK

The Coiled-Coil Motif in Proteins The coiled-coil motif was first identified in 1953 by Linus Pauling, Robert Corey, and Francis Crick as the main structural element of fibrous proteins such as keratin and myosin. Since then, many proteins have been found to contain 1 or more coiled-coil segments or domains. A coiled coil is a bundle of a-helices that are wound into a superhelix. Two, 3, or 4 helical segments may be found in the bundle, and they may be arranged parallel or antiparallel to one another. Coiled coils are characterized by a distinctive and regular packing of side chains in the core of the bundle. This regular meshing of side chains requires that they occupy equivalent positions turn after turn.

This is not possible for undistorted a-helices, which have 3.6 residues per turn. The positions of side chains on their surface shift continuously along the helix surface (see Figure 6.13c). However, giving the right-handed a-helix a left-handed twist reduces the number of residues per turn to 3.5, and because 3.5 * 2 = 7.0, the positions of the side chains repeat after 2 turns (7 residues). Thus, a heptad repeat pattern in the peptide sequence is diagnostic of a coiled-coil structure. The figure shows a sampling of coiled-coil structures (highlighted in colour) in various proteins.

(a) Coiled coil Pitch

(b)

Influenza hemagglutinin

DNA polymerase

GCN4 leucine/isoleucine mutant

Seryl tRNA synthetase

Catabolite activator protein

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Part 1 Building Blocks of the Cell

Fibroin and B-Keratin: B-Sheet Proteins The fibroin proteins found in silk fibres in the cocoons of the silkworm, Bombyx mori, and also in spiderwebs represent another type of fibrous protein. These are composed of stacked antiparallel b-sheets, as shown in Figure 6.14. In the polypeptide sequence of silk proteins, there are large stretches in which every other residue is a glycine. As previously mentioned, the residues of a b-sheet extend alternately above and below the plane of the sheet. As a result, all the glycines end up on one side of the sheet, and the other residues (mainly alanines and serines) compose the opposite surface of the sheet. Pairs of b-sheets can then pack snugly together (glycine surface to glycine surface or alanine–serine surface to alanine–serine surface). The b-keratins found in bird feathers are also made up of stacked b-sheets. Collagen: A Triple Helix Collagen is a rigid, inextensible fibrous protein that is a principal constituent of connective tissue in animals, including tendons, cartilage, bones, teeth, skin, and blood vessels. The high tensile strength of collagen fibres in these structures makes possible the various animal activities such as running and jumping that put severe stresses on joints and skeleton. Broken bones and tendon and cartilage injuries to knees, elbows, and other joints involve tears or hyperextensions of the collagen matrix in these tissues. The basic structural unit of collagen is tropocollagen, which has a molecular weight of 285 000 and consists of 3 intertwined polypeptide chains, each about 1000 amino acids in length. Tropocollagen molecules are about 300 nm long and only about 1.4 nm in diameter. Several kinds of collagen have been identified. Type I collagen, which is the most common, consists of 2 identical peptide chains designated a1(I) and 1 different chain designated a2(I). Type I collagen predominates in bones, tendons, and skin. Type II collagen, found in cartilage, and type III collagen, found in blood vessels, consist of 3 identical polypeptide chains.

Gly

Gly

Gly

Gly Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala Gly

Gly

Gly

Gly

Ala

Gly

Gly

FIGURE 6.14 Silk fibroin consists of a unique stacked array of b-sheets. The primary structure of fibroin molecules consists of long stretches of alternating glycine and alanine or serine residues. When the sheets stack, the more bulky alanine and serine residues on one side of a sheet interdigitate with similar residues on an adjoining sheet. Glycine hydrogens on the alternating faces interdigitate in a similar manner, but with a smaller intersheet spacing. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

N H2C

C

H

C

OH

COO–

O H C

+

O2

+

+

CH2

CH2

CH2

H

C

Proteins: Secondary, Tertiary, and Quaternary Structure

H2C

H COH

O O

H HO

O

OH

COO– Proline

Ascorbic acid

!-Ketoglutarate

Prolyl hydroxylase Fe2+

H C

N H2C H

C

C

OH

COO–

O

+

CO2

+

+

CH2

CH2

CH2

OH

C

H2C

H COH

O O

H O

O

O

O– Hydroxyproline

Succinate

Dehydroascorbate

FIGURE 6.15 Hydroxylation of proline residues is catalyzed by prolyl hydroxylase. The reaction requires a-ketoglutarate and ascorbic acid (vitamin C).

Collagen has an amino acid composition that is unique and is crucial to its 3-dimensional structure and its characteristic physical properties. Nearly 1 in 3 residues is a glycine, and the proline content is also unusually high. Three unusual modified amino acids are also found in collagen: 4-hydroxyproline (Hyp), 3-hydroxyproline, and 5-hydroxylysine (Hyl) (Figure 4.4). Proline and Hyp together compose up to 30% of the residues of collagen. Interestingly, these 3 amino acids are formed from normal proline and lysine after the collagen polypeptides are synthesized. The modifications are effected by 2 enzymes: prolyl hydroxylase and lysyl hydroxylase. The prolyl hydroxylase reaction (Figure 6.15) requires molecular oxygen, a-ketoglutarate, and ascorbic acid (vitamin C) and is activated by Fe2+. The hydroxylation of lysine is similar. Because of their high content of glycine, proline, and hydroxyproline, collagen fibres are incapable of forming traditional structures such as a-helices and b-sheets. Instead, collagen polypeptides intertwine to form a unique right-handed triple helix, with each of the 3 strands arranged in a left-handed helical fashion (Figure 6.16). Compared to the a -helix, the collagen helix is much more extended, with a rise per residue along the triple helix axis of 2.9 Å (versus 1.5 Å for the a-helix). There are about 3.3 residues per turn of each of these helices. The triple helix is a structure that forms to accommodate the unique composition and sequence of collagen. Long stretches of the polypeptide sequence are repeats of a Gly-x-y motif, where x is frequently Pro and y is frequently Pro or Hyp. In the triple helix, every third residue faces or contacts the crowded centre of the structure. This area is so crowded that only Gly can fit, and thus every third residue must be a Gly (as observed). Moreover, the triple helix is a staggered structure, such that Gly residues from the 3 strands stack along the centre of the triple helix, and the Gly from 1 strand lies adjacent to an x residue from the second strand and to a y from the third. This allows the N—H of each Gly residue to hydrogen bond with the C “ O of the adjacent x residue. The triple helix structure is further stabilized and strengthened by the formation of interchain H bonds involving hydroxyproline. Collagen types I, II, and III form strong, organized fibrils, which consist of staggered arrays of tropocollagen molecules (Figure 6.17). The periodic arrangement of triple helices in a head-to-tail fashion results in banded patterns in electron micrographs. The banding pattern typically has a periodicity (repeat distance) of 68 nm. Because collagen triple NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

FIGURE 6.16 Poly(Gly-Pro-Pro), a collagen-like righthanded triple helix composed of 3 left-handed helical chains (pdb id = 1K6F). (Adapted from Miller, M. H., and Scheraga, H. A., 1976. Calculation of the structures of collagen models. Role of interchain interactions in determining the triple-helical coiled-coil conformation. I. Poly(glycyl-prolyl-prolyl). Journal of Polymer Science Symposium 54:171–200.)

Packing of collagen molecules Hole zone 0.6d

J. Gross, Biozentrum/Science Photo Library

J. Gross Biozentrum/Science Photo Library From Trends in Biochemical Sciences Vol. 27, No. 10, page 530. October 2002 SPL P780/0012

Overlap zone 0.4d

FIGURE 6.17 In the electron microscope, collagen fibres exhibit alternating light and dark bands. The dark bands correspond to the 40 nm gaps or “holes” between pairs of aligned collagen triple helices. The repeat distance, d, for the light- and dark-banded pattern is 68 nm. The collagen molecule is 300 nm long, which corresponds to 4.41d. The molecular repeat pattern of 5 staggered collagen molecules corresponds to 5d. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

159

Proteins: Secondary, Tertiary, and Quaternary Structure

helices are 300 nm long, 40 nm gaps occur between adjacent collagen molecules in a row along the long axis of the fibrils and the pattern repeats every 5 rows (5 * 68 nm = 340 nm). The 40 nm gaps are referred to as hole regions, and they are important in at least 2 ways: First, sugars are found covalently attached to 5-hydroxylysine residues in the hole regions of collagen (Figure 6.18). The occurrence of carbohydrate in the hole region has led to the proposal that it plays a role in organizing fibril assembly. Second, the hole regions may play a role in bone formation. Bone consists of microcrystals of hydroxyapatite, Ca5(PO4)3OH, embedded in a matrix of collagen fibrils. When new bone tissue forms, the formation of new hydroxyapatite crystals occurs at intervals of 68 nm. The hole regions of collagen fibrils may be the sites of nucleation for the mineralization of bone.

+ NH3 CH2OH HO Galactose

Fibrous proteins, although interesting for their structural properties, represent only a small percentage of the proteins found in nature. Globular proteins, so named for their approximately spherical shape, are far more numerous. The diversity of protein structures found in nature reflects the remarkable variety of functions performed by proteins: binding, catalysis, regulation, transport, immunity, cellular signalling, and more. The functional diversity and versatility derive in turn from the large number of folded structures that polypeptide chains can adopt, and the varied chemistry of the side chains of the 20 common amino acids. Remarkably, this diversity of structure and function derives from a relatively small number of principles and themes of protein folding and design. The balance of Chapter 6 explores and elaborates these principles and themes.

H OH

H

O

CH2OH H

O H OH

H

H

OH

H

O

CH CH2

H

H

OH

Globular Proteins Mediate Cellular Function

H

CH2

O

N H

CH2

O

C H

C

Hydroxylysine residue

Glucose FIGURE 6.18 A disaccharide of galactose and glucose is covalently linked to the 5-hydroxyl group of hydroxylysines in collagen by the combined action of the enzymes galactosyltransferase and glucosyltransferase.

Helices and Sheets Make Up the Core of Most Globular Proteins Globular proteins exist in an enormous variety of 3-dimensional structures, but nearly all contain substantial amounts of a-helices and b-sheets folded into a compact structure that is stabilized by both polar and non-polar groups. A typical example is bovine ribonuclease A, a small protein (12.6 kD, 124 residues) that contains a few short a-helices, a broad section of antiparallel b-sheet, a few b-turns, and several peptide segments without defined secondary structure (Figure 6.19). The space between the helices and sheets in the protein interior is filled efficiently and tightly with mostly hydrophobic amino acid side chains. Most polar side chains in ribonuclease face the outside of the protein structure and interact with solvent water. With its hydrophobic core and a hydrophilic surface, ribonuclease illustrates the typical properties of many folded globular proteins. The helices and sheets that make up the core of most globular proteins probably represent the starting point for protein folding, as shown later in this chapter. Thus, the folding FIGURE 6.19 The 3-dimensional structure of bovine ribonuclease A (pdb id = 1FS3). (a) Ribbon diagram; ( b) space-filling model. (Jane Richardson.)

(a)

(b)

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Part 1 Building Blocks of the Cell

FIGURE 6.20 The surfaces of proteins are complementary to the molecules they bind. PEP carboxykinase (shown here, pdb id = 1K3D) is an enzyme from the metabolic pathway that synthesizes glucose (gluconeogenesis; see Chapter 21). In the so-called “active site” (yellow) of this enzyme, catalysis depends on complementary binding of substrates. Shown in this image are ADP (brown), a Mg2+ ion (blue), and AlF3- (a phosphate analog, in green, above the Mg2+).

of a globular protein, in its simplest conception, could be viewed reasonably as the condensation of multiple elements of secondary structure. On the other hand, most peptide segments that form helices, sheets, or beta turns in proteins are mostly disordered in small model peptides that contain those amino acid sequences. Thus, hydrophobic interactions and other non-covalent interactions with the rest of the protein must stabilize these relatively unstable helices, sheets, and turns in the whole folded protein. Why should the cores of most globular and membrane proteins consist almost entirely of a-helices and b-sheets? The reason is that the highly polar N—H and C “ O moieties of the peptide backbone must be neutralized in the hydrophobic core of the protein. The extensively H-bonded nature of a-helices and b-sheets is ideal for this purpose, and these structures effectively stabilize the polar groups of the peptide backbone in the protein core. The framework of sheets and helices in the interior of a globular protein is typically constant and conserved in both sequence and structure. The surface of a globular protein is different in several ways. Typically, much of the protein surface is composed of the loops and tight turns that connect the helices and sheets of the protein core, although helices and sheets may also be found on the surface. The result is that the surface of a globular protein is often a complex landscape of different structural elements. These complex surface structures can interact in certain cases with small molecules or even large proteins that have complementary structure or charge (Figure 6.20). These regions of complementary, recognizable structure are formed typically from the peptide segments that connect elements of secondary structure. They are the basis for enzyme–substrate interactions, protein–protein associations in cell signalling pathways, antigen–antibody interactions, and more. The segments of the protein that are not helix, sheet, or turn have traditionally been referred to as coil or random coil. Both of these terms are misleading. Most of these “loop” segments are neither coiled nor random, in any sense of the words. These structures are every bit as organized and stable as the defined secondary structures; they just don’t conform to any frequently recurring pattern. These so-called coil structures are strongly influenced by side-chain interactions with the rest of the protein.

Waters on the Protein Surface Stabilize the Structure A globular protein’s surface structure also includes water molecules. Many of the polar backbone and side-chain groups on the surface of a globular protein make H bonds with solvent water molecules. There are often several such water molecules per amino acid residue, and some are in fixed positions (Figure 6.21). Relatively few water molecules are found inside the protein. In some globular proteins (Figure 6.22), it is common for one face of an a-helix to be exposed to the water solvent, with the other face toward the hydrophobic interior of the protein. The outward face of such an amphiphilic helix consists mainly of polar and charged residues, whereas the inward face contains mostly non-polar, hydrophobic residues. A good example of such a surface helix is that of residues 153–166 of flavodoxin from Anabaena (Figure 6.22a). Note that the helical wheel presentation of this helix readily shows that 1 face contains 4 hydrophobic residues and that the other is almost entirely polar and charged. Less commonly, an a-helix can be completely buried in the protein interior or completely exposed to solvent. Citrate synthase is a dimeric protein in which a-helical segments form part of the subunit–subunit interface. As shown in Figure 6.22b, 1 of these helices (residues 260–270) is highly hydrophobic and contains only 2 polar residues, as would befit a helix in the protein core. On the other hand, Figure 6.22c shows the solvent-exposed helix (residues 74–87) of calmodulin, which consists of 10 charged residues, 2 polar residues, and only 2 non-polar residues. FIGURE 6.21 The surfaces of proteins are ideally suited to form multiple H bonds with water molecules. Shown here are waters (blue and white) associated with actinidin, an enzyme from kiwi fruit that cleaves polypeptide chains at arginine residues (pdb id = 2ACT). The polar backbone atoms and side-chain groups on the surface of actinidin are extensively H-bonded with water.

Packing Considerations The secondary and tertiary structures of ribonuclease A (Figure 6.19) and other globular proteins illustrate the importance of packing in tertiary structures. Secondary structures pack closely to one another and also intercalate with (insert between) extended polypeptide chains. If the sum of the van der Waals volumes of a protein’s constituent amino acids is divided by NEL

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Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

Asp153

(a) Val

8

1

12

Lys

Lys

5

Ile

4

Leu

11

9

Ala Asp

2 Trp

13 Ser

7 Glu

14

6 Ser 3

Arg

10 Glu

#-Helix from flavodoxin (residues 153–166) (b)

Leu260 Asn

1 5

Ala Ala

8

Ala

4

11

Gly

9

Ser 2 Met

7 6 3 Phe

Ala

10 Leu

#-Helix from citrate synthase (residues 260–270) (c)

Arg74 Ser

1

12

Ile

Asp 5

Lys Glu

8

4

11

9

Glu Lys

2 Asp

13 Arg

7 Glu

14

6 Thr 3

Met

10 Glu

#-Helix from calmodulin (residues 74–87)

FIGURE 6.22 The so-called helical wheel presentation can reveal the polar or non-polar character of a-helices. If the helix is viewed end on, and the residues are numbered with residue 1 closest to the viewer, it is easy to see how polar and non-polar residues are distributed to form a wheel. (a) The a-helix consisting of residues 153–166 (red) in flavodoxin from Anabaena is a surface helix and is amphipathic (pdb id = 1RCF). (b) The 2 helices (orange and red) in the interior of the citrate synthase dimer (residues 260–270 in each monomer) are mostly hydrophobic (pdb id = 5CSC). (c) The exposed helix (residues 74–87; red) of calmodulin is entirely accessible to solvent and consists mainly of polar and charged residues (pdb id = 1CLL).

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Part 1 Building Blocks of the Cell

HUMAN BIOCHEMISTRY

Collagen-Related Diseases Collagen provides an ideal case study of the molecular basis of physiology and disease. For example, the nature and extent of collagen cross-linking depends on the age and function of the tissue. Collagen from young animals is predominantly un-cross-linked and can be extracted in soluble form, whereas collagen from older animals is highly cross-linked and thus insoluble. The loss of flexibility of joints with aging is probably due in part to increased cross-linking of collagen. Several serious and debilitating diseases involving collagen abnormalities are known. Lathyrism occurs in animals due to the regular consumption of seeds of Lathyrus odoratus, the sweet pea, and involves weakening and abnormalities in blood vessels, joints, and bones. These conditions are caused by B-aminopropionitrile (see equation), which covalently inactivates lysyl oxidase, preventing intramolecular cross-linking of collagen and causing abnormalities in joints, bones, and blood vessels.

Scurvy results from a dietary vitamin C deficiency and involves the inability to form collagen fibrils properly. This is the result of reduced activity of prolyl hydroxylase, which is vitamin C–dependent, as previously noted. Scurvy leads to lesions in the skin and blood vessels, and in its advanced stages, it can lead to grotesque disfiguration and eventual death. Although rare in the modern world, it was a disease well-known to sea-faring explorers in earlier times who did not appreciate the importance of fresh fruits and vegetables in the diet. A number of rare genetic diseases involve collagen abnormalities, including Marfan syndrome and the Ehlers–Danlos syndromes, which result in hyperextensible joints and skin. The formation of atherosclerotic plaques, which cause arterial blockages in advanced stages, is due in part to the abnormal formation of collagenous structures in blood vessels.

N ‚ C i CH2 i CH2 i NH+3 b-Aminopropionitrile

the volume occupied by the protein, packing densities of 0.72–0.77 are typically obtained. These packing densities are similar to those of a collection of solid spheres. This means that even with close packing, approximately 25% of the total volume of a protein is not occupied by protein atoms. Nearly all this space is in the form of very small cavities. Cavities the size of water molecules or larger do occasionally occur, but they make up only a small fraction of the total protein volume. It is likely that such cavities provide flexibility for proteins and facilitate conformational changes and a wide range of protein dynamics (discussed later).

Protein Domains Are Nature’s Modular Strategy for Protein Design Proteins range in molecular weight from a thousand to more than a million. It is tempting to think that the size of unique, globular, folded structures would increase with molecular weight, but this is not what has been observed. Proteins composed of about 250 amino acids or less often have a simple, compact globular shape. However, larger globular proteins are usually made up of 2 or more recognizable and distinct structures, termed domains or modules—compact, folded protein structures that are usually stable by themselves in aqueous solution. Figure 6.23 shows a 2-domain DNA-binding protein, TonEBP, FIGURE 6.23 TonEBP is a DNA-binding protein consisting of 2 distinct domains. The N-terminal domain is shown here on the right, with DNA (orange) in the middle, and the C-terminal domain on the left (pdb id = 1IMH).

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Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

in which the 2 distinct domains are joined by a short segment of the peptide chain. Most domains consist of a single continuous portion of the protein sequence, but in some proteins the domain sequence is interrupted by a sequence belonging to some other part of the protein that may even form a separate domain (Figure 6.24). In either case, typical domain structures consist of hydrophobic cores with hydrophilic surfaces (as was the case for ribonuclease; Figure 6.19). Importantly, individual domains often possess unique functional behaviours (e.g., the ability to bind a particular ligand with high affinity and specificity), and an individual domain from a larger protein often expresses its unique function within the larger protein in which it is found. Multi-domain proteins typically possess the sum total of functional properties and behaviours of their constituent domains. It is likely that proteins consisting of multiple domains (and thus multiple functions) evolved by the fusion of genes that once coded for separate proteins. This would require gene duplication to be common in nature, and analysis of completed genomes has confirmed that approximately 90% of domains in eukaryotes have been duplicated. Thus, the protein domain is a fundamental unit in evolution. Many proteins have been “assembled” by duplicating domains and then combining them in different ways. Many proteins are assemblies constructed from several individual domains, and some proteins contain multiple copies of the same domain. Figure 6.25 shows the tertiary structures of 9 domains that are frequently duplicated, and Figure 6.26 presents several proteins that contain multiple copies of 1 or more of these domains.

(a)

(b)

(c)

(d)

FIGURE 6.24 Malonyl CoA:ACP transacylase (pdb id = 1NM2) is a metabolic enzyme consisting of 2 subdomains. The large subdomain (blue) includes residues 1–132 and 198–316 and consists of a B-sheet surrounded by 12 A-helices. The small subdomain (gold = residues 133–197) consists of a 4-stranded antiparallel B-sheet and 2 A-helices.

(e)

1 nm

(f)

(g)

(h)

163

(i)

FIGURE 6.25 Ribbon structures of several protein modules used in the construction of complex multi-module proteins. (a) The complement control protein module (pdb id = 1HCC). (b) The immunoglobulin module (pdb id = 1T89). (c) The fibronectin type I module (pdb id = 1Q06). (d) The growth factor module (pdb id = 1FSB). (e) The kringle module (pdb id = 1HPK). (f) The GYF module (pdb id = 1GYF). (g) The g-carboxyglutamate module (pdb id = 1CFI). (h) The FF module (pdb id = 1UZC). (i) The DED domain (pdb id = 1A1W). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Fibronectin

C C C

10

I I I I I

Twitchin

ELAM-1

NGF receptor

IL-2 receptor

I I I I

C

N

N

N

N-CAM

N

F3 F3 I I I I

Plasma membrane

C C C C C C G

LB

C2, factor B

[ ]

Clr,Cls

I I I I I F3 F3 I F3 F3 F3 I F3 F3 I F3 F3 F3 I I F3 F3 I I F3

C C G

K tPA

γCG Factors VII, IX, X and protein C

K F1 G

Factor XII

G G

F2 G F1 G

K

FIGURE 6.26 A sampling of proteins that consist of mosaics of individual protein modules. The modules shown include GCG, a module containing G-carboxyglutamate residues; G, an epidermal growth factor–like module; K, the “kringle” domain–named for a Danish pastry; C, which is found in complement proteins; F1, F2, and F3, first found in fibronectin; I, the immunoglobulin superfamily domain; N, found in some growth factor receptors; E, a module homologous to the calcium-binding E–F hand domain; and LB, a lectin module found in some cell surface proteins. (Adapted from Baron, M., Norman, D., and Campbell, I., 1991. Protein modules. Trends in Biochemical Sciences 16:13–17.)

F1 F1 F1 F1 F1 F1 F2 F2 F1 F1 F1 F3 F3 F3 F3 F3 F3 F3 F3 F3 F3 F3 F3 F3 F3 F3 F1 F1 F1

Part 1 Building Blocks of the Cell

C

164

PDGF receptor

Classification Schemes for the Protein Universe Are Based on Domains The astounding diversity of properties and behaviours in living things can now be explored through the analysis of vast amounts of genomic information. Assessment of sequence and structural data from several million proteins in both protein and genome databases has shown that there is a relatively limited number of structurally distinct domains in proteins. Several comprehensive projects have organized the available information in defined hierarchies or levels of protein structure. The Structural Classification of Proteins database (SCOP, http://scop.mrc-lmb.cam.ac.uk/scop) recognizes 5 overarching classes, which encompass most proteins. SCOP is based on hierarchical levels that embody the evolutionary and structural relationships among known proteins, and protein classification in SCOP is essentially a manual process using visual inspection and comparison of structures. CATH is another hierarchical classification system (www.cathdb.info/) that groups protein NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

165

domain structures into evolutionary families and structural groupings, depending on sequence and structure similarities. CATH differs from SCOP in that it combines manual analysis with automation based on quantitative algorithms to classify protein structures. Figure 6.27 compares the hierarchical structures of SCOP and CATH and defines the different levels of structure. Although the hierarchical names in SCOP and CATH differ somewhat, there are common threads shared in these schemes. Class is determined from the overall composition of secondary structure elements in a domain. A fold describes the number, arrangement, and connections of these secondary structure elements. A superfamily includes domains of similar folds and usually similar functions, thus suggesting a common evolutionary ancestry. A family usually includes domains with closely related amino acid sequences (in addition to folding similarities). Although the numbers of unique folds, superfamilies, and families increase as more genomes are known and analyzed, it has become apparent that the number of protein domains in nature is large but limited. How many proteins can we expect to identify and understand someday? There are approximately 103-105 genes per organism and approximately 13.6 million species of living organisms on earth (and this latter number is likely an underestimate). Thus, there may be approximately (103 * 1.36 * 107), or 1010-1012, different proteins in all organisms on earth. Still, this vast number of

The CATH Hierarchy Class (4) Architecture (40)

FIGURE 6.27 SCOP and CATH are hierarchical classification systems for the known proteins. Proteins are classified in SCOP by a manual process, whereas CATH combines manual and automated procedures. Numbers indicate the population of each category.

Topology (1084) Homologous Superfamily (2091) Sequence Family (7794) Domain (93 885)

The SCOP Hierarchy Class (7) Fold (1086)

Superfamily (1777)

Family (3464)

Domain (97 178)

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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proteins may well consist of only about 105 sequence domain families (Figure 6.27) and approximately 103 protein folds of known structure—a remarkably small number compared to the total number of protein-coding genes (see Table 1.6). It is anticipated that most newly identified proteins will resemble other known proteins and that most structures can be broken into 2 or more domains, which resemble tertiary structures observed in other proteins. Because structure depends on sequence, and because function depends on structure, it is tempting to imagine that all proteins of a similar structure should share a common function, but this is not always true. For example, the TIM barrel is a common protein fold consisting of 8 a-helices and 8 b-strands that alternate along the peptide backbone to form a doughnut-like tertiary structure. The TIM barrel is named for triose phosphate isomerase, an enzyme that interconverts ketone and aldehyde substrates in the breakdown of sugars (see Chapter 17). However, other TIM barrel proteins carry out very different functions (Figure 6.28a), including the reduction of aldose sugars and hydrolysis of phosphate esters. Moreover, not all proteins of similar function possess similar domains. Both proteins shown in Figure 6.28b catalyze the same reaction, but they bear little structural similarity to each other.

Denaturation Leads to Loss of Protein Structure and Function Whereas the primary structure of proteins arises from covalent bonds, the secondary, tertiary, and quaternary levels of protein structure are maintained by weak, non-covalent forces. The environment of a living cell is exquisitely suited to maintain these weak forces and to preserve the structures of its many proteins. However, a variety of external stresses

FIGURE 6.28 (a) Some proteins share similar structural features but carry out different functions (triose phosphate isomerase, pdb id = 8TIM; aldose reductase, pdb id = 1ADS; phosphotriesterase, pdb id = 1DPM). (b) Proteins with different structures can carry out similar functions (yeast aspartate aminotransferase, pdb id = 1YAA); D-amino acid aminotransferase, pdb id = 3DAA).

(a) Same domain type, different functions:

Triose phosphate isomerase

Aldose reductase

Phosphotriesterase

(b) Same function, different structures:

D-amino

acid aminotransferase

Aspartate aminotransferase NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Proteins: Secondary, Tertiary, and Quaternary Structure

167

© Vladimir Glazkov/iStockphoto.com

Chapter 6

Ovalbumin monomer FIGURE 6.29 The proteins of egg white are denatured (as evidenced by their precipitation and aggregation) during cooking. More than half the protein in egg whites is ovalbumin. Ovalbumin pdb id = 1OVA.

H2N

C

NH2

Guanidine HCl O H2N

C Urea

NH2

0.75

0.50

0.25

0.00 20

30

40 50 60 70 Temperature (°C)

1.0

Cl–

0.8 0.6 0.4 0.2 0 0

1

2

3 4 [GdmCl] (M)

5

80

FIGURE 6.30 Proteins can be denatured by heat, with commensurate loss of function. Ribonuclease A (blue) and ribonuclease B (red) lose activity above about 55°C. These 2 enzymes share identical protein structures, but ribonuclease B possesses a carbohydrate chain attached to Asn34. (Adapted from Arnold, U., and Ulbrich-Hofmann, R., 1997. Kinetic and thermodynamic thermal stabilities of ribonuclease A and ribonuclease B. Biochemistry 36:2166–2172.)

(b)

NH + 2

Fraction unfolded

(a)

1.00 Fraction of native protein

(e.g., heat or chemical treatment) can disrupt these weak forces in a process termed denaturation, the loss of protein structure and function. An everyday example is the denaturation of the protein ovalbumin during the cooking of an egg (Figure 6.29 above). About 10% of the mass of an egg white is protein, and 54% of that is ovalbumin. When a chicken egg is cracked open, the “egg white” is a nearly transparent, viscous fluid. Cooking turns this fluid to a solid, white mass. The egg-white proteins have unfolded and have precipitated out of solution, and the unfolded proteins have aggregated into a solid mass. As a typical protein solution is heated slowly, the protein remains in its native state until it approaches a characteristic melting temperature, Tm. As the solution is heated further, the protein denatures over a narrow range of temperatures around Tm (Figure 6.30). Denaturation over a very small temperature range such as this is evidence of a two-state transition between the native and the unfolded states of the protein, and this implies that unfolding is an all-or-none process: When weak forces are disrupted in one part of the protein, the entire structure breaks down. Most proteins can also be denatured below the transition temperature by a variety of chemical agents, including acid or base, organic solvents, detergents, and particular denaturing solutes. Guanidine hydrochloride and urea are examples of the latter (Figure 6.31). Denaturation in all these cases involves disruption of the weak forces that stabilize proteins. Covalent bonds are not affected. Acids and bases cause protonation and deprotonation of dissociable groups on the protein, altering ionic interactions and hydrogen bonds. Organic solvents and detergents disrupt hydrophobic interactions that bury non-polar groups in the protein interior. The effects of guanidine hydrochloride and urea are more

6

FIGURE 6.31 Proteins can be denatured (unfolded) by high concentrations of guanidine-HCl or urea. The denaturation of chymotrypsin is plotted here. (Adapted from Fersht, A., 1999. Structure and Mechanism in Protein Science. New York, W. H. Freeman.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

complex. Recent research indicates that these agents denature proteins by both direct effects (binding to hydrophilic groups on the protein) and indirect effects (altering the structure and dynamics of the water solvent). Also, both guanidine hydrochloride and urea are good H-bond donors and acceptors.

Anfinsen’s Classic Experiment Proved That Sequence Determines Structure As noted earlier (Section 6.2), all the information needed to fold a polypeptide into its native structure is contained in the amino acid sequence. This simple but profound truth of protein structure was confirmed in the 1950s by the elegant studies of denaturation and renaturation of proteins by Christian Anfinsen and his co-workers at the National Institutes of Health. For their pivotal studies, they chose the small enzyme ribonuclease A from bovine pancreas, a protein with 124 residues and 4 disulfide bonds (Figures 6.19 and 6.32).

FIGURE 6.32 Ribonuclease can be unfolded by treatment with urea, and b-mercaptoethanol (MCE) cleaves disulfide bonds. If b-mercaptoethanol is then removed (but not urea) under oxidizing conditions, disulfide bonds re-form in the stillunfolded protein (one possible hypothetical inactive form is shown). If urea is removed in the presence of a small amount of b-mercaptoethanol with gentle warming, ribonuclease returns to its native structure (with the correct set of disulfide bonds), and full enzymatic activity is restored. This experiment demonstrated that the information required for folding of globular proteins is contained in the primary structure.

26

26

95

58

65

110

40

+ MCE + Urea

72

84

40

95

65

– MCE – Urea + Oxygen

110 84

72 58 Active

Inactive

– MCE + Oxygen

Small amount of MCE w/gentle warming

65

26

40

58

84

72 95

110

Hypothetical Inactive Form (Note random formation of disulfides) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

(Ribonuclease cleaves chains of ribonucleic acid. Only ribonuclease in its native structure possesses enzyme activity, so loss of activity in a denaturation experiment was proof of loss of structure.) They treated solutions of ribonuclease with a combination of urea, which unfolded the protein, and mercaptoethanol, which reduced the disulfide bridges. This treatment destroyed all enzymatic activity of ribonuclease. Anfinsen discovered that removing the mercaptoethanol but not the urea restored only 1% of the enzyme activity. This was attributed to the formation of random disulfide bridges by the still-denatured protein. With 8 Cys residues, there are 105 possible ways to make 4 disulfide bridges; thus, a residual activity of 1% made sense to Anfinsen. (The first Cys to form a disulfide has 7 possible partners, the next Cys has 5 possible partners, the next has 3, and the last Cys has only 1 choice; 7 * 5 * 3 * 1 = 105.) However, if Anfinsen removed mercaptoethanol and urea at the same time, the polypeptide was able to fold into its native structure, the correct set of 4 disulfides re-formed, and full enzyme activity was recovered (Figure 6.32). This experiment demonstrated that the information needed for protein folding resided entirely within the amino acid sequence of the protein itself. Many subsequent experiments with a variety of proteins have confirmed this fundamental postulate. For his studies of the relationship of sequence and structure, Anfinsen shared the 1972 Nobel Prize in Chemistry (with William H. Stein and Stanford Moore).

Is There a Single Mechanism for Protein Folding? Christian Anfinsen’s experiments demonstrated that proteins can fold reversibly. A corollary result of Anfinsen’s work is that the native structures of at least some globular proteins are thermodynamically stable states. But the matter of how a given protein achieves such a stable state is a complex one. Cyrus Levinthal pointed out in 1968 that so many conformations are possible for a typical protein that the protein does not have sufficient time to reach its most stable conformational state by sampling all the possible conformations. This argument, termed Levinthal’s paradox, goes as follows: Consider a protein of 100 amino acids. Assume that there are only 2 conformational possibilities per amino acid, or 2100 = 1.27 * 1030 possibilities. Allow 10–13 sec for the protein to test each conformational possibility in search of the overall energy minimum: (10-13 sec)(1.27 * 1030) = 1.27 * 1017 sec = 4 * 109 years. Four billion years is the approximate age of the earth. Levinthal’s paradox led protein chemists to hypothesize that proteins must fold by specific “folding pathways,” and many research efforts have been devoted to the search for these pathways. Several consistent themes have emerged from these studies. Each of them may well play a role in the folding process: • Secondary structures—helices, sheets, and turns—probably form first. • Non-polar residues may aggregate or coalesce in a process termed a hydrophobic collapse. • Subsequent steps probably involve formation of long-range interactions between secondary structures or involving other hydrophobic interactions. • The folding process may involve 1 or more intermediate states, including transition states and what have become known as molten globules. The folding of most globular proteins may well involve several of these themes. For example, even in the denatured state, many proteins appear to possess small amounts of residual structure due to hydrophobic interactions, with strong interresidue contacts between side chains that are relatively distant in the native protein structure. Such interactions, together with small amounts of secondary structure, may act as sites of nucleation for the folding process. A bit further in the folding process, the molten globule is postulated to be a flexible but compact form characterized by significant amounts of secondary structure, virtually no precise tertiary structure, and a loosely packed hydrophobic core. Moreover, it is likely that any one of these themes is more important for some proteins than for others. The process of folding is clearly complex, but sophisticated simulations have NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell Cl2

D (94 ns)

D (70 ns)

I (30 ns)

TS (0.26 ns)

N (0 ns)

Barnase

D (4 ns)

D (2 ns)

I (0.7 ns)

TS (0.15 ns)

N (0 ns)

FIGURE 6.33 Computer simulations of folding and unfolding of proteins can reveal possible folding pathways. Molecular dynamics simulations of the unfolding of small proteins such as chymotrypsin inhibitor 2 (CI2) and barnase are presented here on a reversed time scale to show how folding may occur. D = denatured, I = intermediate, TS = transition state, N = native. (Adapted from Daggett, V., and Fersht, A. R., 2003. Is there a unifying mechanism for protein folding? Trends in Biochemical Sciences 28:18–25. Figures provided by Alan Fersht and Valerie Daggett.)

already provided reasonable models of folding (and unfolding) pathways for many proteins (Figure 6.33). One school of thought suggests that for any given protein there may be multiple folding pathways. For these cases, Ken Dill has suggested that the folding process can be pictured as a funnel of free energies—an energy landscape (Figure 6.34). The rim at the top of the funnel represents the many possible unfolded states for a polypeptide chain, each characterized by high free energy and significant conformational entropy. Polypeptides fall down the wall of the funnel as contacts made between residues establish different folding possibilities. The narrowing of the funnel reflects the smaller number of available states as the protein approaches its final state; bumps or pockets on the funnel walls represent partially stable intermediates in the folding pathway. The most stable (native) folded state of the protein lies at the bottom of the funnel.

What Is the Thermodynamic Driving Force for Folding of Globular Proteins? The free energy change for the folding of a globular protein must be negative if the folded state is more stable than the unfolded state. The free energy change for folding depends, in turn, on changes in enthalpy and entropy for the process: ∆G = ∆H - T∆S. When ∆H, -T∆S, and ∆G are measured separately for the polar side chains and the nonpolar side chains of the protein, an important insight is apparent: The enthalpy and entropy changes for polar residues largely cancel each other out, and the ∆G of folding for the polar residues is approximately zero. To understand the behaviour of the non-polar residues, it is helpful to distinguish the ∆H and -T∆S contributions for the polypeptide chain and for the water solvent. Both ∆H and -T∆S for the non-polar residues of the peptide chain are positive and thus make unfavourable contributions to the folding free energy. However, large numbers of water molecules restricted and immobilized around non-polar residues in the unfolded protein are liberated in the folding process. The burying of non-polar residues in the folded protein’s core produces a dramatic entropy increase for these liberated water molecules. This NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

171

FIGURE 6.34 A model for the steps involved in the folding of globular proteins. The funnel represents a free energy surface or energy landscape for the folding process. The protein folding process is highly cooperative. Rapid and reversible formation of local secondary structures is followed by a slower phase in which establishment of partially folded intermediates leads to the final tertiary structure. Substantial exclusion of water occurs very early in the folding process.

is just enough to make the overall ∆G for folding negative (and thus favourable). The results are crucial: • The largest contribution to the stability of a folded protein is the entropy change for the water molecules associated with the non-polar residues. • The overall free energy change for the folding process is not large—typically -20 to -40 kJ/mol.

Marginal Stability of the Tertiary Structure Makes Proteins Flexible A typical folded protein is only marginally stable. The hundreds of van der Waals interactions and hydrogen bonds in a folded structure are compensated and balanced by a dramatic loss of entropy suffered by the polypeptide as it assumes a compact folded structure. Because stability seems important to protein and cellular function, it is tempting to ask what the advantage of marginal stability might be. The answer appears to lie in flexibility and motion. All chemical bonds undergo a variety of motions, including vibrations and (for single bonds) rotations. This propensity to move, together with the marginal stability of protein structures, means that the many non-covalent interactions within a protein can be interrupted, broken, and rearranged rapidly. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

Motion in Globular Proteins

FIGURE 6.35 Proteins are dynamic structures. The marginal stability of a tertiary structure leads to flexibility and motion in the protein. Determination of structures of proteins (such as the SH3 domain of the A-chain of spectrin, shown here) by nuclear magnetic resonance produces a variety of stable tertiary structures that fit the data. Such structural ensembles provide a glimpse into the range of structures that may be accessible to a flexible, dynamic protein (pdb id = 1M8M).

Proteins are best viewed as dynamic structures. Most globular proteins oscillate and fluctuate continuously about their average or equilibrium structures (Figure 6.35). This flexibility is essential for a variety of protein functions, including ligand binding, enzyme catalysis, and enzyme regulation, as shown throughout the remainder of this text. The motions of proteins may be motions of individual atoms, groups of atoms, or even whole sections of the protein. Furthermore, they may arise from either thermal energy or specific, triggered conformational changes in the protein. Atomic fluctuations such as vibrations typically are random, are very fast, and usually occur over small distances, as shown in Table 6.2. These motions arise from the kinetic energy within the protein and are a function of temperature. In the tightly packed interior of the typical protein, atomic movements of an angstrom or less are typical. The closer to the surface of the protein, the more movement can occur, and on the surface, atomic movements of several angstroms are possible. A class of slower motions, which may extend over larger distances, is collective motions. These are movements of a group of atoms covalently linked in such a way that the group moves as a unit. Such a group can range from a few atoms to hundreds of atoms. These motions are of 2 types: (1) those that occur quickly but infrequently, such as tyrosine ring flips, and (2) those that occur slowly, such as the hinge-bending movement between protein domains. For example, the 2 antigen-binding domains of immunoglobulins move as relatively rigid units to selectively bind separate antigen molecules. These collective motions also arise from thermal energies in the protein and operate on a timescale of 10-12–10-3 sec. It is often important to distinguish the time scale of the motion itself versus the frequency of its occurrence. A tyrosine ring flip takes only a picosecond (1 * 10-12 sec), but such flips occur only about once every millisecond (1 * 10-3 sec). Conformational changes involve motions of groups of atoms (e.g., individual side chains) or even whole sections of proteins. These motions occur on a time scale of 10-9–103 sec, and the distances covered can be as large as 1 nm. These motions may occur in response to specific stimuli or arise from specific interactions within the protein (hydrogen bonding, electrostatic interactions, or ligand binding; see Chapters 13 and 14). The cis–trans isomerization of proline residues in proteins (Figure 6.36) occurs over an even longer time scale—typically 101–104 sec. Conversion of even a single proline from its cis to its trans configuration can alter a protein structure dramatically. Proline cis–trans isomerizations sometimes act as switches to activate a protein or open a channel across a membrane (see Chapter 10).

The Folding Tendencies and Patterns of Globular Proteins Globular proteins adopt the most stable tertiary structure possible. To do this, the peptide chain must both (1) satisfy the constraints inherent in its own structure and (2) fold so as to “bury” the hydrophobic side chains, minimizing their contact with solvent.

TABLE 6.2 Motion and Fluctuations in Proteins Spatial Displacement (Å)

Characteristic Time (sec)

Source of Energy

Atomic vibrations

0.01–1

10-15-10-11

Kinetic energy

Collective motions

0.01–5 or more

10-12-10-3

Kinetic energy

Triggered conformation changes

0.5–10 or more

10-9-103

Interactions with triggering agent

Proline cis–trans isomerization

3–10

101-104

Kinetic energy enzyme driven

Type of Motion

1. Fast: Tyr ring flips; methyl group rotations 2. Slow: hinge bending between domains

Adapted from Petsko, G. A., and Ringe, D., 1984. Fluctuations in protein structure from X-ray diffraction. Annual Review of Biophysics and Bioengineering 13:331–371. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

FIGURE 6.36 The trans and cis configurations of proline residues in peptide chains are almost equally stable. Proline cis-trans isomerizations, often occurring over relatively long time scales, can alter protein structure significantly.

H C#

O C C# H

N H2C

C

CH2

R

N

C# H

trans

H2 C

O

CH2

R

C#

CH2 CH2

H

cis

The polypeptide itself does not usually form simple straight chains. Even in chain segments where helices and sheets are not formed, an extended peptide chain, being composed of L-amino acids, has a tendency to twist slightly in a right-handed direction. As shown in Figure 6.37, this tendency is apparently the basis for the formation of a variety of tertiary structures having a right-handed sense. Principal among these are the right-handed twists in b-sheets and right-handed crossovers in parallel b-sheets. Right-handed twisted b-sheets are found at the centre of a number of proteins (Figure 6.38) and provide an extended, highly stable structural core. Connections between b-strands are of 2 types: hairpins and crossovers. Hairpins, as shown in Figure 6.37, connect adjacent antiparallel b-strands. Crossovers are necessary to connect adjacent (or nearly adjacent) parallel b-strands. Nearly all crossover structures are right-handed. In many crossover structures, the crossover connection itself contains an b-helical segment. This creates a BAB-loop. As shown in Figure 6.37, the strong tendency in nature to form right-handed crossovers, the wide occurrence of b-helices in the crossover connection, and the right-handed twists of b-sheets can all be understood as arising from the tendency of an extended polypeptide chain of L-amino acids to adopt a right-handed twist structure. This is a chiral effect. Proteins composed of D-amino acids would tend to adopt left-handed twist structures. The second driving force that affects the folding of polypeptide chains is the need to bury the hydrophobic residues of the chain, protecting them from solvent water. From a topological viewpoint, then, all globular proteins must have an “inside” where the hydrophobic core can be arranged, and an “outside” toward which the hydrophilic groups must be directed. The sequestration of hydrophobic residues away from water is the dominant force in the arrangement of secondary structures and non-repetitive peptide segments to form a given tertiary structure. Globular proteins can be classified mainly on the basis of

(a)

(b) Antiparallel hairpin

Natural right-handed twist by polypeptide chain

Parallel, right-handed

Crossovers

173

Parallel, left-handed

FIGURE 6.37 (a) The natural right-handed twist exhibited by polypeptide chains, and (b) the types of connections between b-strands. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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the particular kind of core or backbone structure they use to accomplish this goal. The term hydrophobic core, as used here, refers to a region in which hydrophobic side chains cluster together, away from the solvent. Backbone refers to the polypeptide backbone itself, excluding the particular side chains. Globular proteins can be pictured as consisting of “layers” of backbone, with hydrophobic core regions between them. More than half the known globular protein structures have 2 layers of backbone (separated by 1 hydrophobic core). Roughly one-third of the known structures are composed of 3 backbone layers and 2 hydrophobic cores. There are also a few known 4-layer structures and at least one 5-layer structure. A few structures are not easily classified in this way, but it is remarkable that most proteins fit into one of these classes. Examples of each are presented in Figure 6.38.

Most Globular Proteins Belong to One of Four Structural Classes In addition to classification based on layer structure, proteins can be grouped according to the type and arrangement of secondary structure (Figure 6.39). There are 4 such broad groups: all A proteins and all B proteins (in which the structures are dominated by a-helices and b-sheets respectively), A/B proteins (in which helices and sheets are intermingled), and A ! B proteins (in which a-helical and b-sheet domains are separated for the most part).

Layer 1

Layer 2

(a) Cytochrome c#

(d) Triosephosphate isomerase

Hydrophobic residues are buried between layers

(b) Phosphoglycerate kinase (domain 2)

(c) Phosphorylase (domain 2)

FIGURE 6.38 Examples of protein domains with different numbers of layers of backbone structure. (a) Cytochrome c# with 2 layers of a-helix. (b) Domain 2 of phosphoglycerate kinase, composed of a b-sheet layer between 2 layers of helix, 3 layers overall. (c) An unusual 5-layer structure, domain 2 of glycogen phosphorylase, a b-sheet layer sandwiched between 4 layers of a-helix. (d) The concentric “layers” of b-sheet (inside) and a-helix (outside) in triosephosphate isomerase. Hydrophobic residues are buried between these concentric layers in the same manner as in the planar layers of the other proteins. The hydrophobic layers are shaded yellow. (Jane Richardson) NEL

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Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

All # proteins:

Human growth hormone (pdb id = 1HGU)

Leucine-rich repeat variant (pdb id = 1LRV)

Peridinin-chlorophyll protein (a “solenoid”—pdb id = 1PPR)

Endoglucanase A (an #-helical barrel—pdb id = 1CEM)

Cat allergen (pdb id = 1PUO)

All $ proteins:

Mannose-specific aggluttinin (a prism— (pdb id = 1JPC)

Rieske iron protein (a 3-layer $-sandwich— (pdb id = 1RIE)

Hemopexin C-terminal domain (a 4-bladed propellor—pdb id = 1HXN)

Lectin from R. solanacearum (a 6-bladed propellor— pdb id = 1BT9)

Pleckstrin domain of protein kinase B/AKT (pdb id = 1UNQ)

#+$ proteins:

# $ proteins: #/$

Hevamine (a “TIM barrel” —pdb id = 2HVM)

Human bactericidal permeability-increasing protein (pdb id = 1BP1)

MurA (an #–$ prism —pdb id = 1EYN)

Hepatocyte growth factor (N-terminal domain —pdb id = 2HGF)

Equine leukocyte elastase inhibitor (pdb id = 1HLE)

RuvA protein (pdb id = 1CUK)

Ribonuclease H (pdb id = 1RNH)

L-Arginine: glycine amidinotransferase (a metabolic enzyme—pdb id = 4JDW)

Prokaryotic ribosomal protein L9 (pdb id = 1DIV)

Porcine ribonuclease inhibitor (a “horseshoe”—pdb id = 2BNH)

Thymidylate synthase (pdb id = 3TMS)

FIGURE 6.39 Four major classes of protein structure (as defined in the SCOP database). (a) All a proteins, where a-helices dominate the structure; (b) All b proteins, in which b-sheets are the primary feature; (c) a/b proteins, where a-helices and b-sheets are mixed within a domain; (d) a + b proteins, in which a-helical and b-sheet domains are separated to at least some extent. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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It is important to note that the similarities of tertiary structure within these groups do not necessarily reflect similar or even related functions. Instead, functional homology usually depends on structural similarities on a smaller and more intimate scale.

Molecular Chaperones Are Proteins That Help Other Proteins to Fold To a first approximation, all the information necessary to direct the folding of a polypeptide is contained in its primary structure. On the other hand, the high protein concentration inside cells may adversely affect the folding process because hydrophobic interactions may lead to aggregation of some unfolded or partially folded proteins. Also, it may be necessary to suppress or reverse incorrect or premature folding. A family of proteins, known as molecular chaperones, are essential for the correct folding of certain polypeptide chains in vivo; for their assembly into oligomers; and for preventing inappropriate liaisons with other proteins during their synthesis, folding, and transport. Many of these proteins were first identified as heat shock proteins, which are induced in cells by elevated temperature or other stress. The most thoroughly studied proteins are Hsp70, a 70 kD heat shock protein, and the so-called chaperonins, also known as Cpn60s or Hsp60s, a class of 60 kD heat shock proteins. A well-characterized Hsp60 chaperonin is GroEL, an E. coli protein that has been shown to affect the folding of several proteins. The mechanism of action of chaperones is discussed in Chapter 32.

Some Proteins Are Intrinsically Unstructured Remarkably, it is now becoming clear that many proteins exist and function normally in a partially unfolded state. Such proteins, termed intrinsically unstructured proteins (IUPs) or natively unfolded proteins, do not possess uniform structural properties but are nonetheless essential for basic cellular functions. These proteins are characterized by an almost complete lack of folded structure and an extended conformation with high intramolecular flexibility. Intrinsically unstructured proteins contact their targets over a large surface area (Figure 6.40). The p27 protein complexed with cyclin-dependent protein kinase 2 (Cdk2) and cyclin A shows that p27 is in contact with its binding partners across its entire length. It binds in a groove consisting of conserved residues on cyclin A. On Cdk2, it binds to the N-terminal domain and also to the catalytic cleft. One of the most appropriate roles for such long-range interactions is assembly of complexes involved in the transcription of DNA into RNA, where large numbers of proteins must be recruited in macromolecular complexes. Thus, the transactivator domain catenin-binding domain (CBD) of tcf3 is bound to several functional domains of b-catenin (Figure 6.40). Can amino acid sequence information predict the existence of intrinsically unstructured regions on proteins? Intrinsically unstructured proteins are characterized by a unique combination of high net charge and low overall hydrophobicity. Compared with ordered proteins, IUPs have higher levels of E, K, R, G, Q, S, and P, and low amounts of I, L, V, W, F, Y, C, and N. These features provide a rationale for prediction of regions of disorder from amino acid sequence information, and experimental evidence shows that such predictions are better than 80% accurate. Genomic analysis of disordered proteins indicates that the proportion of the genome encoding IUPs and proteins with substantial regions of disorder tends to increase with the complexity of organisms. Thus, predictive analysis of whole genomes indicates that 2% of archaeal, 4.2% of bacterial, and 33% of eukaryotic proteins probably contain long regions of disorder. Some proteins are disordered throughout their length, whereas others may contain stretches of 30–40 residues or more that are disordered and imbedded in an otherwise folded protein. The prevalence of disordered segments in proteins may reflect 2 different cellular needs: (1) Disordered proteins are more malleable and thus can adapt their structures to bind to multiple ligands, including other proteins. Each such interaction could provide a different function in the cell. (2) Compared with compact, folded proteins, disordered segments in proteins appear to be able to form larger intermolecular interfaces to NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 6 (a)

(b)

Proteins: Secondary, Tertiary, and Quaternary Structure

177

(c)

TAFII105 Cdk2

Oct 1 POU SD

Oct 1 POU HD Igκ CycA $-catenin

(d)

(e)

(f)

(g)

FIGURE 6.40 Intrinsically unstructured proteins (IUPs) contact their target proteins over a large surface area. (a) p27Kip1 (yellow) complexed with cyclindependent kinase 2 (Cdk2, blue) and cyclin A (CycA, green). (b) The transactivator domain CBD of Tcf3 (yellow) bound to b-catenin (blue). Note: Part of the b-catenin has been removed for a clear view of the CBD. (c) Bob 1 transcriptional co-activator (yellow) in contact with its 4 partners: TAFII105 (green oval), the Oct 1 domains POU SD and POU HD (green), and the Igk promoter (blue). (From Tompa, P., 2002. Intrinsically unstructured proteins. Trends in Biochemical Sciences 27:527–533.) (d–g) Some intrinsically unstructured proteins (in red and yellow) bind to their targets by wrapping around them. Shown here are (d) SNAP-25 bound to BoNT/A, (e) SARA SBD bound to Smad2 MH2, (f) HIF-1a interaction domain bound to the TAZ1 domain of CBP, and (g) HIF-1a interaction domain bound to asparagine hydroxylase FIH. (Reprinted from Trends in Biochemical Sciences Vol. 27, No. 10, Peter Tompa, Intrinsically unstructured proteins, page 530, 2002, with permission from Elsevier.)

which ligands, such as other proteins, could bind (Figure 6.40). Folded proteins might have to be 2–3 times larger to produce the binding surface possible with a disordered protein. Larger proteins would increase cellular crowding or could increase cell size by 15%–30%. The flexibility of disordered proteins may thus reduce protein, genome, and cell sizes. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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HUMAN BIOCHEMISTRY

A1-Antitrypsin: A Tale of Molecular Mousetraps and a Folding Disease In the human lung, oxygen and CO2 are exchanged across the walls of alveoli—air sacs surrounded by capillaries that connect the pulmonary veins and arteries. The walls of alveoli consist of the elastic protein elastin. Inhalation expands the alveoli, and exhalation compresses them. A pair of human lungs contains 300 million alveoli, and the total area of the alveolar walls in contact with capillaries is about 70 m2—an area about the size of a tennis court! In the lungs, neutrophils (a type of white blood cell) naturally secrete elastase, a protein-cleaving enzyme essential to tissue repair. However, elastase also can attack and break down the elastin of the alveolar walls if it spreads from the site of inflammation repair. To prevent this, the liver secretes into the blood A1-antitrypsin—a 52 kD protein belonging to the serpin (serine protease inhibitor) family—which blocks elastase action, preventing alveolar damage. a1-Antitrypsin is a molecular mousetrap, with a flexible peptide loop (blue in the figure) that contains a Met residue as “bait” for the elastase and that can swing like the arm of a mousetrap. When elastase binds to the loop at the Met residue, it cuts the peptide loop. Now free to move, the loop slides into the middle of a large b-sheet (green), at the same time dragging elastase to the opposite side of the a1-antitrypsin structure. At this new binding site, the elastase structure is distorted, and it cannot complete its reaction and free itself

from the a1-antitrypsin. Cellular scavenger enzymes then attack the elastase–antitrypsin complex and destroy it. By sacrificing itself in this way, the a1-antitrypsin has prevented damage to the alveolar elastin. Defects in a1-antitrypsin can cause serious lung and liver damage. The gene for a1-antitrypsin is polymorphic (i.e., it occurs as many different sequence variants) and many variants of a1-antitrypsin are either poorly secreted by the liver or function poorly in the lungs. Even worse, tobacco smoke oxidizes the critical Met residue in the flexible loop of a1-antitrypsin, and smokers, especially those who carry mutants of this protein, often develop emphysema—the destruction of the elastin connective tissue in the lungs. The flexible loop of a1-antitrypsin—its mousetrap spring—is also its Achilles heel. Mutations in this loop make the protein vulnerable to aberrant conformational changes. The Z-mutation of a1-antitrypsin is an interesting case, with a Lys in place of Glu at residue 342 (indicated by the arrow in M) at the base of the flexible loop. This causes partial loop insertion in the large b-sheet (M*). This induces the modified b-sheet to accept the flexible loop of another a1-antitrypsin, forming a dimer. Repetition of these events forms polymers, which are trapped in the liver (often leading to cirrhosis and death). Z variants that manage to make it to the lungs associate so slowly with elastase that they are ineffective in preventing lung damage. " (a) Elastase (dark grey) is inactivated by binding to a1-anti-

(a)

trypsin. When elastase binds, cleaving the flexible loop at a Met residue, the rest of the loop (the red b-strand) rotates more than 180° and inserts into the green b-sheet, swinging the elastase to the other end of the molecule. At this new location, the elastase is distorted and inactivated. (b) In the Z-mutant of a1-antitrypsin, the flexible loop is only partially inserted in the large b-sheet, promoting polymer formation and trapping a1-antitrypsin at its site of synthesis in the liver. The consequences of this are cirrhosis of the liver, as well as lung damage, since the small amount of a1-antitrypsin that reaches the lungs is ineffective in preventing lung damage. Individual monomers in the a1-antitrypsin polymer are coloured red, blue, and gold (far right). (From Lomas, D. A., et al., 2005. Molecular mousetraps and serpinopathies. Biochem Soc. Transactions 33:321–330. figure provided by David Lomas.)

#1-AT

Elastase

Met-containing loop

Elastase

#1-AT

(b) Z

M

M*

D P NEL

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Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

179

HUMAN BIOCHEMISTRY

Diseases of Protein Folding A number of human diseases are linked to abnormalities of protein folding. Protein misfolding may cause disease by a variety of mechanisms. For example, misfolding may result in loss of function and

the onset of disease. The following table summarizes several other mechanisms and provides an example of each.

Disease

Affected Protein

Mechanism

Alzheimer’s disease

b-Amyloid peptide (derived from amyloid precursor protein)

Misfolded b-amyloid peptide accumulates in human neural tissue, forming deposits known as neuritic plaques.

Familial amyloidotic polyneuropathy

Transthyretin

Aggregation of unfolded proteins. Nerves and other organs are damaged by deposits of insoluble protein products.

Cancer

p53

p53 prevents cells with damaged DNA from dividing. One class of p53 mutations leads to misfolding; the misfolded protein is unstable and is destroyed.

Creutzfeldt-Jakob disease (human equivalent of mad cow disease)

Prion

Prion protein with an altered conformation (PrPSC) may seed conformational transitions in normal PrP (PrPC) molecules.

Hereditary emphysema

a1-Antitrypsin

Mutated forms of this protein fold slowly, allowing its target, elastase, to destroy lung tissue.

Cystic fibrosis

CFTR (cystic fibrosis transmembrane conductance regulator)

Folding intermediates of mutant CFTR forms don’t dissociate freely from chaperones, preventing the CFTR from reaching its destination in the membrane.

HUMAN BIOCHEMISTRY

Structural Genomics The prodigious advances in genome sequencing in recent years, together with advances in techniques for protein structure determination, have not only provided much new information for biochemists but have also spawned a new field of investigation: structural genomics, the large-scale analysis of protein structures and functions based on gene sequences. The scale of this new endeavour is daunting: hundreds of thousands of gene sequences are rapidly being determined, and current estimates suggest that there are probably less than 10 000 distinct and stable polypeptide folding patterns in nature. The feasibility of large-scale, high-throughput structure determination programs is being explored in a variety of pilot studies in Europe, Asia, and North America. These efforts seek to add 20 000 or more new protein structures to our collected knowledge in the near future; from this wealth of new information, it should be possible to predict and determine new structures from sequence information alone. This effort will be vastly more complex and more expensive than the Human Genome Project. It presently costs about $100 000 to determine the structure

6.5

of the typical globular protein, and one of the goals of structural genomics is to reduce this number to $20 000 or less. Advances in techniques for protein crystallization, X-ray diffraction, and NMR spectroscopy, the 3 techniques essential to protein structure determination, will be needed to reach this goal in the near future. The payoffs anticipated from structural genomics are substantial. Access to large amounts of new 3-dimensional structural information should accelerate the development of new families of drugs. The ability to scan databases of chemical entities for activities against drug targets will be enhanced if large numbers of new protein structures are available, especially if complexes of drugs and target proteins can be obtained or predicted. The impact of structural genomics will also extend, however, to functional genomics—the study of the functional relationships of genomic content—which will enable the comparison of the composite functions of whole genomes, leading eventually to a complete biochemical and mechanistic understanding of all organisms, including humans.

How Do Protein Subunits Interact at the Quaternary Level of Protein Structure?

Many proteins exist in nature as oligomers, complexes composed of (often symmetric) non-covalent assemblies of 2 or more monomer subunits. In fact, subunit association is a common feature of macromolecular organization in biology. Most intracellular enzymes are oligomeric and may be composed of either a single type of monomer subunit NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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FIGURE 6.41 The quaternary structure of liver alcohol dehydrogenase. Within each subunit is a 6-stranded parallel sheet. Between the 2 subunits is a 2-stranded antiparallel sheet (pdb id = 1CDO). (Jane Richardson.)

(homomultimers) or several different kinds of subunits (heteromultimers). The simplest case is a protein composed of identical subunits. Liver alcohol dehydrogenase, shown in Figure 6.41 above, is such a protein. Alcohol consumed in beer or a mixed drink is oxidized in the liver by alcohol dehydrogenase. Hormonal signals modulate blood sugar levels by controlling the activity of glycogen phosphorylase, an elegantly regulated homodimeric muscle enzyme. Oxygen is carried in the blood by hemoglobin, which contains 2 each of 2 different subunits (heterotetramer). A counterpoint to these small clusters is made by the proteins that form large polymeric aggregates. Proteins are synthesized on large complexes of many protein units and several RNA molecules called ribosomes. Muscle contraction depends on large polymer clusters of the protein myosin sliding along filamentous polymers of another protein, actin. The way in which separate folded monomeric protein subunits associate to form the oligomeric protein constitutes the quaternary structure of that protein. Table 6.3 lists several proteins and their subunit compositions (see also Table 4.2). Proteins with 2–4 subunits predominate in nature, but many cases of higher numbers exist.

TABLE 6.3 Aggregation Symmetries of Globular Proteins Protein

Number of Subunits

Alcohol dehydrogenase

2

Malate dehydrogenase

2

Superoxide dismutase

2

Triose phosphate isomerase

2

Glycogen phosphorylase

2

Aldolase

3

Bacteriochlorophyll protein

3

Concanavalin A

4

Glyceraldehyde-3-phosphate dehydrogenase

4

Immunoglobulin

4

Lactate dehydrogenase

4

Pre-albumin

4

Pyruvate kinase

4

Phosphoglycerate mutase Hemoglobin Insulin Aspartate transcarbamoylase

4 2+2 6 6+6

Glutamine synthetase

12

TMV protein disc

17

Apoferritin

24

Coat of tomato bushy stunt virus

180 NEL

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Chapter 6 (a) Isologous association

(b) Heterologous association

Proteins: Secondary, Tertiary, and Quaternary Structure

(c) Heterologous tetramer

181

FIGURE 6.42 Isologous and heterologous associations between protein subunits. (a) An isologous interaction between 2 subunits with a 2-fold axis of symmetry perpendicular to the plane of the page. (b) A heterologous interaction that could lead to the formation of a long polymer. (c) A heterologous interaction leading to a closed structure—a tetramer. (d) A tetramer formed by 2 sets of isologous interactions.

(d) Isologous tetramer

Symmetry axis (a) Monomer B

The subunits of an oligomeric protein typically fold independently and then interact with other subunits. The surfaces at which subunits interact are similar in nature to the interiors of the individual subunits—closely packed with both polar and hydrophobic interactions. Interacting surfaces must therefore possess complementary arrangements of polar and hydrophobic groups. Oligomeric associations of protein subunits can be divided into those between identical subunits and those between non-identical subunits. Interactions among identical subunits can be further distinguished as either isologous or heterologous. In isologous interactions, the interacting surfaces are identical and the resulting structure is necessarily dimeric and closed, with a 2-fold axis of symmetry (Figure 6.42 above). If any additional interactions occur to form a trimer or tetramer, these must use different interfaces on the protein’s surface. Many proteins, such as transthyretin, form tetramers by means of 2 sets of isologous interactions (Figure 6.43). Such structures possess 3 different 2-fold axes of symmetry. In contrast, heterologous associations among subunits involve non-identical interfaces. These surfaces must be complementary, but they are generally not symmetric.

Monomer A

(b)

Monomer A′

Monomer B′

There Is Symmetry in Quaternary Structures Many multimeric proteins are symmetric arrangements of asymmetric objects (the monomer subunits). All the polypeptide’s a-carbons are asymmetric, and the polypeptide nearly always folds to form a low-symmetry structure. (The long helical arrays formed by some synthetic polypeptides are an exception.) Thus, protein subunits do not have mirror reflection planes, points, or axes of inversion. The only symmetry operation possible for protein subunits is a rotation. The most common symmetries observed for multisubunit proteins are cyclic symmetry and dihedral symmetry. In cyclic symmetry, the subunits are arranged around a single rotation axis, as shown in Figure 6.44. If there are 2 subunits, the axis is referred to as a 2-fold rotation axis. Rotating the quaternary structure 180° about this axis gives a structure identical to the original one. With 3 subunits arranged about a 3-fold rotation axis, a rotation of 120° about that axis gives an identical structure. Dihedral symmetry occurs when a structure possesses at least one 2-fold rotation axis perpendicular to another n-fold rotation axis. This type of subunit arrangement (Figure 6.44) occurs in annexin XII (where n = 3).

Monomer A Monomer B FIGURE 6.43 Many proteins form tetramers by means of 2 sets of isologous interactions. The dimeric (a) and tetrameric (b) forms of transthyretin (also known as pre-albumin) are shown here (pdb id = 1GKE). The monomers of this protein form a dimer in a manner that extends the large monomer b-sheet. The tetramer is formed by isologous interactions between the large b-sheets of 2 dimers.

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Dimer ARNT PAS-B (pdb id = 2HV1)

Trimer E. blattae acid phosphatase (pdb id = 2EOI)

Cyclic hexamer Circadian clock protein KaiC (pdb id = 1TF7)

Heptamer M. tuberculosis chaperonin-10 (pdb id = 1HX5)

Tetramer B. anthracis dihydrodipicolinate synthase (pdb id = 1XL9)

Trimer of dimers Annexin XII (pdb id = 1DM5)

Octamer Limulus polyphemus SAP-like pentraxin (pdb id = 1QTJ)

Pentamer Shiga-like toxin I B (pdb id = 1CZG)

Bundled hexamer Uridylate kinase (pdb id = 2A1F)

Dodecamer Lactococcus lactis MG1363 DpsB protein (pdb id = 1ZS3)

FIGURE 6.44 Multimeric proteins are symmetric arrangements of asymmetric objects. A variety of symmetries is displayed in these multimeric structures.

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Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

183

Quaternary Association Is Driven by Weak Forces Weak forces stabilize quaternary structures. Typical dissociation constants for simple 2-subunit associations range from 10-8 to 10-16 M. These values correspond to free energies of association of about 50–100 kJ/mol at 37°C. Dimerization of subunits is accompanied by both favourable and unfavourable energy changes. The favourable interactions include van der Waals interactions, hydrogen bonds, ionic bonds, and hydrophobic interactions. However, considerable entropy loss occurs when subunits interact. When 2 subunits move as 1, 3 translational degrees of freedom are lost for 1 subunit because it is constrained to move with the other one. In addition, many peptide residues at the subunit interface, which were previously free to move on the protein surface, now have their movements restricted by the subunit association. This unfavourable energy of association is in the range of 80–120 kJ/mol for temperatures of 25°C37°C. Thus, to achieve stability, the dimerization of 2 subunits must involve approximately 130–220 kJ/mol of favourable interactions.1 van der Waals interactions at protein interfaces are numerous, often running to several hundred for a typical monomer–monomer association. This would account for about 150–200 kJ/mol of favourable free energy of association. However, when solvent is removed from the protein surface to form the subunit–subunit contacts, nearly as many van der Waals associations are lost as are made. One subunit is simply trading water molecules for peptide residues in the other subunit. As a result, the energy of subunit association due to van der Waals interactions actually contributes little to the stability of the dimer. Hydrophobic interactions at the subunit–subunit interface, however, are generally very favourable. For many proteins, the subunit association process effectively buries as much as 20 nm2 of surface area previously exposed to solvent, resulting in as much as 100–200 kJ/mol of favourable hydrophobic interactions. Together with whatever polar interactions occur at the protein–protein interface, this is sufficient to account for the observed stabilization that occurs when 2 protein subunits associate. An additional and important factor contributing to the stability of subunit associations for some proteins is the formation of disulfide bonds between different subunits. All antibodies are a2b2-tetramers composed of 2 heavy chains (53–75 kD) and 2 light chains (23 kD). In addition to intrasubunit disulfide bonds (4 per heavy chain, 2 per light chain), 2 intersubunit disulfide bridges hold the 2 heavy chains together and a disulfide bridge links each of the 2 light chains to a heavy chain (Figure 6.45). N 4.5 nm

S

N

S

An

S

tig

en

S nd in

S

bi

S

g

S

S C SS

S

C S

S

S

S

(CH2O)n addition site

CH2 S S Hinge region

S

CH3

446 S

S

VH N

S

S

S VL

Light N

1For

CH1

S

S

S

An t

ig

en

bi

nd

in

g

S

S CL

21

4

SS

S

S

FIGURE 6.45 Schematic drawing of an immunoglobulin molecule, showing the intermolecular and intramolecular disulfide bonds. Two identical L chains are joined with 2 identical H chains. Each L chain is held to an H chain via an interchain disulfide bond. The variable regions of the 4 polypeptides lie at the ends of the arms of the Y-shaped molecule. These regions are responsible for the antigen recognition function of antibody molecules. For purposes of illustration, some features are shown on only one or the other L chain or H chain, but all features are common to both chains.

Heavy

example, 130 kJ/mol of favourable interaction minus 80 kJ/mol of unfavourable interaction equals a net free energy of association of 50 kJ/mol.

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A DEEPER LOOK

Immunoglobulins: All the Features of Protein Structure Brought Together The immunoglobulin structure in Figure 6.45 represents the confluence of all the details of protein structure that have been thus far discussed. As for all proteins, the primary structure determines other aspects of structure. There are numerous elements of secondary structure, including b-sheets and tight turns. The tertiary structure consists of 12 distinct domains, and the protein adopts a heterotetrameric quaternary structure. To make matters more interesting, both intrasubunit and intersubunit disulfide linkages act to stabilize the discrete domains and to stabilize the tetramer itself. One more level of sophistication awaits. As discussed in Chapter 28, the amino acid sequences of both light and heavy

immunoglobulin chains are not constant! Instead, the primary structure of these chains is highly variable in the N-terminal regions (first 108 residues). Heterogeneity of the amino acid sequence leads to variations in the conformation of these variable regions. This variation accounts for antibody diversity and the ability of antibodies to recognize and bind a virtually limitless range of antigens. This full potential of antibody:antigen recognition enables organisms to mount immunological responses to almost any antigen that might challenge the organism.

Immunoglobulin-Based Techniques We’d be amiss at this critical point in the text if we did not mention an important immunoglobulin-based technique. One general, yet versatile method used today for assaying antibodies and antigens is the enzyme-linked immunosorbent assay (ELISA). Numerous variations of ELISA exist, including indirect, direct competitive, and sandwich ELISA. Both indirect and competitive ELISA employ solid-phase antigen screening by incubation with antibody or antibody:antigen complexes, respectively. Secondary antibodies linked with enzyme are then added. The enzyme portion of the complex is capable of reacting with a fluorogenic substrate, resulting in a detectable coloured product. In sandwich ELISA, however, antigen is incubated with solid-phase bound antibody followed by detection with secondary antibody–enzyme complexes as described above. Sandwich ELISA appears to be the most sensitive of the 3 assay systems and provides a rapid way for assessing the presence or absence of antibody and antigen.

Open Quaternary Structures Can Polymerize All the quaternary structures we have considered to this point have been closed structures, with a limited capacity to associate. Many proteins in nature associate to form open heterologous structures, which can polymerize more or less indefinitely, creating structures that are both esthetically attractive and functionally important to the cells or tissue in which they exist. One such protein is tubulin, the ab-dimeric protein that polymerizes into long, tubular structures that are the structural basis of cilia, flagella, and the cytoskeletal matrix. The microtubule thus formed (Figure 6.46) may be viewed as consisting of 13 parallel filaments arising from end-to-end aggregation of the tubulin dimers. Human immunodeficiency virus, HIV, the causative agent of AIDS (also discussed in Chapter 13), is enveloped by a spherical shell composed of hundreds of coat protein subunits, a large-scale, but closed, quaternary association.

α β

8.0 nm

There Are Structural and Functional Advantages to Quaternary Association There are several important consequences when protein subunits associate in oligomeric structures.

3.5- to 4.0-nm subunit FIGURE 6.46 The structure of a typical microtubule, showing the arrangement of the a- and b-monomers of the tubulin dimer.

Stability One general benefit of subunit association is a favourable reduction of the protein’s surface-to-volume ratio. The surface-to-volume ratio becomes smaller as the radius of any particle or object becomes larger. (This is because surface area is a function of the radius squared and volume is a function of the radius cubed.) Because interactions within the protein usually tend to stabilize the protein energetically, and because the interaction of the protein surface with solvent water is often energetically unfavourable, decreased NEL

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Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

185

surface-to-volume ratios usually result in more stable proteins. Subunit association may also serve to shield hydrophobic residues from solvent water. Subunits that recognize either themselves or other subunits avoid any errors arising in genetic translation by binding mutant forms of the subunits less tightly. Genetic Economy and Efficiency Oligomeric association of protein monomers is genetically economical for an organism. Less DNA is required to code for a monomer that assembles into a homomultimer than for a large polypeptide of the same molecular mass. Another way to look at this is to realize that virtually all the information that determines oligomer assembly and subunit–subunit interaction is contained in the genetic material needed to code for the monomer. For example, HIV protease, an enzyme that is a dimer of identical subunits, performs a catalytic function similar to homologous cellular enzymes that are single polypeptide chains of twice the molecular mass (see Chapter 13).

HUMAN BIOCHEMISTRY

Faster-Acting Insulin: Genetic Engineering Solves a Quaternary Structure Problem Insulin is a peptide hormone secreted by the pancreas that regulates glucose metabolism in the body. Insufficient production of insulin or failure of insulin to stimulate target sites in liver, muscle, and adipose tissue leads to the serious metabolic disorder known as diabetes mellitus. Diabetes afflicts millions of people worldwide. Diabetic individuals typically exhibit high levels of glucose in the blood, but insulin injection therapy allows these individuals to maintain normal levels of blood glucose. Insulin is composed of 2 peptide chains covalently linked by disulfide bonds (see Figure 5.17). This “monomer” of insulin is the active form that binds to receptors in target cells. However, in solution, insulin spontaneously forms dimers, which themselves aggregate to form hexamers. The surface of the insulin molecule that self-associates to form hexamers is also the surface that binds to insulin receptors in target cells. Thus, hexamers of insulin are inactive. Insulin released from the pancreas is monomeric and acts rapidly at target tissues. However, when insulin is administered (by injection) to a diabetic patient, the insulin hexamers dissociate slowly and the patient’s blood glucose levels typically drop slowly (over several hours).

In 1988, G. Dodson showed that insulin could be genetically engineered to prefer the monomeric (active) state. Dodson and his colleagues used recombinant DNA technology (discussed in Chapter 27) to produce insulin with an aspartate residue replacing a proline at the contact interface between adjacent subunits. The negative charge on the Asp side chain creates electrostatic repulsion between subunits and increases the dissociation constant for the hexamer ∆ monomer equilibrium. Injection of this mutant insulin into test animals produced more rapid decreases in blood glucose than did ordinary insulin. This mutant insulin, marketed by the Danish pharmaceutical company Novo as NovoLog in the United States and as NovoRapid in Europe, may eventually replace ordinary insulin in the treatment of diabetes. NovoLog has a faster rate of absorption, a faster onset of action, and a shorter duration of action than regular human insulin. It is particularly suited for mealtime dosing to control postprandial glycemia, the rise in blood sugar following consumption of food. Regular human insulin acts more slowly, so patients must usually administer it 30 minutes before eating.

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

There can be no doubt that one of the greatest medical achievements of the twentieth century was the discovery of insulin. This historic finding was made in Toronto, at the University of Toronto during the early 1920s. The initial studies carried out on diabetic dogs by Canadian scientists Fredrick Banting and Charles Best (pictured) showed that administration of pancreatic extracts could reduce blood sugar levels in these animals. Another Canadian scientist, James Collip, worked actively on purifying the main principle in the pancreas, insulin, which proved to have dramatic effects in diabetics. Large-scale production of insulin to treat thousands of diabetics worldwide was carried out by Connaught Laboratories in Canada and Eli Lilly in the United States. In 1923, the Nobel Prize was awarded for their formidable discovery. Clearly, the breakthrough discovery of insulin by these incredible Canadian scientists and the countless lives insulin saved as a result of this will forever remain a “miracle” finding etched in history.

The Art Archive at Art Resource, NY

Banting and Best: The Great Canadian Discovery

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Part 1 Building Blocks of the Cell

Bringing Catalytic Sites Together Many enzymes (see Chapters 12–14) derive at least some of their catalytic power from oligomeric associations of monomer subunits. This can happen in several ways. The monomer may not constitute a complete enzyme active site. Formation of the oligomer may bring all the necessary catalytic groups together to form an active enzyme. For example, the active sites of bacterial glutamine synthetase are formed from pairs of adjacent subunits. The dissociated monomers are inactive. Oligomeric enzymes may also carry out different but related reactions on different subunits. Thus, tryptophan synthase is a tetramer consisting of pairs of different subunits, a2b2. Purified a-subunits catalyze the following reaction: indoleglycerol phosphate ∆ indole + glyceraldehyde-3-phosphate and the b-subunits catalyze this reaction: indole + L-serine ∆ L-tryptophan. Indole, the product of the a-reaction and the reactant for the b-reaction, is passed directly from the a-subunit to the b-subunit and cannot be detected as a free intermediate. Cooperativity There is another, more important consequence when monomer subunits associate into oligomeric complexes. Most oligomeric enzymes regulate catalytic activity by means of subunit interactions, which may give rise to cooperative phenomena. Multisubunit proteins typically possess multiple binding sites for a given ligand. If the binding of ligand at one site changes the affinity of the protein for ligand at the other binding sites, the binding is said to be cooperative. Information transfer in this manner across long distances in proteins is termed allostery, literally “action at another site.” Increases in affinity at subsequent sites represent positive cooperativity, whereas decreases in affinity correspond to negative cooperativity. The points of contact between protein subunits provide a mechanism for this signal transduction through the protein structure and for communication between the subunits. This in turn provides a way in which the binding of ligand to one subunit can influence the binding behaviour at the other subunits. Such cooperative behaviour, discussed in greater depth in Chapter 14, is the underlying mechanism for regulation of many biological processes.

SUMMARY 6.1 What Non-covalent Interactions Stabilize Protein Structure? Several different kinds of non-covalent interactions are of vital importance in protein structure. Hydrogen bonds, hydrophobic interactions, electrostatic bonds, and van der Waals forces are all non-covalent in nature yet are extremely important influences on protein conformations. The stabilization free energies afforded by each of these interactions are highly dependent on the local environment within the protein. Hydrogen bonds are generally made wherever possible within a given protein structure. Hydrophobic interactions form because nonpolar side chains of amino acids and other non-polar solutes prefer to cluster in a non-polar environment rather than to intercalate in a polar solvent such as water. Electrostatic interactions include the attraction between opposite charges and the repulsion of like charges in the protein. van der Waals interactions involve instantaneous dipoles and induced dipoles that arise because of fluctuations in the electron charge distributions of adjacent non-bonded atoms. 6.2 What Role Does the Amino Acid Sequence Play in Protein Structure? All the information necessary for folding the peptide chain into its “native” structure is contained in the amino acid sequence of the peptide. Just how proteins recognize and interpret the information that is stored in the polypeptide sequence is not yet well understood. It may be assumed that certain loci along the peptide chain act as nucleation points, which initiate folding processes that eventually lead to the correct structures. Regardless of how this process operates, it must take the protein correctly to the

final native structure, without getting trapped in a local energyminimum state, which, although stable, may be different from the native state itself. 6.3 What Are the Elements of Secondary Structure in Proteins, and How Are They Formed? Secondary structure in proteins forms to maximize hydrogen bonding and maintain the planar nature of the peptide bond. Secondary structures include a-helices, b-sheets, and tight turns. 6.4 How Do Polypeptides Fold into Three-Dimensional Protein Structures? First, secondary structures—helices and sheets—form whenever possible as a consequence of the formation of large numbers of hydrogen bonds. Second, a-helices and b-sheets often associate and pack close together in the protein. There are a few common methods for such packing to occur. Third, because the peptide segments between secondary structures in the protein tend to be short and direct, the peptide does not execute complicated twists and knots as it moves from one region of a secondary structure to another. A consequence of these 3 principles is that protein chains are usually folded so that the secondary structures are arranged in one of a few common patterns. For this reason, there are families of proteins that have similar tertiary structure, with little apparent evolutionary or functional relationship among them. Finally, proteins generally fold so as to form the most stable structures possible. The stability of most proteins arises from (1) the formation of large numbers of intramolecular hydrogen bonds and (2) the reduction in the surface area accessible to solvent that occurs upon folding. NEL

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Chapter 6

6.5 How Do Protein Subunits Interact at the Quaternary Level of Protein Structure? The subunits of an oligomeric protein typically fold into apparently independent globular conformations and then interact with other subunits. The particular surfaces at which protein

Proteins: Secondary, Tertiary, and Quaternary Structure

187

subunits interact are similar in nature to the interiors of the individual subunits. These interfaces are closely packed and involve both polar and hydrophobic interactions. Interacting surfaces must therefore possess complementary arrangements of polar and hydrophobic groups.

PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. The central rod domain of a keratin protein is approximately 312 residues in length. What is the length (in Å) of the keratin rod domain? If this same peptide segment were a true a-helix, how long would it be? If the same segment were a b-sheet, what would its length be? 2. A teenager can grow 4 inches in a year during a “growth spurt.” Assuming that the increase in height is due to vertical growth of collagen fibres (in bone), calculate the number of collagen helix turns synthesized per minute. 3. Discuss the potential contributions to hydrophobic and van der Waals interactions and ionic and hydrogen bonds for the side chains of Asp, Leu, Tyr, and His in a protein. 4. Pro is the amino acid least commonly found in a-helices but most commonly found in b-turns. Discuss the reasons for this behaviour. 5. For flavodoxin (pdb id = 5NLL), identify the right-handed crossovers and the left-handed crossovers in the parallel b-sheet. 6. Choose any 3 regions in the Ramachandran plot and discuss the likelihood of observing that combination of f and c in a peptide or protein. Defend your answer using suitable molecular models of a peptide. 7. A new protein of unknown structure has been purified. Gel filtration chromatography reveals that the native protein has a molecular weight of 240 000. Chromatography in the presence of 6 M guanidine hydrochloride yields only a peak for a protein of Mr 60 000. Chromatography in the presence of 6 M guanidine hydrochloride and 10 mM b -mercaptoethanol yields peaks for proteins of Mr 34 000 and 26 000. Explain what can be determined about the structure of this protein from these data. 8. Two polypeptides, A and B, have similar tertiary structures, but A normally exists as a monomer, whereas B exists as a tetramer, B4. What differences might be expected in the amino acid composition of A versus B?

9. The hemagglutinin protein in influenza virus contains a remarkably long a-helix, with 53 residues. a. How long is this a-helix (in nm)? b. How many turns does this helix have? c. Each residue in an a-helix is involved in 2 H bonds. How many H bonds are present in this helix? 10. It is often observed that Gly residues are conserved in proteins to a greater degree than other amino acids. From what you have learned in this chapter, suggest a reason for this observation. 11. Explain how Anfinsen confirmed that the information driving the formation of 3D structure is found in the primary sequence. 12. Which amino acids would be capable of forming H bonds with a lysine residue in a protein? 13. Poly-L-glutamate adopts an a-helical structure at low pH but becomes a random coil above pH 5. Explain this behaviour. 14. Imagine that the dimensions of the alpha helix were such that there were exactly 3.5 amino acids per turn, instead of 3.6. What would be the consequences for coiled-coil structures? 15. Consider the following peptide sequences: EANQIDEMLYNVQCSLTTLEDTVPW LGVHLDITVPLSWTWTLYVKL QQNWGGLVVILTLVWFLM CNMKHGDSQCDERTYP YTREQSDGHIPKMNCDS AGPFGPDGPTIGPK Which of the preceding sequences would be likely to be found in each of the following: a. a parallel b-sheet b. an antiparallel b-sheet c. a tropocollagen molecule d. the helical portions of a protein found in your hair

FURTHER READING General Bliss, M., 1984. Banting: A Biography. Toronto: McClelland and Stewart Limited, The Canadian Publishers. Branden, C., and Tooze, J., 1991. Introduction to Protein Structure. New York: Garland Publishing. Chothia, C., 1984. Principles that determine the structure of proteins. Annual Review of Biochemistry 53:537–572. Fink, A., 2005. Natively unfolded proteins. Current Opinion in Structural Biology 15:35–41.

Greene, L., Lewis, T., et al., 2006. The CATH domain structure database: New protocols and classification levels give a more comprehensive resource for exploring evolution. Nucleic Acids Research 35:D291–D297. Hardie, D. G., and Coggins, J. R., Eds., 1986. Multidomain Proteins: Structure and Evolution. New York: Elsevier. Harper, E., and Rose, G. D., 1993. Helix stop signals in proteins and peptides: The capping box. Biochemistry 32:7605–7609. Hornbeck, P., 1991. Assays for antibody production. Current Protocols in Immunology 2.1.1–2.1.22.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

Judson, H. F., 1979. The Eighth Day of Creation. New York: Simon and Schuster.

Mok, K., Kuhn, L., et al., 2007. A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein. Nature 447:106–109.

Laskowski, R. A., and Thornton, J. M., 2008. Understanding the molecular machinery of genetics through 3D structures. Nature Reviews Genetics 9:141–151.

Murphy, K. P., 2001. Protein Structure, Stability, and Folding. New Jersey: Humana Press.

Lupas, A., 1996. Coiled coils: New structures and new functions. Trends in Biochemical Sciences 21:375–382. Petsko, G., and Ringe, D., 2004. Protein Structure and Function. London: New Science Press. Richardson, J. S., 1981. The anatomy and taxonomy of protein structure. Advances in Protein Chemistry 34:167–339. Schulze, A. J., Huber, R., et al., 1994. Structural aspects of serpin inhibition. FEBS Letters 344:117–124. Smith, T., 2000. Structural genomics—special supplement. Nature Structural Biology Volume 7, Issue 11S. [This entire supplemental issue is devoted to structural genomics and contains a trove of information about this burgeoning field.] Tompa, P., 2002. Intrinsically unstructured proteins. Trends in Biochemical Sciences 27:527–533. Tompa, P., Szasz, C., et al., 2005. Structural disorder throws new light on moonlighting. Trends in Biochemical Sciences 30:484–489. Uversky, V. N., 2002. Natively unfolded proteins: A point where biology waits for physics. Protein Science 11:739–756. Webster, D. M., 2000. Protein Structure Prediction—Methods and Protocols. New Jersey: Humana Press. Protein Folding Aurora, R., Creamer, T., et al., 1997. Local interactions in protein folding: Lessons from the a-helix. The Journal of Biological Chemistry 272:1413–1416. Baker, D., 2000. A surprising simplicity to protein folding. Nature 405:39–42. Creighton, T. E., 1997. How important is the molten globule for correct protein folding? Trends in Biochemical Sciences 22:6–11. Deber, C. M., and Therien, A. G., 2002. Putting the b-breaks on membrane protein misfolding. Nature Structural Biology 9:318–319.

Myers, J. K., and Oas, T. G., 2002. Mechanisms of fast protein folding. Annual Review of Biochemistry 71:783–815. Orengo, C., and Thornton, J., 2005. Protein families and their evolution— a structural perspective. Annual Review of Biochemistry 74:867–900. Radford, S. E., 2000. Protein folding: Progress made and promises ahead. Trends in Biochemical Sciences 25:611–618. Raschke, T. M., and Marqusee, S., 1997. The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions. Nature Structural Biology 4:298–304. Srinivasan, R., and Rose, G. D., 1995. LINUS: A hierarchic procedure to predict the fold of a protein. Proteins: Structure, Function and Genetics 22:81–99. Secondary Structure Xiong, H., Buckwalter, B., et al., 1995. Periodicity of polar and nonpolar amino acids is the major determinant of secondary structure in selfassembling oligomeric peptides. Proceedings of the National Academy of Sciences 92:6349–6353. Structural Studies Bradley, P., Misura, K., et al., 2005. Toward high-resolution de novo structure prediction for small proteins. Science 309:1868–1871. Hadley, C., and Jones, D., 1999. A systematic comparison of protein structure classifications: SCOP, CATH, and FSSP. Structure 7:1099–1112. Lomas, D., Belorgey, D., et al., 2005. Molecular mousetraps and the serpinopathies. Biochemical Society Transactions 33(part 2):321–330. Wagner, G., Hyberts, S., et al., 1992. NMR structure determination in solution: A critique and comparison with X-ray crystallography. Annual Review of Biophysics and Biomolecular Structure 21:167–242. Wand, A. J., 2001. Dynamic activation of protein function: A view emerging from NMR spectroscopy. Nature Structural Biology 8:926–931.

Dill, K. A., and Chan, H. S., 1997. From Levinthal to pathways to funnels. Nature Structural Biology 4:10–19.

Diseases of Protein Folding

Dinner, A. R., Sali, A., et al., 2001. Understanding protein folding via free-energy surfaces from theory and experiment. Trends in Biochemical Sciences 25:331–339.

Bucciantini, M., Giannoni E., et al., 2002. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416:507–511.

Han, J.-H., Batey, S., et al., 2007. The folding and evolution of multidomain proteins. Nature Reviews Molecular Cell Biology 8:319–330.

Sifers, R. M., 1995. Defective protein folding as a cause of disease. Nature Structural Biology 2:355–367.

Kelly, J., 2005. Structural biology: Form and function instructions. Nature 437:486–487.

Stein, P. E., and Carrell, R. W., 1995. What do dysfunctional serpins tell us about molecular mobility and disease? Nature Structural Biology 2:96–113.

Mirny, L., and Shakhnovich, E., 2001. Protein folding theory: From lattice to all-atom models. Annual Review of Biophysics and Biomolecular Structure 30:361–396.

Thomas, P. J., Qu, B-H. et al., 1995. Defective protein folding as a basis of human disease. Trends in Biochemical Sciences 20:456–459.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

7

Carbohydrates and the Glycoconjugates of Cell Surfaces

! Discovery of Honey by Bacchus—Piero di Cosimo

Sugar in the gourd and honey in the horn, I never was so happy since the hour I was born. Turkey in the Straw, stanza 6 (classic American folk tune)

KEY QUESTIONS What Are Carbohydrates, and How Are They Named?

7.2

What Is the Structure and Chemistry of Monosaccharides?

7.3

What Is the Structure and Chemistry of Oligosaccharides?

7.4

What Is the Structure and Chemistry of Polysaccharides?

7.5

What Are Glycoproteins, and How Do They Function in Cells?

ESSENTIAL QUESTION

7.6

Carbohydrates are a versatile class of molecules of the formula (CH2O)n. They are a major form of stored energy in organisms, and they are the metabolic precursors of virtually all other biomolecules. Conjugates of carbohydrates with proteins and lipids perform a variety of functions, including recognition events that are important in cell growth, transformation, and other processes.

How Do Proteoglycans Modulate Processes in Cells and Organisms?

7.7

Do Carbohydrates Provide a Structural Code?

Burstein Collection/Corbis

7.1

What is the structure, chemistry, and biological function of carbohydrates?

WHY IT MATTERS Before we begin our discussion on the structure and function of carbohydrates, we pose the question: “Why are carbohydrates important to the cell?” Carbohydrates, also known as sugars, are used for energy (e.g., glycogen, starch) and structure (e.g., cellulose, chitin), and they assist in cell signalling (e.g., lectins). Carbohydrates, when linked to proteins, can target them to membranes and also anchor them there. Also, specific proteins recognize and bind to carbohydrates, and mediate processes such as cell adhesion, growth regulation, inflammation, immunity, and cancer metastasis. Thus, the attachment of carbohydrates to proteins represents an important post-translational modification of proteins. Carbohydrates are the single most abundant class of organic molecules found in nature. Energy from the sun captured by green plants, algae, and some bacteria during photosynthesis (see Chapter 20) converts more than 250 billion kilograms of carbon dioxide into carbohydrates every day on earth. In turn, carbohydrates are the metabolic precursors of virtually all other biomolecules. Breakdown of carbohydrates provides the energy that sustains animal life. In addition, carbohydrates are covalently linked with a variety of other molecules. These glycoconjugates are important components of cell walls and extracellular structures in plants, animals, and bacteria. In addition to the structural roles such molecules play, they also serve in a variety of processes involving

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

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190

Part 1 Building Blocks of the Cell

BIOCHEMISTRY: A CANADIAN CONTEXT

Dr. Raymond Urgel Lemieux was a Canadian organic chemist, born in 1920 in Lac La Biche, Alberta, Canada, who pioneered a number of discoveries in the field of carbohydrate chemistry. He majored in Chemistry at the University of Alberta and received his Ph.D. at McGill University. In 1946, he was awarded a post-doctoral scholarship to study at Ohio State University, where Bristol Laboratories Inc. sponsored his research on the structure of streptomycin. In 1953, together with fellow researcher George Huber, Lemieux was the first to successfully synthesize sucrose in the laboratory. This breakthrough happened in 1953, while Lemieux was working for the National Research Council in Saskatoon. A  year later, he was invited to help develop the Chemistry Department at the University of Ottawa, where he served as the Dean of Pure and Applied Science. He then went on to develop methods for the synthesis of oligosaccharides, which led to improved treatments for leukemia and hemophilia, as well as the development of new antibiotics, blood reagents, and organ anti-rejection drugs. While in Alberta, Lemieux established a number of biochemical companies, including R&L Molecular Research Ltd., Raylo Chemicals Ltd., and Chembiomed. In 1990, Dr. Lemieux published an autobiography entitled Explorations with Sugars: How Sweet It Was. He was the first recipient of the Gerhard Herzberg Canada Gold Medal for Science and Engineering and also received 5 other international awards, including the Albert Einstein World Award in Science in 1992 and the Gairdner Foundation International Award in 1985. In 1999, Dr. Lemieux was named Companion of the Order of Canada. His research helped to establish the rapidly growing field of glycobiology.

Courtesy, University of Alberta Archives (R. Lemieux Biographical file)

The Synthesis of Sucrose

recognition between cell types, or recognition of cellular structures by other molecules. Recognition events are important in normal cell growth, fertilization, transformation of cells, and other processes. All these functions are made possible by the characteristic chemical features of carbohydrates: • • • •

the existence of at least 1 and often 2 or more asymmetric centres; the ability to exist in either linear or ring structures; the capacity to form polymeric structures via glycosidic bonds; and the potential to form multiple hydrogen bonds with water or other molecules in their environment.

7.1

What Are Carbohydrates, and How Are They Named?

The name carbohydrate arises from the basic molecular formula (CH2O)n, where n = 3 or more. (CH2O)n can be rewritten (C·H2O)n to show that these substances are polyhydroxylated aldehydes or ketones containing 3 or more carbons. Carbohydrates are generally classified into 3 groups: monosaccharides (and their derivatives), oligosaccharides, and polysaccharides. The monosaccharides are also called simple sugars and have the formula (CH2O)n. Monosaccharides cannot be broken down into smaller sugars under mild conditions. Oligosaccharides derive their name from the Greek word oligo, meaning “few,” and consist of 2–10 simple sugar residues. Disaccharides are common in nature, and trisaccharides also occur frequently. Four- to six-sugar–unit oligosaccharides are usually bound covalently to other molecules, including glycoproteins. As their name suggests, polysaccharides are polymers of the simple sugars and their derivatives. They may be either linear or branched polymers and may contain hundreds or even thousands of monosaccharide units. Their molecular weights range up to 1 million or more. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

7.2

What Is the Structure and Chemistry of Monosaccharides?

O

H

H

C HO

C

Monosaccharides Are Classified as Aldoses and Ketoses

O C

H or H

CH2OH

CH2OH

C

Dihydroxyacetone

CHO

ALDOTRIOSE

Carbon 2 number

HCOH

3

CH2OH

Glyceraldehyde

HO

CHO

H

C

H

C

Carbon number

CHO

2

HCOH

3

L-Glyceraldehyde

D-Glyceraldehyde

FIGURE 7.1 Structure of a simple aldose (glyceraldehyde) and a simple ketose (dihydroxyacetone).

CH2OH

CHO HOCH ALDOTETROSES

HCOH

4

HCOH

CH2OH

CH2OH

D-Erythrose

D-Threose

1

CHO

2

HCOH

Carbon number 3

HCOH

HCOH

4

HCOH

HCOH

5

CHO

D-Ribose

CHO

HOCH

CH2OH

(Ara)

2

HCOH

3 Carbon number 4

HCOH

HCOH

HCOH

HCOH

HCOH

HCOH

5

HCOH

HCOH

HCOH

HCOH

CH2OH D-Allose

CH2OH D-Altrose

HCOH HOCH

CHO

D-Xylose

HCOH

HOCH

HCOH

CH2OH

D-Glucose

D-Mannose

(Man)

CH2OH

(Xyl)

CHO

HOCH

CH2OH (Glc)

HCOH

CH2OH

CHO

HOCH

HOCH

HCOH

D-Arabinose

CHO

HOCH

ALDOPENTOSES HOCH

1

6

CHO

CHO

HCOH

CH2OH

(Rib)

OH

CH2OH

D-Glyceraldehyde

1

O

D-Isomer

CH2OH

CHO

C

OH

CH2OH

L-Isomer

Monosaccharides consist typically of 3–7 carbon atoms and are described as either aldoses or ketoses, depending on whether the molecule contains an aldehyde function or a ketone group. The simplest aldose is glyceraldehyde, and the simplest ketose is dihydroxyacetone (Figure 7.1). These 2 simple sugars are termed trioses because they each contain 3 carbon atoms. The structures and names of a family of aldoses and ketoses with 3, 4, 5, and 6 carbons are shown in Figures 7.2 and 7.3. Hexoses are the

1

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Carbohydrates and the Glycoconjugates of Cell Surfaces

HOCH HCOH

CHO HOCH HCOH HOCH HCOH

CH2OH D-Gulose

CH2OH D-Idose

D-Lyxose

CHO HCOH

(Lyx)

CHO HOCH

HOCH

HOCH

HOCH

HOCH

HCOH CH2OH D-Galactose

(Gal)

HCOH CH2OH D-Talose

ALDOHEXOSES FIGURE 7.2 The structure and stereochemical relationships of D-aldoses with 3–6 carbons. The configuration in each case is determined by the highest numbered asymmetric carbon (shown in pink). In each row, the “new” asymmetric carbon is shown in yellow. Blue highlights indicate the most common aldoses. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

FIGURE 7.3 The structure and stereochemical relationships of D-ketoses with 3–6 carbons. The configuration in each case is determined by the highest numbered asymmetric carbon (shown in pink). In each row, the “new” asymmetric carbon is shown in yellow. Blue highlights indicate the most common ketoses.

CH2OH

1 Carbon 2 number

C

O

KETOTRIOSE

CH2OH

3

Dihydroxyacetone

Carbon number

1

CH2OH

2

C

O

KETOTETROSE

3 HCOH CH2OH

4

D-Erythrulose

1

CH2OH

CH2OH

2

C

C

O

Carbon 3 HCOH number

HOCH

4 HCOH 5

Carbon number

KETOPENTOSES

HCOH

CH2OH

CH2OH

D-Ribulose

D-Xylulose

1

CH2OH

CH2OH

CH2OH

CH2OH

2

C

C

C

C

O

3 HCOH

O

HOCH

4 HCOH

HCOH

5 HCOH

HCOH

6

Glyco: A generic term relating to sugars.

O

O

HCOH HOCH HCOH

O

HOCH HOCH

KETOHEXOSES

HCOH

CH2OH

CH2OH

CH2OH

CH2OH

D-Psicose

D-Fructose

D-Sorbose

D-Tagatose

most abundant sugars in nature. Nevertheless, sugars from all these classes are important in metabolism. Monosaccharides, either aldoses or ketoses, are often given more detailed generic names to describe both the important functional groups and the total number of carbon atoms. Thus, one can refer to aldotetroses and ketotetroses, aldopentoses and ketopentoses, aldohexoses and ketohexoses, and so on. Sometimes the ketone-containing monosaccharides are named simply by inserting the letters -ul- into the simple generic terms, such as tetruloses, pentuloses, hexuloses, heptuloses, and so on. The simplest monosaccharides are water soluble, and most taste sweet.

Stereochemistry Is a Prominent Feature of Monosaccharides Aldoses with at least 3 carbons and ketoses with at least 4 carbons contain chiral centres (see Chapter 4). The nomenclature for such molecules must specify the configuration about each asymmetric centre, and drawings of these molecules must be based on a system that clearly specifies these configurations. As noted in Chapter 4, the Fischer projection system is used almost universally for this purpose today. The structures shown in Figures 7.2 and 7.3 are NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

Fischer projections. For monosaccharides with 2 or more asymmetric carbons, the prefix D- or L- refers to the configuration of the highest numbered asymmetric carbon (the asymmetric carbon farthest from the carbonyl carbon). A monosaccharide is designated D if the hydroxyl group on the highest numbered asymmetric carbon is drawn to the right in a Fischer projection, as in D-glyceraldehyde (Figure 7.1). Note that the designation D or L merely relates the configuration of a given molecule to that of glyceraldehyde and does not specify the sign of rotation of plane-polarized light. If the sign of optical rotation is to be specified in the name, the convention of D or L designations may be used along with a + (plus) or - (minus) sign. Thus, D-glucose (Figure 7.2) may also be called D(+)-glucose because it is dextrorotatory, whereas D-fructose (Figure 7.3), which is levorotatory, can also be named D(-)-fructose. All the structures shown in Figures 7.2 and 7.3 are D-configurations, and the D-forms of monosaccharides predominate in nature, just as L-amino acids do. These preferences, established in apparently random choices early in evolution, persist uniformly in nature because of the stereospecificity of the enzymes that synthesize and metabolize these small molecules. L-Monosaccharides do exist in nature, serving a few relatively specialized roles. L-Galactose is a constituent of certain polysaccharides, and L-arabinose is a constituent of bacterial cell walls. According to convention, the D- and L-forms of a monosaccharide are mirror images of each other, as shown in Figure 7.4 for fructose. Stereoisomers that are non-superimposable mirror images of each other are called enantiomers, or sometimes enantiomeric pairs. For molecules that possess 2 or more chiral centres, more than 2 stereoisomers can exist. Pairs of isomers that have opposite configurations at 1 or more of the chiral centres but that are not mirror images of each other are called diastereomers or diastereomeric pairs. Any 2 structures in a given row in Figures 7.2 and 7.3 are diastereomeric pairs. Two sugars that differ in configuration at only 1 chiral centre are described as epimers. For example, D-mannose and D-talose are epimers and D-glucose and D-mannose are epimers, whereas D-glucose and D-talose are not epimers but merely diastereomers.

CH2OH

CH2OH

C

O

C

O

HO

C

H

H

C

OH

H

C

OH

C

H

H

C

HO Mirror-image OH configurations HO

C

H

Enantiomers

CH2OH

CH2OH

D-Fructose

L-Fructose

FIGURE 7.4 D-Fructose and L-fructose, an enantiomeric pair. Note that changing the configuration only at C5 would change D-fructose to L-sorbose.

H H HO

Monosaccharides Exist in Cyclic and Anomeric Forms

H

Although Fischer projections are useful for presenting the structures of particular monosaccharides and their stereoisomers, they discount one of the most interesting facets of sugar structure—the ability to form cyclic structures with formation of an additional asymmetric centre. Alcohols react readily with aldehydes to form hemiacetals (Figure 7.5). The H R

H

+

O

O

C

H C

R' Alcohol

R

O

R'

Aldehyde

OH

Hemiacetal

CH2OH H

O 1

H HO H H

2 3 4 5 6

H

C C C

6

OH H

C

OH

C

OH

CH2OH

D-Glucose

5C

H C HO

C CH2OH

4

H OH C

HO

H O

3

2

H

H C OH

O

Pyran

H 1

C

C

O

H OH

H

C

C

H C OH

H

1 2 3 4 5

C

OH

C

OH

C

H

C

OH

O

C

6

CH2OH !-D-Glucopyranose HO

C

H

H

C

OH

HO

C

H

H

C

OH

H

C

O

CH2OH "-D-Glucopyranose FISCHER PROJECTION FORMULAS

H OH !-D-Glucopyranose

Cyclization O

CH2OH H C HO

C

O

H OH

H

C

C

193

Carbohydrates and the Glycoconjugates of Cell Surfaces

OH C H

OH H "-D-Glucopyranose HAWORTH PROJECTION FORMULAS

"-D-Glucopyranose

FIGURE 7.5 The linear form of D-glucose undergoes an intramolecular reaction to form a cyclic hemiacetal. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

194

Part 1 Building Blocks of the Cell R''

H R

+

O

O

C R'

Alcohol

R

R''

HOH2C

C O

R'

Ketone

HO

OH

Hemiketal

H O

HOH2C 1 2

HO H H

3 4 5 6

H

CH2OH C

O

C

H

C

OH

C

OH

C H

6

1

O 5

H C

HO 4

3

OH

HO

CH2OH OH

Cyclization

C O

O

HOH2C O

Furan

3 4 5

C

OH

C

H

C

OH

O

C

6

CH2OH !-D-Fructofuranose

CH2OH

H

CH2OH

D-Fructose

C

2

H

H

2

OH H !-D-Fructofuranose

H HOH2C

1

H

H

HO

OH CH2OH

HO

C

CH2OH

HO

C

H

H

C

OH

H

C

O

OH H "-D-Fructofuranose

CH2OH "-D-Fructofuranose

HAWORTH PROJECTION FORMULAS

FISCHER PROJECTION FORMULAS

FIGURE 7.6 The linear form of D-fructose undergoes an intramolecular reaction to form a cyclic hemiketal.

"-D-Fructofuranose

British carbohydrate chemist Sir Norman Haworth showed that the linear form of glucose (and other aldohexoses) could undergo a similar intramolecular reaction to form a cyclic hemiacetal. The resulting 6-membered oxygen-containing ring is similar to pyran and is designated a pyranose. The reaction is catalyzed by acid (H+) or base (OH-) and is readily reversible. In a similar manner, ketones can react with alcohols to form hemiketals. The analogous intramolecular reaction of a ketose sugar such as fructose yields a cyclic hemiketal (Figure 7.6). The 5-membered ring thus formed is reminiscent of furan and is referred to as a furanose. The cyclic pyranose and furanose forms are the preferred structures for monosaccharides in aqueous solution. At equilibrium, the linear aldehyde or ketone structure is only a minor component of the mixture (generally much less than 1%). When hemiacetals and hemiketals are formed, the carbon atom that carried the carbonyl function becomes an asymmetric carbon atom. Isomers of monosaccharides that differ only in their configuration about that carbon atom are called anomers, designated as a or b, as shown in Figure 7.5, and the carbonyl carbon is thus called the anomeric carbon. When the hydroxyl group at the anomeric carbon is on the same side of a Fischer projection as the oxygen atom at the highest numbered asymmetric carbon, the configuration at the anomeric carbon is a, as in a-D-glucose. When the anomeric hydroxyl is on the opposite side of the Fischer projection, the configuration is b, as in b-D-glucopyranose (Figure 7.5). The addition of this asymmetric centre upon hemiacetal and hemiketal formation alters the optical rotation properties of monosaccharides, and the original assignment of the a and b notations arose from studies of these properties. Early carbohydrate chemists frequently observed that the optical rotation of glucose (and other sugar) solutions could change with time, a process called mutarotation. This indicated that a  structural change was occurring. It was eventually found that a-D-glucose has a specific optical rotation, [a]d20, of 112.2°, and that b-D-glucose has a specific optical rotation of 18.7°. Mutarotation involves interconversion of a- and b-forms of the monosaccharide with intermediate formation of the linear aldehyde or ketone, as shown in Figures 7.5 and 7.6. NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

Carbohydrates and the Glycoconjugates of Cell Surfaces

Haworth Projections Are a Convenient Device for Drawing Sugars Another of Haworth’s lasting contributions to the field of carbohydrate chemistry was his proposal to represent pyranose and furanose structures as hexagonal and pentagonal rings lying perpendicular to the plane of the paper, with thickened lines indicating the side of the ring closest to the reader. Such Haworth projections, which are now widely used to represent saccharide structures (Figures 7.5 and 7.6), show substituent groups extending either above or below the ring. Substituents drawn to the left in a Fischer projection are drawn above the ring in the corresponding Haworth projection. Substituents drawn to the right in a Fischer projection are below the ring in a Haworth projection. Exceptions to these rules occur in the formation of furanose forms of pentoses and the formation of furanose or pyranose forms of hexoses. In these cases, the structure must be redrawn with a rotation about the carbon whose hydroxyl group is involved in the formation of the cyclic form (Figure 7.7) in order to orient the appropriate hydroxyl group for ring formation. This is merely for illustrative purposes and involves no change in configuration of the saccharide molecule. The rules previously mentioned for assignment of a- and b-configurations can be readily applied to Haworth projection formulas. For the D-sugars, the anomeric hydroxyl group is below the ring in the a-anomer and above the ring in the b-anomer. For L-sugars, the opposite relationship holds. As Figure 7.7 implies, in most monosaccharides there are 2 or more hydroxyl groups that can react with an aldehyde or ketone at the other end of the molecule to form a hemiacetal or hemiketal. Consider the possibilities for glucose, as shown in Figure 7.7. If the C-4 hydroxyl group reacts with the aldehyde of glucose, a 5-membered ring is formed, whereas if the C-5 hydroxyl reacts, a 6-membered ring is formed. The C-6 hydroxyl does not react effectively because a 7-membered ring is too strained to form a stable hemiacetal. The same is true for the C-2 and C-3 hydroxyls, and thus 5- and 6-membered rings are by far the most likely to be formed from 6-membered monosaccharides. D-Ribose, with 5 carbons, readily forms either 5-membered rings (a- or b-D-ribofuranose) or 6-membered rings (a- or b-D-ribopyranose) (Figure 7.7). In general, aldoses and ketoses with 5 or more carbons can form either furanose or pyranose rings; the more stable form depends on structural factors. The nature of the substituent groups on the carbonyl and hydroxyl groups and the configuration about the asymmetric carbon will determine whether a given monosaccharide prefers the pyranose or the furanose structure. In general, the pyranose form is favoured over the furanose

CH2OH O HO

O

OH

OH

HO

CH2OH OH

HC

Pyranose form

OH

CHOH O

OH CH2

OH OH

CH2OH

OH

OH D-Glucose

OH

H

OH

OH

Pyranose form

OH

H C

C

O

O OH

OH

CH2OH

D-Ribose

O

OH

OH OH Furanose form

OH

OH

Furanose form

FIGURE 7.7 D-Glucose, D-ribose, and other simple sugars can cyclize in 2 ways, forming either furanose or pyranose structures. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

195

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Part 1 Building Blocks of the Cell

FIGURE 7.8 (a) Chair and boat conformations of a pyranose sugar. (b) Two possible chair conformations of b-D-glucose.

(a)

Axis

109° e

Axis a

a e a

O a

a

e

e

a e

e a = axial bond e = equatorial bond

e

e

e a

a

Chair

e

O

a

a Boat

(b) H

CH2OH O

HO

H

CH2OH H H

OH OH

HO H

H

OH

H OH

H

O

OH H

H OH

ring for aldohexose sugars, although, as we shall see, furanose structures are more stable for ketohexoses. Although Haworth projections are convenient for displaying monosaccharide structures, they do not accurately portray the conformations of pyranose and furanose rings. Given C i C i C tetrahedral bond angles of 109° and C i O i C angles of 111°, neither pyranose nor furanose rings can adopt true planar structures. Instead, they take on puckered conformations, and in the case of pyranose rings, the 2 favoured structures are the chair conformation and the boat conformation, shown in Figure 7.8. Note that the ring substituents in these structures can be equatorial, which means approximately coplanar with the ring, or axial, that is, parallel to an axis drawn through the ring, as shown. Two general rules dictate the conformation to be adopted by a given saccharide unit. First, bulky substituent groups on such rings are more stable when they occupy equatorial positions rather than axial positions, and second, chair conformations are slightly more stable than boat conformations. For a typical pyranose, such as b-D-glucose, there are 2  possible chair conformations (Figure 7.8). Of all the D-aldohexoses, b-D-glucose is the only one that can adopt a conformation with all its bulky groups in an equatorial position. With this advantage of stability, it may come as no surprise that b-D-glucose is the most widely occurring organic group in nature and the central hexose in carbohydrate metabolism.

Monosaccharides Can Be Converted to Several Derivative Forms A variety of chemical and enzymatic reactions produce derivatives of the simple sugars. These modifications produce a diverse array of saccharide derivatives. Some of the most common derivations are discussed here. Sugar Acids Sugars with free anomeric carbon atoms are reasonably good reducing agents and will reduce hydrogen peroxide, ferricyanide, certain metals (Cu2+ and Ag+), and other oxidizing agents. Such reactions convert the sugar to a sugar acid. For example, addition of alkaline CuSO4 (called Fehling’s solution) to an aldose sugar produces a red cuprous oxide (Cu2O) precipitate

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

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Carbohydrates and the Glycoconjugates of Cell Surfaces

COOH

Oxidation at C-1

O

H

H

C

OH

HO

C

H

H

C

OH

H

C

OH

C

H HO

CH2OH D-Gluconic acid

O

C H

CH2OH

CH2OH OH

H OH

H

H

OH

D-Gluconic

O

O

H

C O–

HO

acid

H OH

H

H

OH

O

+

OH–

D- #-Gluconolactone

Note: D-Gluconic acid and other aldonic acids exist in equilibrium with lactone structures.

H C

OH

HO

C

H

H

C

OH

H

C

OH

H

Oxidation at C-6

OH

HO

C

H

H

C

OH

H

C

OH

CH2OH D-Glucose

C

COOH D-Glucuronic

H HO

COOH

H O H

HO H

H HO

H

OH H

OH

D-Glucuronic acid (GlcUA)

O H H

COOH HO

OH H

OH

D-Iduronic

acid (IdUA)

acid (GlcUA) Oxidation at C-1 and C-6

COOH H

C

OH

HO

C

H

H

C

OH

H

C

OH

COOH D-Glucaric

acid

FIGURE 7.9 Oxidation of D-glucose to sugar acids.

and converts the aldose to an aldonic acid, such as gluconic acid (Figure 7.9). Formation of a precipitate of red Cu2O constitutes a positive test for an aldehyde. Carbohydrates that can reduce oxidizing agents in this way are referred to as reducing sugars. By quantifying the amount of oxidizing agent reduced by a sugar solution, one can accurately determine the concentration of the sugar. Diabetes mellitus is a condition that causes high levels of glucose in urine and blood, and frequent analysis of reducing sugars in diabetic patients is an important part of the diagnosis and treatment of this disease. Over-the-counter kits for the easy and rapid determination of reducing sugars have made this procedure a simple one for diabetic persons. Monosaccharides can be oxidized enzymatically at C-6, yielding uronic acids such as D-glucuronic and L-iduronic acids (Figure 7.9). L-Iduronic acid is similar to D-glucuronic acid except it has an opposite configuration at C-5. Oxidation at both C-1 and C-6 produces aldaric acids, such as D-glucaric acid. Sugar Alcohols Sugar alcohols, another class of sugar derivative, can be prepared by the mild reduction (with NaBH4 or similar agents) of the carbonyl groups of aldoses and ketoses. Sugar alcohols, or alditols, are designated by the addition of -itol to the name of the parent sugar (Figure 7.10). The alditols are linear molecules that cannot cyclize in the

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

CH2OH

CH2OH

H

C

OH

HO

C

H

HO

C

H

HO

C

H

CH2OH H

C

C

OH

H

C

OH

HO

C

H

H

C

OH

H

C

OH

H

C

OH

CH2OH (sorbitol)

HO

OH

H

D-Glucitol

CH2OH

H

CH2OH H

C

CH2OH

CH2OH

4

OH

HO

CH2OH

D-Xylitol

D-Mannitol

OH 3

H OH 5

H

D-Glycerol

2

OH

H H

1

6

H

H

C

OH

H

C

OH

H

C

OH

OH

CH2OH D-Ribitol

myo-Inositol

© Steven Lunetta Photography, 2012

FIGURE 7.10 Structures of some sugar alcohols. (Note that myo-inositol is a polyhydroxy cyclohexane, not a sugar alcohol.)

FIGURE 7.11 Sugar alcohols such as sorbitol, mannitol, and xylitol sweeten many “sugarless” gums and candies.

manner of aldoses. Nonetheless, alditols are characteristically sweet tasting, and sorbitol, mannitol, and xylitol are widely used to sweeten sugarless gum and mints (Figure 7.11). Sorbitol buildup in the eyes of diabetic persons is implicated in cataract formation. Glycerol and myo-inositol, a cyclic alcohol, are components of lipids (see Chapter 8). There are 9 different stereoisomers of inositol; the one shown in Figure 7.10 was first isolated from heart muscle and thus has the prefix myo- for muscle. Ribitol is a constituent of flavin coenzymes (see Chapter 16). Deoxy Sugars The deoxy sugars are monosaccharides with 1 or more hydroxyl groups replaced by hydrogens. 2-Deoxy-D-ribose (Figure 7.12), whose systematic name is 2-deoxyD-erythro-pentose, is a constituent of DNA in all living things (see Chapter 24). Deoxy sugars also occur frequently in glycoproteins and polysaccharides. L-Fucose and L-rhamnose, both 6-deoxy sugars, are components of some cell walls, and rhamnose is a component of ouabain, a highly toxic cardiac glycoside found in the bark and root of the ouabaio tree. Ouabain is used by the East African Somalis as an arrow poison. The sugar moiety is not the toxic part of the molecule (see Chapter 10). Sugar Esters Phosphate esters of glucose, fructose, and other monosaccharides are important metabolic intermediates, and the ribose moiety of nucleotides such as ATP and GTP is phosphorylated at the 5!-position (Figure 7.13).

H O

HOH2C H

H

H H

HO

OH

H

H O OH

CH3 H H

H

O OH CH3 H HO HO H H

OH H

OH OH

OH H

2-Deoxy-!-D-ribose

!-L-Rhamnose (Rha)

!-L-Fucose (Fuc)

FIGURE 7.12 Several deoxy sugars. Hydrogen and carbon atoms highlighted in red are “deoxy” positions.

H HO

CH2OH O H OH H

H OPO32–

H OH !-D-Glucose-1-phosphate

O

2–O

3POH2C

H

H

HO

CH2OPO32– OH

OH H !-D-Fructose-1,6-bisphosphate

O

2–O

3POH2C

H

H

H

H

OH

OH

OH

!-D-Ribose-5-phosphate

FIGURE 7.13 Several sugar esters important in metabolism. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

199

Carbohydrates and the Glycoconjugates of Cell Surfaces

A DEEPER LOOK

Honey: An Ancestral Carbohydrate Treat fructose respectively. In polysaccharides, fructose invariably prefers the furanose form, but free fructose (and crystalline fructose) is predominantly b-fructopyranose. Source: White, J. W., 1978. Honey. Advances in Food Research 24:287–374; and Prince, R. C., Gunson, D. E., Leigh, J. S., and McDonald, G. G., 1982. The predominant form of fructose is a pyranose, not a furanose ring. Trends in Biochemical Sciences 7:239–240. 1

6

OH

5

HO

6

O CH2OH 3

4

2

HO

OH

OH

1

O OH

5 4

3

3 1CH2OH

4

OH

!-D-Fructopyranose

HOH2C

O OH OH 2

5

"-D-Fructopyranose

CH2OH

O

HOH2C

2

OH

4

OH

OH

!-D-Fructofuranose

OH OH

5

3

2

CH2OH 1

"-D-Fructofuranose

Honey

© Scott Camazine / Photo Researchers, Inc.

Honey, the first sweet known to humankind, is the only sweetening agent that can be stored and used exactly as produced in nature. Bees process the nectar of flowers so that their final product is able to survive long-term storage at ambient temperature. Used as a ceremonial material and medicinal agent in earliest times, honey was not regarded as a food until the Greeks and Romans tried it. Only in modern times have cane and beet sugar surpassed honey as the most frequently used sweetener. What is the chemical nature of this magical, viscous substance? Bees’ processing of honey consists of (1) reducing the water content of the nectar (30%–60%) to the self-preserving range of 15%–19%, (2) hydrolyzing the significant amount of sucrose in nectar to glucose and fructose by the action of the enzyme invertase, and (3) producing small amounts of gluconic acid from glucose by the action of the enzyme glucose oxidase. Most of the sugar in the final product is glucose and fructose, and the final product is supersaturated with respect to these monosaccharides. Honey actually consists of an emulsion of microscopic glucose hydrate and fructose hydrate crystals in a thick syrup. Sucrose accounts for only about 1% of the sugar in the final product, with fructose at about 38% and glucose at 31% by weight. The accompanying figure shows a 13C nuclear magnetic resonance spectrum of honey from a mixture of wildflowers in southeastern Pennsylvania. Interestingly, 5 major hexose species contribute to this spectrum. Although most textbooks show fructose exclusively in its furanose form, the predominant form of fructose (67% of total fructose) is b-D-fructopyranose, with the b- and a-fructofuranose forms accounting for 27% and 6% of the

Amino Sugars Amino sugars, including D-glucosamine and D-galactosamine (Figure 7.14), contain an amino group (instead of a hydroxyl group) at the C-2 position. They are found in many oligosaccharides and polysaccharides, including chitin, a polysaccharide in the exoskeletons of crustaceans and insects. Muramic acid and neuraminic acid, which are components of the polysaccharides of cell membranes of higher organisms and also bacterial cell walls, are glycosamines linked to 3-carbon acids at the C-1 or C-3 positions. In muramic acid (thus named as an amine isolated from bacterial cell wall polysaccharides; murus is Latin for “wall”), the hydroxyl group of a lactic acid moiety makes an ether linkage to the C-3 of glucosamine. Neuraminic acid (an amine isolated from neural tissue) forms a C i C bond between the C-1 of N-acetylmannosamine and the C-3 of pyruvic acid (Figure 7.15). The N-acetyl and N-glycolyl derivatives of neuraminic acid are collectively known as sialic acids and are distributed widely in bacteria and animal systems. Acetals, Ketals, and Glycosides Hemiacetals and hemiketals can react with alcohols in the presence of acid to form acetals and ketals, as shown in Figure 7.16. This reaction is

!-D-Glucopyranose "-D-Glucopyranose !-D-Fructofuranose "-D-Fructofuranose "-D-Fructopyranose

H HO

CH2OH O H OH H H

OH H

HO H

NH2

"-D-Glucosamine

CH2OH O H OH H H

OH H

NH2

"-D-Galactosamine

FIGURE 7.14 Structures of D-glucosamine and D-galactosamine.

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200

Part 1 Building Blocks of the Cell (a) H HO

(b)

CH2OH O H O H H CH3

H

C

OH O

COOH

Muramic acid

O

CH3

C

H

C

CH3

C

H

C

H

H

C

OH

H

C

OH

C

HCOH

H N

COOH HCOH

H

OH

N H HO

O

O

Pyruvic acid

CH2

NH2 CH

H

COOH

OH

CH2OH H

H

OH

H

N-Acetylmannosamine

CH2OH N-Acetyl-D-neuraminic acid (NeuNAc), a sialic acid FIGURE 7.15 Structures of (a) muramic acid and (b) several depictions of a sialic acid.

R

O

R

H

+

C

R''

O

R'

R''' C

R' OH Hemiketal

R

+

R''

H C

R' OH Hemiacetal R

O

OH

O Acetal

O

OH

+

H2O

+

H2O

R''

R''' C

R'

O

R''

Ketal

FIGURE 7.16 Acetals and ketals can be formed from hemiacetals and hemiketals respectively.

another example of a dehydration synthesis and is similar in this respect to the reactions undergone by amino acids to form peptides and nucleotides to form nucleic acids. The pyranose and furanose forms of monosaccharides react with alcohols in this way to form glycosides with retention of the a- or b-configuration at the C-1 carbon. The new bond between the anomeric carbon atom and the oxygen atom of the alcohol is called a glycosidic bond. Glycosides are named according to the parent monosaccharide. For example, methylb-D-glucoside (Figure 7.17) can be considered a derivative of b-D-glucose.

H HO

CH2OH O H OH H

H O CH3

H OH Methyl-!-D-glucoside

H HO

CH2OH O H OH H

O CH3

7.3

What Is the Structure and Chemistry of Oligosaccharides?

Given the relative complexity of oligosaccharides and polysaccharides in higher organisms, it is perhaps surprising that these molecules are formed from relatively few different monosaccharide units. (In this respect, the oligosaccharides and polysaccharides are similar to proteins: both form complicated structures based on a small number of different building blocks.) Monosaccharide units include the hexoses glucose, fructose, mannose, and galactose and the pentoses ribose and xylose.

H

H OH Methyl-"-D-glucoside

FIGURE 7.17 The anomeric forms of methyl-D-glucoside.

Disaccharides Are the Simplest Oligosaccharides The simplest oligosaccharides are the disaccharides, which consist of 2 monosaccharide units linked by a glycosidic bond. As in proteins and nucleic acids, each individual unit in an oligosaccharide is termed a residue. All the disaccharides shown in Figure 7.18 are NEL

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Chapter 7

Carbohydrates and the Glycoconjugates of Cell Surfaces

Free anomeric carbon (reducing end) CH2OH O HO OH

CH2OH O O

Simple sugars CH2OH O

HOH

OH

HO

OH OH Lactose (galactose-"-1,4-glucose)

OH

Glucose

CH2OH O O

OH

Galactose

HOH

HO

OH

CH2OH O

OH OH Maltose (glucose-!-1,4-glucose)

O

HO

CH2OH O

H CH2OH

OH OH Sucrose (glucose-1!-"2-fructose)

HO

OH

OH

OH

O OH

CH2OH O O

HOH

OH OH Cellobiose (glucose-"-1,4-glucose)

Fructose

CH2OH O HO

CH2OH O

201

HO

CH2 OH

O HOH

OH Isomaltose (glucose-!-1,6-glucose)

FIGURE 7.18 The structures of several important disaccharides. Note that the notation i HOH means that the configuration can be either a or b. If the i OH group is above the ring, the configuration is termed b. The configuration is a if the i OH group is below the ring. Also note that sucrose has no free anomeric carbon atom.

common in nature, with sucrose, maltose, and lactose being the most common. Each is a mixed acetal, with 1 hydroxyl group provided intramolecularly and 1 hydroxyl from the other monosaccharide. Except for sucrose, each of these structures possesses 1 free unsubstituted anomeric carbon atom, and thus each of these disaccharides is a reducing sugar. The end of the molecule containing the free anomeric carbon is called the reducing end, and the other end is called the non-reducing end. In the case of sucrose, both of the anomeric carbon atoms are substituted, that is, neither has a free —OH group. The substituted anomeric carbons cannot be converted to the aldehyde configuration and thus cannot participate in the oxidation–reduction reactions characteristic of reducing sugars. Thus, sucrose is not a reducing sugar. Maltose, isomaltose, and cellobiose are all homodisaccharides because they each contain only one kind of monosaccharide, namely, glucose. Maltose is produced from starch (a polymer of a-D-glucose produced by plants) by the action of amylase enzymes and is a component of malt, a substance obtained by allowing grain (particularly barley) to soften in water and germinate. The enzyme diastase, produced during the germination process, catalyzes the hydrolysis of starch to maltose. Maltose is used in beverages (e.g., malted milk), and because it is fermented readily by yeast, it is important in the brewing of beer. In both maltose and cellobiose, the glucose units are 1 S 4 linked, meaning that the C-1 of 1 glucose is linked by a glycosidic bond to the C-4 oxygen of the other glucose. The only difference between them is in the configuration at the glycosidic bond. Maltose exists in the a-configuration, whereas cellobiose is a b-configuration. Isomaltose is obtained in the hydrolysis of some polysaccharides (such as dextran), and cellobiose is obtained from the acid hydrolysis of cellulose. Isomaltose also consists of 2 glucose units in a glycosidic bond, but in this case, C-1 of one glucose is linked to C-6 of the other, and the configuration is a. The complete structures of these disaccharides can be specified in shorthand notation using abbreviations for each monosaccharide, a or b, to denote configuration, and appropriate numbers to indicate the nature of the linkage. Thus, cellobiose is Glcb1–4Glc, whereas isomaltose is Glca1–6Glc. Often the glycosidic linkage is written with an arrow so that cellobiose and isomaltose would be Glcb1 S 4Glc and Glca1 S 6Glc respectively. Because the linkage carbon on the first sugar is always C-1, a newer trend is to drop the 1– or 1 S and describe these simply as Glcb4Glc and Glca6Glc respectively. More complete names can also be used, however; for example, maltose would be O-a-Dglucopyranosyl-(1 S 4)-D-glucopyranose. Cellobiose, because of its b-glycosidic linkage, is formally O-b-D-glucopyranosyl-(1 S 4)-D-glucopyranose. !-D-Lactose (O-b-D-galactopyranosyl-(1 S 4)-D-glucopyranose) (Figure 7.18) is the principal carbohydrate in milk and is of critical nutritional importance to

Sucrose

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202

Part 1 Building Blocks of the Cell

A DEEPER LOOK

Trehalose: A Natural Protectant for Bugs Insects use an open circulatory system to circulate hemolymph (insect blood). The “blood sugar” is not glucose but rather trehalose, an unusual, non-reducing disaccharide (see figure). Trehalose is found typically in organisms that are naturally subject to temperature variations and other environmental stresses—bacterial spores, fungi, yeast, and many insects. (Interestingly, honeybees do not have trehalose in their hemolymph, perhaps because they practise a colonial, rather than solitary, lifestyle. Bee colonies maintain a constant temperature of 18°C, protecting the residents from large temperature changes.) What might explain this correlation between trehalose utilization and environmentally stressful lifestyles? Konrad Bloch* suggests that trehalose may act as a natural cryoprotectant. Freezing

and thawing of biological tissues frequently causes irreversible structural changes, destroying biological activity. High concentrations of polyhydroxy compounds, such as sucrose and glycerol, can protect biological materials from such damage. Trehalose is particularly well-suited for this purpose and has been shown to be superior to other polyhydroxy compounds, especially at low concentrations. Support for this novel idea comes from studies by Paul Attfield,** which show that trehalose levels in the yeast Saccharomyces cerevisiae increase significantly during exposure to high salt and high growth temperatures—the same conditions that elicit the production of heat shock proteins! H

H

CH2OH H O

OH

HO *Bloch, K., 1994. Blondes in Venetian Paintings, the Nine-Banded Armadillo, and Other Essays in Biochemistry. New Haven: Yale University Press. **Attfield, P. V., 1987. Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock responses. FEBS Letters 225:259.

H

HO H

OH

OH

H

H OH

O

O

H

CH2OH H H

mammals in the early stages of their lives. It is formed from D-galactose and D-glucose via a b(1 S 4) link, and because it has a free anomeric carbon, it is capable of mutarotation and is a reducing sugar. It is an interesting quirk of nature that lactose cannot be absorbed directly into the bloodstream. It must first be broken down into galactose and glucose by lactase, an intestinal enzyme that exists in young, nursing mammals but  is not produced in significant quantities in the mature mammal. Most adult humans, with the exception of certain groups in Africa and northern Europe, produce only low levels of lactase. For most individuals, this is not a problem, but some cannot tolerate lactose and experience intestinal pain and diarrhea upon consumption of milk. Sucrose, in contrast, is a disaccharide of almost universal appeal and tolerance. Produced by many higher plants and commonly known as table sugar, it is one of the products of photosynthesis and is composed of fructose and glucose. Sucrose has a specific optical rotation, [a]d20, of + 66.5°, but an equimolar mixture of its component monosaccharides has a net negative rotation ([a]D20 of glucose is +52.5° and of fructose is -92°). Sucrose is hydrolyzed by the enzyme invertase, so named for the inversion of optical rotation accompanying this reaction. Sucrose is also easily hydrolyzed by dilute acid, apparently because the fructose in sucrose is in the relatively unstable furanose form. Although sucrose and maltose are important to the human diet, they are not taken up directly in the body. In a manner similar to lactose, they are first hydrolyzed by sucrase and maltase, respectively, in the human intestine.

A Variety of Higher Oligosaccharides Occur in Nature In addition to the simple disaccharides, many other oligosaccharides are found in both prokaryotic and eukaryotic organisms, either as naturally occurring substances or as hydrolysis products of natural materials. Oligosaccharides also occur widely as components (via glycosidic bonds) of antibiotics derived from various sources. Figure 7.19 shows the structures of 2 representative carbohydrate-containing antibiotics.

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Chapter 7 Erythromycin

Streptomycin (a broad-spectrum antibiotic) NH

O H3C H3C

CH3

HO

HO O

HO H

H O

CH3 OCH3 H CH3

CH3

O

H H

H3C

O

N(CH3)2 H H

H

O

HO

OH

H

NHCNH2

CHO OH

H

HO

O

O

O CH3

OH

HO

OH

CH3 O

NH

H2NCNH

CH3

H

Carbohydrates and the Glycoconjugates of Cell Surfaces

O

CH2OH CH3NH

OH

FIGURE 7.19 Some antibiotics are oligosaccharides or contain oligosaccharide groups.

7.4

What Is the Structure and Chemistry of Polysaccharides?

Nomenclature for Polysaccharides Is Based on Their Composition and Structure By far the majority of carbohydrate material in nature occurs in the form of polysaccharides. By our definition, polysaccharides include not only those substances composed only of glycosidically linked sugar residues but also molecules that contain polymeric saccharide structures linked via covalent bonds to amino acids, peptides, proteins, lipids, and other structures. Polysaccharides, also called glycans, consist of monosaccharides and their derivatives. If a polysaccharide contains only one kind of monosaccharide molecule, it is a homopolysaccharide, or homoglycan, whereas those containing more than one kind of monosaccharide are heteropolysaccharides. The most common constituent of polysaccharides is D-glucose, but D-fructose, D-galactose, L-galactose, D-mannose, L-arabinose, and D-xylose are also common. Common monosaccharide derivatives in polysaccharides include the amino sugars (D-glucosamine and D-galactosamine), their derivatives (N-acetylneuraminic acid and N-acetylmuramic acid), and simple sugar acids (glucuronic and iduronic acids). Polysaccharides differ not only in the nature of their component monosaccharides but also in the length of their chains and in the amount of chain branching that occurs. Although a given sugar residue has only 1 anomeric carbon and thus can form only 1 glycosidic linkage with hydroxyl groups on other molecules, each sugar residue carries several hydroxyls, 1 or more of which may be an acceptor of glycosyl substituents (Figure 7.20). This ability to form branched structures distinguishes polysaccharides from proteins and nucleic acids, which occur only as linear polymers.

Polysaccharides Serve Energy Storage, Structure, and Protection Functions Polysaccharides function as storage materials, structural components, or protective substances. Thus, starch, glycogen, and other storage polysaccharides, as readily metabolizable food, provide energy reserves for cells. Chitin and cellulose provide strong support for the skeletons of arthropods and green plants respectively. Mucopolysaccharides, such as the hyaluronic acids, form protective coats on animal cells. In each of these cases, the relevant NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

203

204

Part 1 Building Blocks of the Cell CH2OH O

CH2OH O O

CH2OH O

CH2OH O

O

CH2OH O

O

O. . .

O

Amylose

CH2OH O

CH2OH O O

CH2OH O O O

CH2OH O

CH2OH O O

CH2

CH2OH O

O

O

O

CH2OH O O

O...

Amylopectin FIGURE 7.20 Amylose and amylopectin are the 2 forms of starch. Note that the linear linkages are a(1 S 4) but the branches in amylopectin are a(1 S 6). Branches in polysaccharides can involve any of the hydroxyl groups on the monosaccharide components. Amylopectin is a highly branched structure, with branches occurring every 12–30 residues.

polysaccharide is either a homopolymer or a polymer of small repeating units. Recent research indicates, however, that oligosaccharides and polysaccharides with varied structures may also be involved in much more sophisticated tasks in cells, including a variety of cellular recognition and intercellular communication events, as discussed later.

Polysaccharides Provide Stores of Energy

I

I

I

I

I

I

FIGURE 7.21 Suspensions of amylose in water adopt a helical conformation. Iodine (I2) can insert into the middle of the amylose helix to give a blue colour that is characteristic and diagnostic for starch.

Organisms store carbohydrates in the form of polysaccharides rather than as monosaccharides to lower the osmotic pressure of the sugar reserves. Because osmotic pressures depend only on numbers of molecules, the osmotic pressure is greatly reduced by formation of a few polysaccharide molecules out of thousands (or even millions) of monosaccharide units. Starch By far the most common storage polysaccharide in plants is starch, which exists in 2 forms: A-amylose and amylopectin (Figure 7.20). Most forms of starch in nature are 10%–30% a-amylose and 70%–90% amylopectin. a-Amylose is composed of linear chains of D-glucose in a(1 S 4) linkages. The chains are of varying length, having molecular weights from several thousand to half a million. As can be seen from the structure in Figure 7.20, the chain has a reducing end and a non-reducing end. Although poorly soluble in water, a-amylose forms micelles in which the polysaccharide chain adopts a helical conformation (Figure 7.21). Iodine reacts with a-amylose to give a characteristic blue colour, which arises from the insertion of iodine into the middle of the hydrophobic amylose helix. Amylopectin is a highly branched chain of glucose units (Figure 7.20). Branches occur in these chains every 12–30 residues. The average branch length is between 24 and 30 residues, and molecular weights of amylopectin molecules can range up to 100 million. The linear linkages in amylopectin are a(1 S 4), whereas the branch linkages are a(1 S 6). As is the case for a-amylose, amylopectin forms micellar suspensions in water; iodine reacts with such suspensions to produce a red–violet colour. Starch is stored in plant cells in the form of granules in the stroma of plastids (plant cell organelles). When starch is to be mobilized and used by the plant that stored it, it is split into its monosaccharide elements by stepwise phosphorolytic cleavage of glucose units, a reaction catalyzed by starch phosphorylase (Figure 7.22). The products are 1 molecule of glucose-1-phosphate and a starch molecule with 1 less glucose unit. In a-amylose, this process continues all along the chain until the end is reached. In animals, digestion and use of plant starches begin in the mouth with salivary "-amylase [a(1 S 4)-glucan 4-glucanohydrolase], the major enzyme secreted by the salivary glands. Although the capability of making and secreting salivary a-amylases is widespread in the animal world, some animals (such as cats, dogs, birds, and horses) do not NEL

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Chapter 7 CH2OH O

CH2OH O O

CH2OH O

Carbohydrates and the Glycoconjugates of Cell Surfaces

FIGURE 7.22 The starch phosphorylase reaction cleaves glucose residues from amylose, producing a-D-glucose-1-phosphate.

CH2OH O

O

O

OH n

Amylose

Non-reducing end

Reducing end HPO42–

CH2OH O OPO32–

+

CH2OH O

CH2OH O O

!-D-Glucose-1-phosphate

205

CH2OH O O n–1

OH

secrete them. Salivary a-amylase is an endoamylase that splits a(1 S 4) glycosidic linkages only within the chain. Raw starch is not very susceptible to salivary endoamylase. However, when suspensions of starch granules are heated, the granules swell, taking up water and causing the polymers to become more accessible to enzymes. Thus, cooked starch is more digestible. Most starch digestion occurs in the small intestine via glycohydrolases. Glycogen The major form of storage polysaccharide in animals is glycogen. Glycogen is found mainly in the liver (where it may amount to as much as 10% of liver mass) and skeletal muscle (where it accounts for 1%–2% of muscle mass). Liver glycogen consists of granules containing highly branched molecules, with a(1 S 6) branches occurring every 8–12 glucose units. Like amylopectin, glycogen yields a red–violet colour with iodine. Glycogen can be hydrolyzed by both a- and b-amylases, yielding glucose and maltose, respectively, as products and can also be hydrolyzed by glycogen phosphorylase, an enzyme present in liver and muscle tissue, to release glucose-1-phosphate. Dextran Another important family of storage polysaccharides is the dextrans, which are a(1 S 6)-linked polysaccharides of D-glucose with branched chains found in yeast and bacteria. Because the main polymer chain is a(1 S 6) linked, the repeating unit is isomaltose, Glca1 S 6Glc. The branch points may be 1 S 2, 1 S 3, or 1 S 4 in various species. The degree of branching and the average chain length between branches depend on the species and strain of the organism. Bacteria growing on the surfaces of teeth produce extracellular accumulations of dextrans, an important component of dental plaque.

Polysaccharides Provide Physical Structure and Strength to Organisms Cellulose The structural polysaccharides have properties that are dramatically different from those of the storage polysaccharides, even though the compositions of these 2 classes are similar. The structural polysaccharide cellulose is the most abundant natural polymer in the world. Found in the cell walls of nearly all plants, cellulose is one of the principal components providing physical structure and strength. The wood and bark of trees are insoluble, highly organized structures formed from cellulose and also from lignin (see Figure 23.35). It is awe-inspiring to look at a large tree and realize the amount of weight supported by polymeric structures derived from sugars and organic alcohols. Cellulose also has its delicate side, however. Cotton, whose woven fibres make some of our most comfortable clothing fabrics, is almost pure cellulose. Derivatives of cellulose have found wide use in our society. Cellulose acetates are produced by the action of acetic anhydride on cellulose in the presence of sulfuric acid and can be spun into a variety of fabrics with particular properties. Referred to simply as acetates, they have a silky appearance, a luxuriously soft feel, and a deep lustre and are used in dresses, lingerie, linings, and blouses. Cellulose is a linear homopolymer of D-glucose units, just as in a-amylose. The structural difference, which completely alters the properties of the polymer, is that in cellulose the glucose units are linked by b(1 S 4)-glycosidic bonds, whereas in a-amylose the NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

206

Part 1 Building Blocks of the Cell

OH

O

OH

O

O OH

O

OH

HO OH

HO

O O

HO

OH

OH

HO

O

O

O

OH

O

O

!-1,4-Linked D-glucose units

OH "-1,4-Linked D-glucose units

(a)

(b)

HO

O

OH

FIGURE 7.23 (a) Amylose, composed exclusively of the relatively bent a(1 S 4) linkages, prefers to adopt a helical conformation, whereas (b) cellulose, withb(1 S 4)-glycosidic linkages, can adopt a fully extended conformation with alternating 180° flips of the glucose units. The hydrogen bonding inherent in such extended structures is responsible for the great strength of tree trunks and other cellulose-based materials.

linkage is a(1 S 4). The conformational difference between these 2 structures is shown in Figure 7.23 above. The a(1 S 4)-linkage sites of amylose are naturally bent, conferring a gradual turn to the polymer chain, which results in the helical conformation already described (Figure 7.21). The most stable conformation about the b(1 S 4) linkage involves alternating 180° flips of the glucose units along the chain so that the chain adopts a fully extended conformation, referred to as an extended ribbon. Juxtaposition of several such chains permits efficient interchain hydrogen bonding, the basis of much of the strength of cellulose. The structure of one form of cellulose, determined by X-ray and electron diffraction data, is shown in Figure 7.24. The flattened sheets of the chains lie side by side and are joined by hydrogen bonds. These sheets are laid on top of one another in a way that staggers the chains, just as bricks are staggered to give strength and stability to a wall. Cellulose is extremely resistant to hydrolysis, whether by acid or by the digestive tract amylases described earlier. As a result, most animals (including humans) cannot digest cellulose to any significant degree. Ruminant animals, such as cattle, deer, giraffes, and

Intrachain hydrogen bond

Interchain hydrogen bond

Intersheet hydrogen bond

FIGURE 7.24 The structure of cellulose, showing the hydrogen bonds (blue) between the sheets, which strengthen the structure. Intrachain hydrogen bonds are in red, and interchain hydrogen bonds are in green. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

Carbohydrates and the Glycoconjugates of Cell Surfaces

207

camels, are an exception because bacteria that live in the rumen (Figure 7.25) secrete the enzyme cellulase, a b-glucosidase effective in the hydrolysis of cellulose. The resulting glucose is then metabolized in a fermentation process to the benefit of the host animal. Termites and shipworms (Teredo navalis) similarly digest cellulose because their digestive tracts also contain bacteria that secrete cellulase. Chitin A polysaccharide that is similar to cellulose, both in its biological function and its primary, secondary, and tertiary structure, is chitin. Chitin is present in the cell walls of fungi and is the fundamental material in the exoskeletons of crustaceans, insects, and spiders. The structure of chitin, an extended ribbon, is identical to that of cellulose, except that the —OH group on each C-2 is replaced by —NHCOCH3, so the repeating units are N-acetyl-D-glucosamines in b(1 S 4) linkage. Like cellulose (Figure 7.24), the chains of chitin form extended ribbons (Figure 7.26) and pack side by side in a crystalline, strongly hydrogen-bonded form. One significant difference between cellulose and chitin is whether the chains are arranged in parallel (all the reducing ends together at one end of a packed bundle and all the non-reducing ends together at the other end) or antiparallel (each sheet of chains having the chains arranged oppositely from the sheets above and below). Natural cellulose seems to occur only in parallel arrangements. Chitin, however, can occur in 3 forms, sometimes all in the same organism. a-Chitin is an all-parallel arrangement of the chains, whereas b-chitin is an antiparallel arrangement. In @-chitin, the structure is thought to involve pairs of parallel sheets separated by single antiparallel sheets. Chitin is Earth’s second most abundant carbohydrate polymer (after cellulose), and its ready availability and abundance offer opportunities for industrial and commercial applications. Chitin-based coatings can extend the shelf life of fruits, and a chitin derivative that binds to iron atoms in meat has been found to slow the reactions that cause rancidity and flavour loss. Without such a coating, the iron in meats activates oxygen from the air, forming reactive free radicals that attack and oxidize polyunsaturated lipids, causing most of the flavour loss associated with rancidity. Chitin-based coatings coordinate the iron atoms, preventing their interaction with oxygen.

Esophagus

Omasum Small intestine

Reticulum Abomasum Rumen

FIGURE 7.25 Giraffes, cattle, deer, and camels are ruminant animals that are able to metabolize cellulose, thanks to bacterial cellulase in the rumen, a large first compartment in the stomach of a ruminant.

Agarose An important polysaccharide mixture isolated from marine red algae (Rhodophyceae) is agar, which consists of 2 components: agarose and agaropectin. Agarose Cellulose

OH CH 2 O O

OH CH 2 O

OH

HO

O

O HO

CH

HO

2 OH

O

OH

HO

O

O HO

CH

HO

2 OH

CH3 C

Chitin

OH CH 2 O O

CH

HN C

O

C

OH CH 2 O

2 OH

O

O

O

NH

HO

O

O HO

CH3

NH

HO

O

O

O HO

CH

HN

N-Acetylglucosamine units

CH3

C

2 OH

O

O

CH3

Mannan

OH CH 2 O HO

O HO

OH CH 2 O

HO O

HO

CH

2 OH

O

HO

O HO

HO O

HO

CH

2 OH

O

O

Mannose units FIGURE 7.26 Like cellulose, chitin and mannan form extended ribbons and pack together efficiently, taking advantage of multiple hydrogen bonds. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

A DEEPER LOOK

A Complex Polysaccharide in Red Wine: The Strange Story of Rhamnogalacturonan II For many years, cotton and grape growers and other farmers have known that boron is an essential trace element for their crops. Until recently, however, the role or roles of boron in sustaining plant growth were unknown. Recent reports show that at least 1 role for boron in plants is that of cross-linking an unusual polysaccharide called rhamnogalacturonan II (RGII). RGII is a lowmolecular-weight (5–10 kDa) polysaccharide, but it is thought to be the most complex polysaccharide on Earth, comprised as it is of 11 different sugar monomers. It can be released from plant cell walls by treatment with a galacturonase, and it is also present in red wine. Part of the structure of RGII is shown in the accompanying figure. The nature of the borate ester cross-links (also indicated in the figure) was elucidated by Malcolm O’Neill and his colleagues, who used a combination of chemical methods and boron-11 NMR. Why is rhamnogalacturonan II essential for the structure and growth of plant walls? Plant walls are extremely sophisticated composite materials, composed of networks of protein, polysaccharides, and phenolic compounds. Cellulose microfibrils as strong as steel provide a load-bearing framework for the plant. These microfibrils

are tiny wires made of crystalline arrays of b-1,4-linked chains of glucose residues, which are extruded from hexameric spinnerets in the plasma membrane of the plant cell, surrounding the growing plant cell like hoops around a barrel. These microfibrils thus constrain the directions of cell expansion and determine the shapes of the plant cells and the plant as well. The separation of the barrel hoops is controlled by hemicelluloses, such as xyloglucans, which form H-bonded cross-links with the cellulose microfibrils. The hemicellulose network is embedded in a hydrated gel inside the plant wall. This gel consists of complex galacturonic acid–rich polysaccharides, including RGII—it provides a dynamic operating environment for cell wall processes. It is interesting to note that the tiny spinnerets of plant cells are nature’s version of the viscose process, developed in 1910, for the production of rayon fibres. In this process, viscose—literally a viscous solution of cellulose—is forced through a spinneret (a device resembling a shower head with many tiny holes). Each hole produces a fine filament of viscose. The fibres precipitate in an acid bath and are stretched to form the interchain H bonds that give the filaments the properties essential for use as textile fibres. RGII monomer

OH

O HO

OH

OH

O O

OH

HO

CH3 O O

C ! HCOH O

HO

O OH HO

HO HOCH2

O

O OH

C

O!

C! O

HO O O

HO

O O

O ! OH O C

O

O

O

HO

O

O

O

O C OH O! O OH

H3C H3C O

O

O

OH O O

OCH3

!

O

O

O

O O

O O C OH O ! O

HO

Site of boron attachment

CH2 OH

CH2OH

O

O C ! O O O ! OH O C HO

O

OH OH O C OH O ! OO

CH2

OH

O

O

OH

OH O

OH

CH3 C

O

O

O O

O C O !O

OH

CH3

O

HO

OH

CH

O

OH

O ! O C

O

O

OH

C

OH HO OH

O

O

O C ! O O O ! OH O C

O

C ! O

O C ! O CH2OHOCH3

O O O

OH O

RGII dimer

O

OH

H3C

O

O

CH3 O C OH O

CH2OH

O OH

O HO

OH

O HO H

CH3 OH

B

Methyl groups Acetyl groups Source: Hofte, H., 2001. A baroque residue in red wine. Science 294:795–797.

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Chapter 7

Carbohydrates and the Glycoconjugates of Cell Surfaces

(Figure 7.27) is a chain of alternating D-galactose and 3,6-anhydro-L-galactose, with side chains of 6-methyl-D-galactose. Agaropectin is similar but, in addition, it contains sulfate ester side chains and D-glucuronic acid. The 3-dimensional structure of agarose is a double helix with a 3-fold screw axis, as shown in Figure 7.27. The central cavity is large enough to accommodate water molecules. Agarose and agaropectin readily form gels containing large amounts (up to 99.5%) of water. Glycosaminoglycans A class of polysaccharides known as glycosaminoglycans are involved in a variety of extracellular (and sometimes intracellular) functions. Glycosaminoglycans consist of linear chains of repeating disaccharides in which 1 of the monosaccharide units is an amino sugar and 1 (or both) of the monosaccharide units contains at least 1 negatively charged sulfate or carboxylate group. The repeating disaccharide structures found commonly in glycosaminoglycans are shown in Figure 7.28. Heparin, with the highest net negative charge of the disaccharides shown, is a natural anticoagulant substance. It binds strongly to antithrombin III (a protein involved in terminating the clotting process) and inhibits blood clotting. Hyaluronate molecules may consist of as many as 25 000 disaccharide units, with molecular weights of up to 107. Hyaluronates are important components of the vitreous humor in the eye and of synovial fluid, the lubricant fluid of joints in the body. The chondroitins and keratan sulfate are found in tendons, cartilage, and other connective tissue; dermatan sulfate, as its name implies, is a component of the extracellular matrix of skin. Glycosaminoglycans are fundamental constituents of proteoglycans (discussed later).

–O SO 3

H 4

COO– O H OH H H

H β

O

1

4

CH2OH O H H 3

H

O

H

NHCCH3 O

H

OH

β 1

N-Acetyl-

D-Glucuronate

D-galactosamine-4-sulfate

COO– O H H H 4 OH H 1

H

4

COO– O H OH H

D-glucosamine-6-sulfate

2-sulfate

H

Heparin

CH2OSO3– O β HO O H 4 H 1 H H β

1

O

H

NHCCH3 O

H

OH

N-Acetyl-

D-galactosamine-6-sulfate

D-Glucuronate

COO– O H H 4 OH H H

CH2OH O β H O H H 1 3 H HO β

1

H

β

1

O

H

OH

L-Iduronate

NHCCH3 O

H

N-Acetyl-Dgalactosamine-4-sulfate

Dermatan sulfate

H

NHCCH3 O

OH

N-Acetyl-

D-Glucuronate

D-glucosamine

Hyaluronate

CH2OH –O SO O β 3 O H 4 H 1 3 H H

O H – 4 COO OH H

O

H

Chondroitin-6-sulfate

H

NHSO3–

N-Sulfo-

D-Glucuronate-

Chondroitin-4-sulfate

H

O O CH2OH HO O O HO O O CH2 n OH 3,6-Anhydro bridge

2

OSO3–

H

Agarose

CH2OSO3– O H H H 4 OH H 1 α O

O

α

2

209

CH2OSO3– O β H O H 4 OH H 1 H 6

CH2OH O HO H H H H 3 H

OH

D-Galactose

β

O

H

Agarose double helix

NHCCH3 O

N-Acetyl-

FIGURE 7.27 The favoured conformation of agarose in water is a double helix with a 3-fold screw axis.

D-glucosamine-6-sulfate

Keratan sulfate

FIGURE 7.28 Glycosaminoglycans are formed from repeating disaccharide arrays. Glycosaminoglycans are components of proteoglycans.

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Part 1 Building Blocks of the Cell

A DEEPER LOOK

Billiard Balls, Exploding Teeth, and Dynamite: The Colourful History of Cellulose Although humans cannot digest it and most people’s acquaintance with cellulose is limited to comfortable cotton clothing, cellulose has enjoyed a colourful and varied history of utilization. In 1838, Théophile Pelouze in France found that paper or cotton could be made explosive if dipped in concentrated nitric acid. Christian Schönbein, a professor of chemistry at the University of Basel, prepared “nitrocotton” in 1845 by dipping cotton in a mixture of nitric and sulfuric acids and then washing the material to remove excess acid. In 1860, Major E. Schultze of the Prussian Army used the same material, now called guncotton, as a propellant replacement for gunpowder, and its preparation in brass cartridges quickly made it popular for this purpose. The only problem was that it was too explosive and could detonate unpredictably in factories where it was produced. The entire town of Faversham, England, was destroyed in such an accident. In 1868, Alfred Nobel mixed guncotton with ether and alcohol, thus preparing nitrocellulose, and in turn mixed this with nitroglycerine and sawdust to produce dynamite. Nobel’s income from dynamite and also from his profitable development of the Russian oil fields in Baku eventually formed the endowment for the Nobel Prizes.

In 1869, concerned over the precipitous decline (from hunting) of the elephant population in Africa, the billiard ball manufacturers Phelan and Collender offered a prize of $10 000 for production of a substitute for ivory. Brothers Isaiah and John Hyatt in Albany, New York, produced a substitute for ivory by mixing guncotton with camphor, then heating and squeezing it to produce celluloid. This product found immediate uses well beyond billiard balls. It  was easy to shape, strong, and resilient, and it exhibited a high tensile strength. Celluloid was eventually used to make dolls, combs, musical instruments, fountain pens, piano keys, and a variety of other products. The Hyatt brothers eventually formed the Albany Dental Company to make false teeth from celluloid. Because camphor was used in their production, the company advertised that their teeth smelled “clean,” but as reported in the New York Times in 1875, the teeth also occasionally exploded! Portions adapted from Burke, J., 1996. The Pinball Effect: How Renaissance Water Gardens Made the Carburetor Possible and Other Journeys Through Knowledge. New York: Little, Brown, & Company.

Polysaccharides Provide Strength and Rigidity to Bacterial Cell Walls Some of nature’s most interesting polysaccharide structures are found in bacterial cell walls. Given the strength and rigidity provided by polysaccharide structures, it is not surprising that bacteria use such structures to provide protection for their cellular contents. Bacteria normally exhibit high internal osmotic pressures and frequently encounter variable, often hypotonic exterior conditions. The rigid cell walls synthesized by bacteria maintain cell shape and size and prevent swelling or shrinkage that would inevitably accompany variations in solution osmotic strength.

Peptidoglycan Is the Polysaccharide of Bacterial Cell Walls Bacteria are conveniently classified as either gram-positive or gram-negative depending on their response to the so-called Gram stain. Despite substantial differences in the various structures surrounding these 2 types of cells, nearly all bacterial cell walls have a strong, protective peptide–polysaccharide layer called peptidoglycan. Gram-positive bacteria have a thick (approximately 25 nm) cell wall consisting of multiple layers of peptidoglycan. This thick cell wall surrounds the bacterial plasma membrane. Gram-negative bacteria, in contrast, have a much thinner (2–3 nm) cell wall consisting of a single layer of peptidoglycan sandwiched between the inner and outer lipid bilayer membranes. In either case, peptidoglycan, sometimes called murein (from the Latin murus, meaning “wall”), is a continuous cross-linked structure— in essence, a single molecule—built around the cell. The structure is shown in Figure 7.29. The backbone is a b(1 S 4)-linked polymer of N-acetyl glucosamine and N-acetylmuramic acid units. This part of the structure is similar to that of chitin, but it is joined to a tetrapeptide, usually L-Ala · D-Glu · L-Lys · D-Ala, in which the L-lysine is linked to the g-COOH of D-glutamate. The peptide is linked to the N-acetylmuramic acid units via its D-lactate moiety. The e-amino group of lysine in this peptide is linked to the —COOH of D-alanine of an adjacent tetrapeptide. In gram-negative cell walls, the lysine e-amino group forms a direct amide bond with this D-alanine carboxyl (Figure 7.29). In gram-positive cell walls, a pentaglycine chain bridges the lysine e-amino group and the D-Ala carboxyl group. Gram-negative cell walls are also covered with highly complex lipopolysaccharides (Figure 7.30 on page 212). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7 (a)

Carbohydrates and the Glycoconjugates of Cell Surfaces

(b) Gram-positive cell wall

H

H O

CH2OH O H OH H H

N-Acetylmuramic acid (NAM)

CH2OH O H H

H O H

NHCOCH3

H

(NAG)

O

NHCOCH3 (NAM)

N-Acetylglucosamine (NAG)

H

H n

O H3C

CH

C

O

NH L-Ala

CH C

L-Ala D-Glu

CH3

L-Lys

D-Ala

O

Pentaglycine crosslink

NH COO–

CH Isoglutamate

CH2

(c) Gram-negative cell wall

CH2 $ -Carboxyl linkage to L-Lys L-Lys

C NH

(c) (CH2)4

CH C

O

NH D-Ala

O

O

CH

N H (b)

Gram-negative

C D-Ala O

(C

O CH2

)

N H

5

C D-Ala

Grampositive

CH3

COO–

L-Ala D-Glu L-Lys

D-Ala

Direct crosslink

FIGURE 7.29 (a) The structure of peptidoglycan. The tetrapeptides linking adjacent backbone chains contain an unusual g-carboxyl linkage. (b) The cross-link in gram-positive cell walls is a pentaglycine bridge. (c) In gram-negative cell walls, the linkage between the tetrapeptides of adjacent carbohydrate chains in peptidoglycan involves a direct amide bond between the lysine side chain of one tetrapeptide and D-alanine of the other.

Cell Walls of Gram-Negative Bacteria In gram-negative bacteria, the peptidoglycan wall is the rigid framework around which is built an elaborate membrane structure (Figure 7.30). The peptidoglycan layer encloses the periplasmic space and is attached to the outer membrane via a group of hydrophobic proteins. As shown in Figure 7.31, the outer membrane of gram-negative bacteria is coated with a highly complex lipopolysaccharide, which consists of a lipid group (anchored in the outer membrane) joined to a polysaccharide made up of long chains with many different and characteristic repeating structures (Figure 7.31 on page 213). These many different unique units determine the antigenicity of the bacteria; that is, animal immune systems recognize them as foreign substances and raise antibodies against them. As a group, these antigenic determinants are called the O antigens, and there are thousands of different ones. The Salmonella bacteria alone have well over a thousand known O antigens, which have been organized into 17 different groups. The great variation in these O antigen NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

211

212

Part 1 Building Blocks of the Cell (a)

Gram-positive bacteria

Polysaccharide coat

Peptidoglycan layers (cell wall)

(b) Gram-negative bacteria

Lipopolysaccharide

Outer lipid bilayer membrane Cell wall

Peptidoglycan Inner lipid bilayer membrane

FIGURE 7.30 The structures of the cell wall and membrane(s) in gram-positive and gram-negative bacteria. The gram-positive cell wall is thicker than that in gram-negative bacteria, compensating for the absence of a second (outer) bilayer membrane.

structures apparently plays a role in the recognition of one type of cell by another and in evasion of the host immune system. Cell Walls of Gram-Positive Bacteria In gram-positive bacteria, the cell exterior is less complex than for gram-negative cells. Having no outer membrane, gram-positive cells compensate with a thicker wall. Covalently attached to the peptidoglycan layer are teichoic acids, which often account for 50% of the dry weight of the cell wall. The teichoic acids are polymers of ribitol phosphate or glycerol phosphate linked by phosphodiester bonds.

Animals Display a Variety of Cell Surface Polysaccharides Compared to bacterial cells, which are identical within a given cell type (except for O antigen variations), animal cells display a wondrous diversity of structure, constitution, and function. Although each animal cell contains, in its genetic material, the instructions to replicate the entire organism, each differentiated animal cell carefully controls its NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

composition and behaviour within the organism. A great part of each cell’s uniqueness begins at the cell surface. This surface uniqueness is critical to each animal cell because cells spend their entire life span in intimate contact with other cells and must therefore communicate with one another. That cells are able to pass information among themselves is evidenced by numerous experiments. For example, heart myocytes, when grown in culture (in glass dishes), establish synchrony when they make contact, so that they “beat” or contract in unison. If they are removed from the culture and separated, they lose their synchronous behaviour, but if allowed to reestablish cell-to-cell contact, they spontaneously restore their synchronous contractions. As these and many other related phenomena show, it is clear that molecular structures on one cell are recognizing and responding to molecules on the adjacent cell or to molecules in the extracellular matrix, the complex “soup” of connective proteins and other molecules that exists outside and among cells. Many of these interactions involve glycoproteins on the cell surface and proteoglycans in the extracellular matrix. The “information” held in these special carbohydrate-containing molecules is not encoded directly in the genes (as with proteins), but is determined instead by expression of the appropriate enzymes that assemble carbohydrate units in a characteristic way on these molecules. Also, by virtue of the several hydroxyl linkages that can be formed with each carbohydrate monomer, these structures are arguably more information-rich than proteins and nucleic acids, which can form only linear polymers. A few of these glycoproteins and their unique properties are described in the following sections.

7.5

213

Carbohydrates and the Glycoconjugates of Cell Surfaces Lipopolysaccharide

Mannose O antigen Abequose Rhamnose

D -Galactose

What Are Glycoproteins, and How Do They Function in Cells?

Many proteins found in nature are glycoproteins because they contain covalently linked oligosaccharide and polysaccharide groups. The list of known glycoproteins includes structural proteins, enzymes, membrane receptors, transport proteins, and immunoglobulins, among others. In most cases, the precise function of the bound carbohydrate moiety is not understood. Carbohydrate groups may be linked to polypeptide chains via the hydroxyl groups of serine, threonine, or hydroxylysine residues (in O-linked saccharides) (Figure 7.32a) or via the amide nitrogen of an asparagine residue (in N-linked saccharides) (Figure 7.32b). The carbohydrate residue linked to the protein in O-linked saccharides is usually an N-acetyl galactosamine, but mannose, galactose, and xylose residues linked to protein hydroxyls are also found (Figure 7.32a). Oligosaccharides O-linked to glycophorin (see Figure 10.10) involve N-acetyl galactosamine linkages and are rich in sialic acid residues. N-linked saccharides always have a unique core structure composed of 2 N-acetyl glucosamine residues linked to a branched mannose triad (Figure 7.32b, c). Many other sugar units may be linked to each of the mannose residues of this branched core. The enzymes that link the sugar moieties to amino acid side chains are the glycosyltransferases. They also catalyze the linkage of monosaccharides to lipids. These enzymes are classified according to the type of carbohydrate groups that they link to acceptors, and include the hexosyltransferases (glucosyl-, galactosyl-, glucuronosyl-, fucosyl-, and mannosyltransferases), the pentosyltransferases, and the sialyltransferases. The ABO blood group system is determined by the type of glucosyltransferases expressed in the body. The ABO gene locus expresses 3 main allelic forms of the glucosyltransferases. The A and B alleles encode the glucosyltransferases that bond a-N-acetyl galactosamine and a-D-galactose to the D-galactose end of the H antigen, producing the A and B antigens respectively. The O allele contains a frameshift mutation that results in the translation of a completely different protein from the glucosyltransferase and that lacks enzymatic activity. When this results, the H antigen remains unchanged. The combination of glucosyltransferases, produced by both alleles present in each person, determines whether the blood type of the person is AB, A, B, or O. O-linked saccharides are often found in cell surface glycoproteins and in mucins, the large glycoproteins that coat and protect mucous membranes in the respiratory and

Core oligosaccharide

Heptose KDO NAG O

P P

O

P P

P P

Protein

Lipopolysaccharides

Outer cell wall Peptidoglycan Plasma membrane

Proteins

FIGURE 7.31 Lipopolysaccharide (LPS) coats the outer membrane of gram-negative bacteria. The lipid portion of the LPS is embedded in the outer membrane and is linked to a complex polysaccharide.

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214

Part 1 Building Blocks of the Cell

FIGURE 7.32 The carbohydrate moieties of glycoproteins may be linked to the protein via (a) serine or threonine residues (in the O-linked saccharides) or (b) asparagine residues (in the N-linked saccharides). (c) N-linked glycoproteins are of 3 types: high mannose, complex, and hybrid, the latter of which combines structures found in the high mannose and complex saccharides.

(a)

O-linked saccharides CH2OH

HO H

CH2OH

O

H OH

H

H

HO

O

O

H

H

H

H

OH

H

H

C CH2

O

O

C

NHCCH3

Ser

H

NH

O "-Galactosyl-1,3-!-N-acetylgalactosyl-serine

HOCH2

CH2OH

O CH3 C

OH

CH

O

O H

C

OH

H

O H OH HO

HO

Thr

H

NH

C CH2

O

H

!-Xylosyl-threonine

(b)

H

O Ser

H

C NH

!-Mannosyl-serine

Core oligosaccharides in N-linked glycoproteins HOCH2 O OH HO

HO

Man

HOCH2

O ! 1,6 CH2

HOCH2

O HO

O

OH HO

HO O ! 1,3

Man

(c)

HO

O O " 1,4

O O " 1,4

OH HN GlcNAc

Man

O

HOCH2

C

NH

CH2

C

OH

CH3

O

HN GlcNAc

C

O

C

H

N

H

Asn

CH3

C O

N-linked glycoproteins Man ! 1,2 Man ! 1,2

Man ! 1,2

! 1,2

Man ! 1,3

Man ! 1,3

Man ! 1,6 Man

Man " 1,4

Sia

Man

! 1,6

GlcNAc " 1,4 GlcNAc Asn

High mannose

Sia ! 2,3 or 6

! 2,3 or 6 Gal " 1,4

Gal " 1,4

GlcNAc " 1,2

GlcNAc " 1,2

Man

Man

! 1,3

! 1,6

Gal " 1,4 GlcNAc " 1,2

Man

Man

! 1,3

! 1,6

Man

Man

! 1,3

! 1,6

Man " 1,4

Man " 1,4

GlcNAc " 1,4

GlcNAc " 1,4

GlcNAc

GlcNAc

Asn

Asn

Complex

Hybrid

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Chapter 7

Carbohydrates and the Glycoconjugates of Cell Surfaces

gastrointestinal tracts in the body. Certain viral glycoproteins also contain O-linked sugars. O-linked saccharides in glycoproteins are often found clustered in richly glycosylated domains of the polypeptide chain. Physical studies on mucins show that they adopt rigid, extended structures. An individual mucin molecule (Mr = 107) may extend over a distance of 150–200 nm in solution. Inherent steric interactions between the sugar residues and the protein residues in these cluster regions cause the peptide core to fold into an extended and relatively rigid conformation. This interesting effect may be related to the function of O-linked saccharides in glycoproteins. It allows aggregates of mucin molecules to form extensive, intertwined networks, even at low concentrations. These viscous networks protect the mucosal surface of the respiratory and gastrointestinal tracts from harmful environmental agents. There appear to be 2 structural motifs for membrane glycoproteins containing O-linked saccharides. Certain glycoproteins, such as leukosialin, are O-glycosylated throughout much or most of their extracellular domain (Figure 7.33). Leukosialin, like mucin, adopts a highly extended conformation, allowing it to project great distances above the membrane surface, perhaps protecting the cell from unwanted interactions with macromolecules or other cells. The second structural motif is exemplified by the low-density lipoprotein (LDL) receptor and by decay-accelerating factor (DAF). These proteins contain a highly O-glycosylated stem region that separates the transmembrane domain from the globular, functional extracellular domain. The O-glycosylated stem serves to raise the functional domain of the protein far enough above the membrane surface to make it accessible to the extracellular macromolecules with which it interacts.

Polar Fish Depend on Antifreeze Glycoproteins A unique family of O-linked glycoproteins permit fish to live in the icy seawater of the Arctic and Antarctic regions, where water temperature may reach as low as -1.9°C. Antifreeze glycoproteins (AFGPs) are found in the blood of nearly all Antarctic fish and at least 5 Arctic fish. These glycoproteins have the peptide structure [Ala-Ala-Thr]n-Ala-Ala,

Leukosialin

Decay-accelerating factor (DAF)

O-linked saccharides

LDL receptor

Globular protein heads

Glycocalyx (10 nm)

Plasma membrane FIGURE 7.33 The O-linked saccharides of glycoproteins appear in many cases to adopt extended conformations that serve to extend the functional domains of these proteins above the membrane surface. (Adapted from Jentoft, N., 1990. Why are proteins O-glycosylated? Trends in Biochemical Sciences 15:291–294.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

A DEEPER LOOK

Drug Research Finds a Sweet Spot A variety of diseases are being successfully treated with sugar-based therapies. As this table shows, several carbohydrate-based drugs are

either on the market or at various stages of clinical trials. Some of these drugs are enzymes, whereas others are glycoconjugates.

Drug

Description

Manufacturer

Cerezyme (imiglucerase)

This enzyme degrades glycolipids, compensating for an enzyme deficiency that causes Gaucher’s disease.

Genzyme, Cambridge, MA

Vancocin (vancomycin)

A very potent glycopeptide antibiotic that is used typically against antibiotic-resistant infections. It inhibits synthesis of peptidoglycan in the bacterial cell wall.

Eli Lilly, Indianapolis, IN

Vevesca (OGT 918)

A sugar analog that inhibits synthesis of the glycolipid that accumulates in Gaucher’s disease.

Oxford GlycoSciences, Abingdon, UK

GMK

A vaccine containing ganglioside GM2; it triggers an immune response against cancer cells carrying GM2.

Progenics Pharmaceuticals, Tarrytown, NY

StaphVAX

A vaccine that is a protein with a linked bacterial sugar; it is intended to treat Staphylococcus infection.

NABI Biopharmaceuticals, Boca Raton, FL

Bimosiamose (TBC1269)

A sugar analog that inhibits selectin-based inflammation in blood vessels.

Texas Biotechnology, Houston, TX

GCS-100

A sugar that blocks action of a sugar-binding protein on tumours.

GlycoGenesys, Boston, MA

GD0039 (swainsonine)

A sugar analog that inhibits synthesis of carbohydrates essential to tumour metastasis.

GlycoDesign, Toronto, Canada

PI-88

A sugar that inhibits growth factor–dependent angiogenesis and enzymes that promote metastasis.

Progen, Darra, Australia

Adapted from Maeder, T., 2002. Sweet medicines. Scientific American 287:40–47. Additional references: Alper, J., 2001. Searching for medicine’s sweet spot. Science 291:2338–2343; Borman, S., 2007. Sugar medicine. Chemical & Engineering News 85:19–30.

where n can be 4, 5, 6, 12, 17, 28, 35, 45, or 50. Each of the threonine residues is glycosylated with the disaccharide b-galactosyl-(1 S 3)-a-N-acetyl galactosamine (Figure 7.34). This glycoprotein adopts a flexible rod conformation with regions of 3-fold left-handed helix. The evidence suggests that antifreeze glycoproteins may inhibit the formation of ice in the fish by binding specifically to the growth sites of ice crystals, inhibiting further growth of the crystals.

N-Linked Oligosaccharides Can Affect the Physical Properties and Functions of a Protein N-linked oligosaccharides are found in many different proteins, including immunoglobulins G and M, ribonuclease B, ovalbumin, and peptide hormones. Many different functions are known or suspected for N-glycosylation of proteins. Glycosylation can affect the physical and chemical properties of proteins, altering solubility, mass, and electrical charge. Carbohydrate moieties have been shown to stabilize protein conformations and protect proteins against proteolysis. Eukaryotic organisms use post-translational additions of N-linked oligosaccharides to direct selected proteins to various membrane compartments. Recent evidence indicates that N-linked oligosaccharides promote the proper folding of newly synthesized polypeptides in the endoplasmic reticulum (see A Deeper Look on page 218).

Oligosaccharide Cleavage Can Serve as a Timing Device for Protein Degradation The slow cleavage of monosaccharide residues from N-linked glycoproteins circulating in the blood targets these proteins for degradation by the organism. The liver contains specific NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

N H3C

C

H

N

H O

HOCH2 HOCH2 HO

HO O

CH

O NH

OH

C

OH

CH3

FIGURE 7.34 The structure of the repeating unit of antifreeze glycoproteins, a disaccharide consisting of b-galactosyl-(1 S 3)-a-N-acetyl galactosamine in glycosidic linkage to a threonine residue.

Ala

H C

O

C

CH3

N

H

217

Ala

C

O

O

H

Carbohydrates and the Glycoconjugates of Cell Surfaces

C

H C

Thr

O

O

CH3

"-Galactosyl-1,3-!-N-acetylgalactosamine Repeating unit of antifreeze glycoproteins

receptor proteins that recognize and bind glycoproteins that are ready to be degraded and recycled. Newly synthesized serum glycoproteins contain N-linked triantennary (3-chain) oligosaccharides having structures similar to those in Figure 7.35, in which sialic acid residues cap galactose residues. As these glycoproteins circulate, enzymes on the blood vessel walls cleave off the sialic acid groups, exposing the galactose residues. In the liver, the asialoglycoprotein receptor binds the exposed galactose residues of these glycoproteins with very high affinity (Kd = 10-9-10-8 M). The complex of receptor and glycoprotein is then taken into the cell by endocytosis, and the glycoprotein is degraded in cellular lysosomes. Highest affinity binding of glycoprotein to the asialoglycoprotein receptor requires 3 free galactose residues. Oligosaccharides with only 1 or 2 exposed galactose residues bind less tightly. This is an elegant way for the body to keep track of how long glycoproteins have GlcNAc

Man

Sia

Gal

GlcNAc

Man

FIGURE 7.35 Progressive cleavage of sialic acid residues exposes galactose residues. Binding to the asialoglycoprotein receptor in the liver becomes progressively more likely as more Gal residues are exposed.

GlcNAc

GlcNAc

Asn

...

Man Gal GlcNAc Sia (Does not bind)

Man Man

Gal

GlcNAc

Sia Gal GlcNAc (Binds poorly)

GlcNAc

Man

Asn

...

Sia

GlcNAc

Sialic acid

Gal

GlcNAc

Man

Gal

GlcNAc

Man

GlcNAc

GlcNAc

Asn

...

Man Sia Gal GlcNAc (Binds moderately)

Sialic acid

Gal

GlcNAc

Man

Gal

GlcNAc

Man

...

GlcNAc

...

Gal

...

Sialic acid

Man

GlcNAc

GlcNAc

Asn

...

Gal

...

Sia

Gal GlcNAc (Binds tightly to liver asialoglycoprotein receptor) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

A DEEPER LOOK

N-Linked Oligosaccharides Help Proteins Fold One important effect of N-linked oligosaccharides in eukaryotic organisms may be their contribution to the correct folding of certain globular proteins. This adaptation of saccharide function allows cells to produce and secrete larger and more complex proteins at high levels. Inhibition of glycosylation leads to production of misfolded, aggregated proteins that lack function. Certain proteins are highly dependent on glycosylation, whereas others are much less dependent, and certain glycosylation sites are more important for protein folding than are others.

Studies with model peptides show that oligosaccharides can alter the conformational preferences near the glycosylation sites. In addition, the presence of polar saccharides may serve to orient that portion of a peptide toward the surface of protein domains. However, it has also been found that saccharides usually are not essential for maintaining the overall folded structure after a glycoprotein has reached its native, folded structure. Helenius, A., and Aebi, M., 2001. Intracellular functions of N-linked glycans. Science 291:2364–2369.

been in circulation. Over a period of time—anywhere from a few hours to weeks—the sialic acid groups are cleaved one by one. The longer the glycoprotein circulates and the more sialic acid residues are removed, the more galactose residues become exposed so that the glycoprotein is eventually bound to the liver receptor. Glycoside hydrolases (glycosidases) catalyze the hydrolysis of glycosidic linkages to release sugar hemiacetals or hemiketals from the free aglycone. They can catalyze the hydrolysis of O-, N-, and S-linked glycosides. This family of enzymes is classified as either endo- or exo-glycoside hydrolases depending upon their ability to cleave the glycosidic linkage in the middle or the end (either the non-reducing or reducing end) of a carbohydrate chain respectively. Retaining and inverting glycoside hydrolases catalyze hydrolysis with a net retention or a net inversion of the anomeric configuration of the released sugars respectively. The glycoside hydrolases are typically named after the substrate upon which they act; these include xylanases, which remove hemicelluloses from paper pulp; invertases, for the manufacture of invert sugar (used as a sweetener); amylase, for the production of maltodextrins in the food industry; and cellulases, for degrading cellulose to glucose, which can be used for ethanol production.

7.6

How Do Proteoglycans Modulate Processes in Cells and Organisms?

Proteoglycans are a family of glycoproteins whose carbohydrate moieties are predominantly glycosaminoglycans. The structures of only a few proteoglycans are known, and even these few display considerable diversity (Figure 7.36). Those known range in size from serglycin, having 104 amino acid residues (10.2 kD), to versican, having 2409 residues (265 kD). Each of these proteoglycans contains 1 or 2 types of covalently linked glycosaminoglycans. In the known proteoglycans, the glycosaminoglycan units are O-linked to serine residues of Ser-Gly dipeptide sequences. Serglycin is named for a unique central domain of 49 amino acids composed of alternating serine and glycine residues. The cartilage matrix proteoglycan contains 117 Ser-Gly pairs to which chondroitin sulfates attach. Decorin, a small proteoglycan secreted by fibroblasts and found in the extracellular matrix of connective tissues, contains only 3 Ser-Gly pairs, only 1 of which is normally glycosylated. In addition to glycosaminoglycan units, proteoglycans may also contain other N-linked and O-linked oligosaccharide groups.

Functions of Proteoglycans Involve Binding to Other Proteins Proteoglycans may be soluble and located in the extracellular matrix, as is the case for serglycin, versican, and the cartilage matrix proteoglycan, or they may be integral transmembrane proteins, such as syndecan. Both types of proteoglycan appear to function by interacting with a variety of other molecules through their glycosaminoglycan components and through specific receptor domains in the polypeptide itself. For example, syndecan (from the Greek syndein, meaning “to bind together”) is a transmembrane proteoglycan NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

Carbohydrates and the Glycoconjugates of Cell Surfaces (e) Rat cartilage proteoglycan

(a) Versican

(b) Serglycin NH+

NH+ 3

3

Hyaluronic acid– binding domain (link-protein-like)

Chondroitin sulfate

Ser/Gly protein core

COO– Chondroitin sulfate

Chondroitin sulfate

(c) Decorin NH+ Protein core

3

Chondroitin/dermatan sulfate chain O-linked oligosaccharides

COO–

(d) Syndecan Heparan sulfate NH+ 3

Epidermal growth factor–like domains COO–

Extracellular domain

Chondroitin sulfate Keratan sulfate

COO–

Cytoplasmic domain

Transmembrane domain

N-linked oligosaccharides

FIGURE 7.36 The known proteoglycans include a variety of structures. The carbohydrate groups of proteoglycans are predominantly glycosaminoglycans O-linked to serine residues. Proteoglycans include both soluble proteins and integral transmembrane proteins.

that associates intracellularly with the actin cytoskeleton (see Chapter 15). Outside the cell, it interacts with fibronectin, an extracellular protein that binds to several cell surface proteins and to components of the extracellular matrix. The ability of syndecan to participate in multiple interactions with these target molecules allows them to act as a sort of “glue” in the extracellular space, linking components of the extracellular matrix, facilitating the binding of cells to the matrix, and mediating the binding of growth factors and other soluble molecules to the matrix and to cell surfaces (Figure 7.37). Many of the functions of proteoglycans involve the binding of specific proteins to the glycosaminoglycan groups of the proteoglycan. The glycosaminoglycan-binding sites on these specific proteins contain multiple basic amino acid residues. The amino acid sequences NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

FIGURE 7.37 Proteoglycans serve a variety of functions on the cytoplasmic and extracellular surfaces of the plasma membrane. Many of these functions appear to involve the binding of specific proteins to the glycosaminoglycan groups.

(Outside)

Extracellular matrix

Fibronectin

Chondroiton sulfate proteoglycan

Binding site Binding site

Growth factor bound to heparan sulfate in matrix

Integrin receptor for fibronectin

Membrane heparan sulfate proteoglycan Growth factor receptor

Cytoskeleton (actin)

(Inside)

BBXB and BBBXXB (where B is a basic amino acid and X is any amino acid) recur repeatedly in these binding domains. Basic amino acids such as lysine and arginine provide charge neutralization for the negative charges of glycosaminoglycan residues, and in many cases, the binding of extracellular matrix proteins to glycosaminoglycans is primarily charge dependent. For example, more highly sulfated glycosaminoglycans bind more tightly to fibronectin. However, certain protein–glycosaminoglycan interactions require a specific carbohydrate sequence. A particular pentasaccharide sequence in heparin, for example, binds tightly to antithrombin III (Figure 7.38), accounting for the anticoagulant properties of heparin. Other glycosaminoglycans interact much more weakly.

Proteoglycans May Modulate Cell Growth Processes

FIGURE 7.38 A portion of the structure of heparin, a carbohydrate having anticoagulant properties. It is used by blood banks to prevent the clotting of blood during donation and storage and also by physicians to prevent the formation of life-threatening blood clots in patients recovering from serious injury or surgery. This sulfated pentasaccharide sequence in heparin binds with high affinity to antithrombin III, accounting for this anticoagulant activity. The 3-O-sulfate marked by an asterisk is essential for high-affinity binding of heparin to antithrombin III.

Several lines of evidence raise the possibility of modulation or regulation of cell growth processes by proteoglycans. First, heparin and heparan sulfate are known to inhibit cell proliferation in a process involving internalization of the glycosaminoglycan moiety and its migration to the cell nucleus. Second, fibroblast growth factor binds tightly to heparin and other glycosaminoglycans, and the heparin–growth factor complex protects the growth factor from degradative enzymes, thus enhancing its activity. There is evidence that binding of fibroblast growth factors by proteoglycans and glycosaminoglycans in the extracellular matrix creates a reservoir of growth factors for cells to use. Third, transforming growth factor b has been shown to stimulate the synthesis and secretion of proteoglycans in certain cells. Fourth, several proteoglycan core proteins, including versican and lymphocyte homing receptor, have domains similar in sequence to those of epidermal growth factor and

OSO3– O OH

COO– O O

HNR''

OH

O

OR' O (*) OSO – 3

O – HNSO3

OSO3– O O COO– OH

O

OH

O HNSO3–

OSO3–

OH NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

Carbohydrates and the Glycoconjugates of Cell Surfaces

221

complement regulatory factor. These growth factor domains may interact specifically with growth factor receptors in the cell membrane in processes that are not yet understood.

Proteoglycans Make Cartilage Flexible and Resilient Cartilage matrix proteoglycan is responsible for the flexibility and resilience of cartilage tissue in the body. In cartilage, long filaments of hyaluronic acid are studded or coated with proteoglycan molecules, as shown in Figure 7.39. The hyaluronate chains can be as long as 4 μm and can coordinate 100 or more proteoglycan units. Cartilage proteoglycan possesses Carboxylate group

Proteoglycan

Core protein Link protein Hyaluronic acid

FIGURE 7.39 Hyaluronate (see Figure 7.28) forms the backbone of proteoglycan structures, such as those found in cartilage. The proteoglycan subunits consist of a core protein containing numerous O-linked and N-linked glycosaminoglycans. In cartilage, these highly hydrated proteoglycan structures are enmeshed in a network of collagen fibres. Release (and subsequent reabsorption) of water by these structures during compression accounts for the shock-absorbing qualities of cartilaginous tissue.

Core protein

Link protein O-linked oligosaccharides

N-linked oligosaccharides

Ser

Ser

Ser

Asn

O

O

O

N

Xyl

GalNAc

GalNAc

GlcNAc

Gal

Gal

Gal

GlcNAc

Gal

GlcNAc

Gal

NeuNAc

NeuNAc

Gal

NeuNAc

Man

O

O

GluA

Gal

O

O

GluNAc

GluNAc

O

O

GluA

Gal

O

O

NeuNAc

Man

Man

GlcNAc GlcNAc Keratan sulfate

Chondroitin sulfate

Sulfate group

Hyaluronic acid

Gal

Gal

NeuNAc NeuNAc

GluNAc O

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Part 1 Building Blocks of the Cell

a hyaluronic acid–binding domain on the NH2-terminal portion of the polypeptide, which binds to hyaluronate with the assistance of a link protein. The proteoglycan–hyaluronate aggregates can have molecular weights of 2 million or more. The proteoglycan–hyaluronate aggregates are highly hydrated by virtue of strong interactions between water molecules and the polyanionic complex. When cartilage is compressed (such as when joints absorb the impact of walking or running), water is briefly squeezed out of the cartilage tissue and then reabsorbed when the stress is diminished. This reversible hydration gives cartilage its flexible, shock-absorbing qualities and cushions the joints during physical activities that might otherwise injure the involved tissues.

7.7

Do Carbohydrates Provide a Structural Code?

The surprisingly low number of genes in the genomes of complex multicellular organisms has led biochemists to consider other explanations for biological complexity and diversity. Oligosaccharides and polysaccharides, endowed with an unsurpassed variability of structures, are information carriers, and glycoconjugates—complexes of proteins with oligosaccharides and polysaccharides—are the mediators of information transfer by these carbohydrate structures. Individual sugar units are the “letters” of the sugar code, and the “words” and “sentences” of this code are synthesized by glycosyltransferases, glycosidases, and other enzymes. The total number of permutations for a 6-unit polymer formed from an alphabet of 20 hexose monosaccharides is a staggering 1.44 * 1015, whereas only 6.4 * 107 hexamers can be formed from 20 amino acids, and only 4096 hexanucleotides can be formed from the 4 nucleotides of DNA. The vast array of possible glycan structures adds a glycomic dimension to the genomic complexity achieved by protein expression in organisms. The processes of cell migration, cell–cell interaction, immune response, and blood clotting, along with many other biological processes, depend on information transfer modulated by glycoconjugates. Many of the proteins involved in glycoconjugate formation belong to the lectins—a class of proteins that bind carbohydrates with high specificity and affinity. Lectins are the translators of the sugar code. Table 7.1 describes a few of the many known lectins, their carbohydrate affinities, and their functions. A few examples of lectin– carbohydrate complexes and their roles in biological information transfer will illustrate the nature of these important and complex interactions.

Selectins, Rolling Leukocytes, and the Inflammatory Response Human bodies are constantly exposed to a plethora of bacteria, viruses, and other inflammatory substances. To combat these infectious and toxic agents, the body has developed a carefully regulated inflammatory response system. Part of that response is the orderly migration of leukocytes to sites of inflammation. Leukocytes literally roll along the vascular wall and into the tissue site of inflammation. This rolling movement is mediated by reversible adhesive interactions between the leukocytes and the vascular surface.

TABLE 7.1

Specificities and Functions of Some Animal Lectins

Lectin Family

Carbohydrate Specificity

Function

Calnexins

Glucose

Ligand-selective molecular chaperones in ER

C-type lectins

Variable

Cell-type specific endocytosis and other functions

ERGIC-53

Mannose

Intracellular routing of glycoproteins and vesicles

Galectins

Galactose/lactose

Cellular growth regulation and cell–matrix interactions

Pentraxins

Variable

Anti-inflammatory action

Selectins

Variable

Cell migration and routing NEL

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Carbohydrates and the Glycoconjugates of Cell Surfaces

223

These interactions involve adhesion proteins called selectins, which are found on both the rolling leukocytes and the endothelial cells of the vascular walls. Selectins have a characteristic domain structure, consisting of an N-terminal extracellular lectin (LEC) domain, a single epidermal growth factor (E) domain, a series of 2–9 short consensus repeat (SCR) domains, a single transmembrane segment, and a short cytoplasmic domain. The lectin domains bind carbohydrates with high affinity and specificity. Selectins of 3  types are known: E-selectins, L-selectins, and P-selectins. L-Selectin is found on the surfaces of leukocytes, including neutrophils and lymphocytes, and binds to carbohydrate ligands on endothelial cells (Figure 7.40). The presence of L-selectin is a necessary component of leukocyte rolling. P-Selectin and E-selectin are located on the vascular endothelium and bind with carbohydrate ligands on leukocytes. Typical neutrophil cells possess 10 000–20 000 P-selectin–binding sites. Selectins are expressed on the surfaces of their respective cells by exposure to inflammatory signal molecules, such as histamine, hydrogen peroxide, and bacterial endotoxins. P-Selectins, for example, are stored in intracellular granules and are transported to the cell membrane within seconds to minutes of exposure to a triggering agent. Substantial evidence supports the hypothesis that selectin–carbohydrate ligand interactions modulate the rolling of leukocytes along the vascular wall. Studies with L-selectin–deficient and P-selectin–deficient leukocytes show that L-selectins mediate weaker adherence of the leukocyte to the vascular wall and promote faster rolling along the wall. Conversely, P-selectins promote stronger adherence and slower rolling. Thus, leukocyte rolling velocity in the inflammatory response could be modulated by variable exposure of P-selectins and L-selectins at the surfaces of endothelial cells and leukocytes respectively.

Galectins: Mediators of Inflammation, Immunity, and Cancer The galectins are a very conserved family of proteins with carbohydrate recognition domains (CRDs) of about 135 amino acids that bind b-galactosides specifically. Galectins occur in both vertebrates and invertebrates, and they participate in processes such as cell adhesion, growth regulation, inflammation, immunity, and cancer metastasis. In humans, one galectin is associated with increased risk of heart attacks and another is implicated in inflammatory bowel disease. The structure of human galectin-1 is a dimer of antiparallel beta-sandwich subunits (Figure 7.41a). Lactose binds at opposite ends of the dimer. Structural studies of the protein in the presence and absence of ligand reveal that the amino acid residues implicated in galactose binding are kept in their proper orientation in the absence of ligand by a hydrogen-bonded network of 4 water molecules (Figure 7.41b). FIGURE 7.40 (a) The interactions of selectins with their receptors. (b) The selectin family of adhesion proteins.

(a) L-Selectin

Selectin receptors

Leukocyte

(b) SCR repeat P-Selectin

LEC E SCR repeat

Selectin receptor

E-Selectin

LEC E SCR repeat

E-Selectin L-Selectin

Endothelial cell

LEC E

P-Selectin NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

224

Part 1 Building Blocks of the Cell (a)

(b)

FIGURE 7.41 (a) Structure of the human galectin-1 dimer. The lactose-binding sites are at opposite ends of the dimer. (b) The carbohydrate recognition site of human galectin-1, showing the amino acids involved in galactose binding and the network of water molecules (red circles) that orient these residues.

C-Reactive Protein: A Lectin That Limits Inflammation Damage The pentraxins are lectins that adopt an unusual quaternary structure in which 5 identical subunits combine to form a planar ring with a central hole (Figure 7.42a). C-reactive protein is a pentraxin that functions to limit tissue damage, acute inflammation, and autoimmune reactions. C-reactive protein acts by binding to phosphocholine moieties on damaged membranes. Binding of the protein to phosphocholine is apparently mediated through a bound calcium ion and a hydrophobic pocket centred on Phe66 (Figure 7.42b).

(a)

(b)

FIGURE 7.42 (a) The C-reactive protein pentamer. (b) The phosphocholine-binding site of C-reactive protein contains 2 bound Ca2+ ions (blue) and a hydrophobic pocket. Phe66 is shown in purple.

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Chapter 7

Carbohydrates and the Glycoconjugates of Cell Surfaces

225

SUMMARY Carbohydrates are a versatile class of molecules of the formula (CH2O)n. They are a major form of stored energy in organisms, and they are the metabolic precursors of virtually all other biomolecules. Carbohydrates linked to lipids (glycolipids) are components of biological membranes. Carbohydrates linked to proteins (glycoproteins) are important components of cell membranes and function in recognition between cell types and recognition of cells by other molecules. Recognition events are important in cell growth, differentiation, fertilization, tissue formation, transformation of cells, and other processes. 7.1 What Are Carbohydrates, and How Are They Named? Carbohydrates are classified into 3 groups: monosaccharides, oligosaccharides, and polysaccharides. Monosaccharides cannot be broken down into smaller sugars under mild conditions. Oligosaccharides consist of 2–10 simple sugar molecules. Polysaccharides are polymers of simple sugars and their derivatives and may be branched or linear. Their molecular weights range up to 1 million or more. 7.2 What Is the Structure and Chemistry of Monosaccharides? Monosaccharides consist typically of 3–7 carbon atoms and are described as either aldoses or ketoses. Aldoses with at least 3 carbons and ketoses with at least 4 carbons contain chiral centres. The prefixes D- and L- are often used to indicate the configuration of the highest numbered asymmetric carbon. The D- and L-forms of a monosaccharide are mirror images of each other, called enantiomers. Pairs of isomers that have opposite configurations at 1 or more chiral centres, but are not mirror images of each other, are called diastereomers. Sugars that differ in configuration at only 1 chiral centre are epimers. An interesting feature of carbohydrates is their ability to form cyclic structures with formation of an additional asymmetric centre. Aldoses and ketoses with 5 or more carbons can form either furanose or pyranose rings, and the more stable form depends on structural factors. A variety of chemical and enzymatic reactions produce derivatives of simple sugars, such as sugar acids, sugar alcohols, deoxy sugars, sugar esters, amino sugars, acetals, ketals, and glycosides. 7.3 What Is the Structure and Chemistry of Oligosaccharides? The complex array of oligosaccharides in higher organisms is formed from relatively few different monosaccharide units, particularly glucose, fructose, mannose, galactose, ribose, and xylose. Disaccharides consist of 2 monosaccharide units linked by a glycosidic bond, and each unit is termed a residue. The most common disaccharides in nature are sucrose, maltose, and lactose. The anomeric carbons of oligosaccharides may be substituted or unsubstituted. Disaccharides with a free, unsubstituted anomeric carbon can reduce oxidizing agents and thus are termed reducing sugars. 7.4 What Is the Structure and Chemistry of Polysaccharides? Polysaccharides are formed from monosaccharides and their

derivatives. If a polysaccharide consists of only one kind of monosaccharide, it is a homopolysaccharide, whereas those with more than one kind of monosaccharide are heteropolysaccharides. Polysaccharides may function as energy storage materials, structural components of organisms, or protective substances. Starch and glycogen are readily metabolizable and provide energy reserves for cells. Chitin and cellulose provide strong support for the skeletons of arthropods and green plants respectively. Mucopolysaccharides such as hyaluronic acid form protective coats on animal cells. Peptidoglycan, the strong protective macromolecule of bacterial cell walls, is composed of peptidelinked glycan chains. 7.5 What Are Glycoproteins, and How Do They Function in Cells? Glycoproteins are proteins that contain covalently linked oligosaccharides and polysaccharides. Carbohydrate groups may be linked to proteins via the hydroxyl groups of serine, threonine, or hydroxylysine residues (in O-linked saccharides) or via the amide nitrogen of an asparagine residue (in N-linked saccharides). O-Glycosylated stems of certain proteins raise the functional domain of the protein above the membrane surface and the associated glycocalyx, making these domains accessible to interacting proteins. N-Glycosylation confers a variety of functions to proteins. N-linked oligosaccharides promote the proper folding of newly synthesized polypeptides in the endoplasmic reticulum of eukaryotic cells. 7.6 How Do Proteoglycans Modulate Processes in Cells and Organisms? Proteoglycans are a family of glycoproteins whose carbohydrate moieties are predominantly glycosaminoglycans. Proteoglycans may be soluble and located in the extracellular matrix, as for serglycin, versican, and cartilage matrix proteoglycans, or they may be integral transmembrane proteins, such as syndecan. Both types appear to function by interacting with a variety of other molecules through their glycosaminoglycan components and through specific receptor domains in the polypeptide itself. Proteoglycans modulate cell growth processes and are also responsible for the flexibility and resilience of cartilage tissue in the body. 7.7 Do Carbohydrates Provide a Structural Code? Oligosaccharides and polysaccharides are information carriers, and glycoconjugates are the mediators of information transfer by these carbohydrate structures. The vast array of possible glycan structures adds a glycomic dimension to the genomic complexity achieved by protein expression in organisms. The processes of cell migration, cell–cell interaction, immune response, and blood clotting, along with many other biological processes, depend on information transfer modulated by glycoconjugates. Many of the proteins involved in glycoconjugate formation belong to the lectins—a class of proteins that bind carbohydrates with high specificity and affinity.

PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. Draw Haworth structures for the 2 possible isomers of D-altrose (Figure 7.2) and D-psicose (Figure 7.3). 2. (Integrates with Chapters 4 and 5) Consider the peptide DGNILSR, where N has a covalently linked galactose and S has a covalently linked glucose. Draw the structure of this glycopeptide, and also draw titration curves for the glycopeptide and for the free peptide that would result from hydrolysis of the 2 sugar residues. 3. (Integrates with Chapters 5 and 6) Human hemoglobin can react with sugars in the blood (usually glucose) to form covalent adducts. The a-amino groups of N-terminal valine in the Hb b-subunits react with

the C-1 (aldehyde) carbons of monosaccharides to form aldimine adducts, which rearrange to form very stable ketoamine products. Quantitation of this “glycated hemoglobin” is important clinically, especially for diabetic individuals. Suggest at least 3 methods by which glycated Hb could be separated from normal Hb and quantitated. 4. Trehalose, a disaccharide produced in fungi, has the following structure:

H HO

CH2OH O OH

H

H

OH

H H

H O

OH

OH H H HOCH2 OH O H

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Part 1 Building Blocks of the Cell

a. What is the systematic name for this disaccharide? b. Is trehalose a reducing sugar? Explain. 5. Draw a Fischer projection structure for L-sorbose (D-sorbose is shown in Figure 7.3). 6. a-D-Glucose has a specific rotation, [a]d20, of +112.2°, whereas b-D-glucose has a specific rotation of +18.7°. What is the composition of a mixture of a-D- and b-D-glucose, which has a specific rotation of 83.0°? 7. Use the information in the Critical Developments in Biochemistry box titled “Rules for Description of Chiral Centres in the (R,S) System” (Chapter 4) to name D-galactose using (R,S) nomenclature. Do the same for L-altrose. 8. A 0.2 g sample of amylopectin was analyzed to determine the fraction of the total glucose residues that are branch points in the structure. The sample was exhaustively methylated and then digested, yielding 50 μmol of 2,3-dimethyl glucose and 0.4 μmol of 1,2,3,6-tetramethyl glucose. a. What fraction of the total residues are branch points? b. How many reducing ends does this sample of amylopectin have? 9. (Integrates with Chapters 5, 6, and 10) Consider the sequence of glycophorin (see Figure 10.10) and imagine subjecting glycophorin, and also a sample of glycophorin treated to remove all sugars, to treatment with trypsin and chymotrypsin. Would the presence of sugars in the native glycophorin make any difference to the results? 10. (Integrates with Chapters 4, 5, and 22) The caloric content of protein and carbohydrate are similar, at approximately 16–17 kJ/g, whereas that of fat is much higher, at 38 kJ/g. Discuss the chemical basis for the similarity of the values for carbohydrate and protein. 11. Write a reasonable chemical mechanism for the starch phosphorylase reaction (Figure 7.22). 12. Laetrile is a glycoside found in bitter almonds and peach pits. Laetrile treatment is offered in some countries as a cancer therapy. This procedure is dangerous, and there is no valid clinical evidence of its efficacy. Look up the structure of laetrile and suggest at least one reason that laetrile treatment could be dangerous for human patients. 13. Treatment with chondroitin and glucosamine is offered as one popular remedy for arthritis pain. Suggest an argument for the efficacy of this treatment, and then comment on its validity, based on what you know of polysaccharide chemistry. 14. Certain foods, particularly beans and legumes, contain substances that are indigestible (at least in part) by the human stomach, but which are metabolized readily by intestinal microorganisms, producing flatulence. One of the components of such foods is stachyose. HO

Beano is a commercial product that can prevent flatulence. Describe the likely breakdown of stachyose in the human stomach and intestines and how Beano could contribute to this process. What would be an appropriate name for the active ingredient in Beano? 15. Give the systematic name for stachyose. 16. Prolonged exposure of amylopectin to starch phosphorylase yields a substance called a limit dextrin. Describe the chemical composition of limit dextrins, and draw a mechanism for the enzyme-catalyzed reaction that can begin the breakdown of a limit dextrin. 17. Biochemist Joseph Owades revolutionized the production of beer in the United States by developing a simple treatment with an enzyme that converted regular beer into “light beer,” which was marketed aggressively as a beverage that “tastes great,” even though it is “less filling.” What was the enzyme-catalyzed reaction that Owades used to modify the fermentation process so cleverly, and how is regular beer different from light beer? 18. Amateur brewers of beer, who do not have access to the enzyme described in Problem 17, have managed to brew light beers using a readily available commercial product. What is that product, and how does it work? 19. Kudzu is a vine that grows prolifically in the southern and southeastern United States. A native of Japan, China, and India, kudzu was brought to the United States in 1876 to the Centennial Exposition in Philadelphia. During the Great Depression of the 1930s, the Soil Conservation Service promoted kudzu for erosion control, and farmers were paid to plant it. Today, however, kudzu is a universal nuisance, spreading rapidly and covering and destroying trees in large numbers. Already covering 7–10 million acres in the United States, kudzu grows at the rate of a foot per day. Assume that the kudzu vine consists almost entirely of cellulose fibres, and assume that the fibres lie parallel to the vine axis. Calculate the rate of the cellulose synthase reaction that adds glucose units to the growing cellulose molecules. Use the structures in your text to make a reasonable estimate of the unit length of a cellulose molecule (from one glucose monomer to the next). 20. Heparin has a characteristic pattern of hydroxy and anionic functions. What amino acid side chains on antithrombin III might be the basis for the strong interactions between this protein and the anticoagulant heparin? 21. What properties of hyaluronate, chondroitin sulfate, and keratan sulfate make them ideal components of cartilage?

CH2OH O OH

O CH2 O OH HO OH

O

CH2

OH Stachyose

HO

O

OH OH

CH2OH O HO

O

CH2OH

OH

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 7

Carbohydrates and the Glycoconjugates of Cell Surfaces

227

FURTHER READING Carbohydrate Structure and Chemistry

Proteoglycans

Collins, P. M., 1987. Carbohydrates. London: Chapman and Hall.

Day, A. J., and Prestwich, G. D., 2002. Hyaluronan-binding proteins: Tying up the giant. Journal of Biological Chemistry 277:4585–4588.

Davison, E. A., 1967. Carbohydrate Chemistry. New York: Holt, Rinehart and Winston. Pigman, W., and Horton, D., 1972. The Carbohydrates. New York: Academic Press.

Kjellen, L., and Lindahl, U., 1991. Proteoglycans: Structures and interactions. Annual Review of Biochemistry 60:443–475.

Sharon, N., 1980. Carbohydrates. Scientific American 243:90–102.

Lennarz, W. J., 1980. The Biochemistry of Glycoproteins and Proteoglycans. New York: Plenum Press.

Polysaccharides

Ruoslahti, E., 1989. Proteoglycans in cell regulation. Journal of Biological Chemistry 264:13369–13372.

Aspinall, G. O., 1982. The Polysaccharides, Vols. 1 and 2. New York: Academic Press.

Glycobiology

Höfte, H., 2001. A baroque residue in red wine. Science 294:795–797.

Bertozzi, C. R., and Kiessling, L. L., 2001. Chemical glycobiology. Science 291:2357–2363.

McNeil, M., Darvill, A. G., et al., 1984. Structure and function of the primary cell walls of plants. Annual Review of Biochemistry 53:625–664. O’Neill, M. A., Eberhard, S., et al., 2002. Requirements of borate crosslinking of cell wall rhamnogalacturonan II for Arabidopsis growth. Science 294:846–849. Glycoproteins Feeney, R. E., Burcham, T. S., et al., 1986. Antifreeze glycoproteins from polar fish blood. Annual Review of Biophysical Chemistry 15:59–78. Helenius, A., and Aebi, M., 2001. Intracellular functions of N-linked glycans. Science 291:2364–2369. Jentoft, N., 1990. Why are proteins O-glycosylated? Trends in Biochemical Sciences 155:291–294.

Lodish, H. F., 1991. Recognition of complex oligosaccharides by the multisubunit asialoglycoprotein receptor. Trends in Biochemical Sciences 16:374–377. Maeder, T., 2002. Sweet medicines. Scientific American 287:40–47. Miyamoto, S., 2006. Clinical applications of glycomic approaches for the detection of cancer and other diseases. Current Opinion in Molecular Therapies 8:507–513. Rademacher, T. W., Parekh, R. B., et al., 1988. Glycobiology. Annual Review of Biochemistry 57:785–838. Timmer, M., Stocker, B., et al., 2007. Probing glycomics. Current Opinion in Chemical Biology 11:59–65.

Sharon, N., 1984. Glycoproteins. Trends in Biochemical Sciences 9:198–202.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

8

Lipids

! “The mighty whales which swim in a sea

of water, and have a sea of oil swimming in them.” Herman Melville, “Extracts.” Moby Dick. New York: Penguin Books, 1972. (Humpback whale [Megaptera novaeangliae] breaching, Cape Cod, MA)

A feast of fat things, a feast of wines on the lees. Isaiah 25:6

8.1

What Are the Structures and Chemistry of Fatty Acids?

8.2

What Are the Structures and Chemistry of Triacylglycerols?

8.3

What Are the Structures and Chemistry of Glycerophospholipids?

8.4

What Are Sphingolipids, and How Are They Important for Higher Animals?

8.5

What Are Waxes, and How Are They Used?

8.6

What Are Terpenes, and What Is Their Relevance to Biological Systems?

8.7

What Are Steroids, and What Are Their Cellular Functions?

8.8

How Do Lipids and Their Metabolites Act as Biological Signals?

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

© Brandon D. Cole/CORBIS

KEY QUESTIONS

ESSENTIAL QUESTION Lipids are a class of biological molecules defined by low solubility in water and high solubility in non-polar solvents. As molecules that are largely hydrocarbon in nature, lipids represent highly reduced forms of carbon and, upon oxidation in metabolism, yield large amounts of energy. Lipids are thus the molecules of choice for metabolic energy storage. What are the structure, chemistry, and biological function of lipids?

WHY IT MATTERS Before we begin our discussion of lipids, we pose the question: why do lipids matter? As we will see in this chapter and in Chapter 9, the major constituents of lipids (or fats as they are commonly referred to) are fatty acids. The extreme dietary consumption of particular fatty acids deemed “bad” by scientists can have profound effects on our overall well-being and can be catalysts in promoting atherosclerosis, cardiovascular disease, and stroke. Such diseases have led us to rethink dietary lipid intake and have even brought back the famous “butter versus margarine” debate. The lipids found in biological systems are either hydrophobic (containing only non-polar groups) or amphipathic (possessing both polar and non-polar groups). The hydrophobic nature of lipid molecules allows membranes to act as effective barriers to more polar molecules. In this chapter, we discuss the chemical and physical properties of the various classes of lipid molecules. Chapter 10 considers membranes, whose properties depend intimately on their lipid constituents (see Chapter 9 for lipid biosynthesis).

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 8

Lipids

Thinkstock

" “Butter versus Margarine”

8.1

What Are the Structures and Chemistry of Fatty Acids?

A fatty acid is composed of a long hydrocarbon chain (“tail”) and a terminal carboxyl group (or “head”). The carboxyl group is normally ionized under physiological conditions. Fatty acids occur in large amounts in biological systems but only rarely in the free, uncomplexed state. They are typically esterified to glycerol or other backbone structures. Most of the fatty acids found in nature have an even number of carbon atoms (usually 14–24). Certain marine organisms, however, contain substantial amounts of fatty acids with odd numbers of carbon atoms. Fatty acids are either saturated (all carbon–carbon bonds are single bonds) or unsaturated (with 1 or more double bonds in the hydrocarbon chain). If a fatty acid has a single double bond, it is said to be monounsaturated, and if it has more than 1, polyunsaturated. Fatty acids can be named or described in at least 3 ways, as shown in Table 8.1. For example, a fatty acid composed of an 18-carbon chain with no double bonds can be called by its systematic name (octadecanoic acid); its common name (stearic acid); or its shorthand notation, in which the number of carbons is followed by a colon and the number of double bonds in the molecule (18:0 for stearic acid). The structures of several common fatty acids are given in Figure 8.1. Stearic acid (18:0) and palmitic acid (16:0) are the most common saturated fatty acids in nature. Free rotation around each of the carbon–carbon bonds makes saturated fatty acids extremely flexible molecules. Owing to steric constraints, however, the fully extended conformation (Figure 8.1) is the most stable for saturated fatty acids. Nonetheless, the degree of stabilization is slight, and (as will be seen) saturated fatty acid chains adopt a variety of conformations. Unsaturated fatty acids are slightly more abundant in nature than saturated fatty acids, especially in higher plants. The most common unsaturated fatty acid is oleic acid, or 18:1!9, with the number beside the delta (!) symbol indicating that the double bond is between carbons 9 and 10. The number of double bonds in an unsaturated fatty acid typically varies from 1 to 4, but in the fatty acids found in most bacteria, this number rarely exceeds 1. The double bonds found in fatty acids are nearly always in the cis configuration. As shown in Figure 8.1, this causes a bend or “kink” in the fatty acid chain. This bend has very important consequences for the structure of biological membranes. Saturated fatty acid chains can pack closely together to form ordered, rigid arrays under certain conditions, but unsaturated fatty acids prevent such close packing and produce flexible, fluid aggregates. Some fatty acids are not synthesized by mammals and yet are necessary for normal growth and life. These essential fatty acids include linoleic and g-linolenic acids. These must be obtained by mammals in their diet (specifically from plant sources). Arachidonic acid, which NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

229

230

Part 1 Building Blocks of the Cell

TABLE 8.1 Common Biological Fatty Acids Number of Carbons

Common Name

Systematic Name

Symbol

Structure

12

Lauric acid

Dodecanoic acid

12:0

CH3(CH2)10COOH

14

Myristic acid

Tetradecanoic acid

14:0

CH3(CH2)12COOH

16

Palmitic acid

Hexadecanoic acid

16:0

CH3(CH2)14COOH

18

Stearic acid

Octadecanoic acid

18:0

CH3(CH2)16COOH

20

Arachidic acid

Eicosanoic acid

20:0

CH3(CH2)18COOH

22

Behenic acid

Docosanoic acid

22:0

CH3(CH2)20COOH

24

Lignoceric acid

Tetracosanoic acid

24:0

CH3(CH2)22COOH

Saturated fatty acids

Unsaturated fatty acids (all double bonds are cis) 16

Palmitoleic acid

9-hexadecenoic acid

16:1*

CH3(CH2)5CH “ CH(CH2)7COOH

18

Oleic acid

9-octadecenoic acid

18:1

CH2(CH2)7CH “ CH(CH2)7COOH

18

Linoleic acid

9,12-octadecadienoic acid

18:2

CH3(CH2)4(CH “ CHCH2)2(CH2)6COOH

18

a-Linolenic acid

9,12,15-octadecatrienoic acid

18:3

CH3CH2(CH “ CHCH2)3(CH2)6COOH

18

g-Linolenic acid

6,9,12-octadecatrienoic acid

18:3

CH3(CH2)4(CH “ CHCH2)3(CH2)3COOH

20

Arachidonic acid

5,8,11,14-eicosatetraenoic acid

20:4

CH3(CH2)4(CH “ CHCH2)4(CH2)2COOH

24

Nervonic acid

15-tetracosenoic acid

24:1

CH3(CH2)7CH “ CH(CH2)13COOH

*Palmitoleic acid can also be described as 16:1!9, in a convention used to indicate the position of the double bond.

O

OH

O

C

OH

O

C

OH

O

C

OH C

CH2 H2C CH2 H2C CH2 H2C CH2 H2C CH2

Palmitic acid

Stearic acid

Oleic acid

H2C CH2 H2C CH2 H2C CH3 O

OH C

Linoleic acid

O

OH C

!-Linolenic acid

O

OH C

Arachidonic acid

FIGURE 8.1 The structures of some typical fatty acids. Note that most natural fatty acids contain an even number of carbon atoms and that the double bonds are nearly always cis and rarely conjugated. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 8

231

Lipids

TABLE 8.2 Fatty Acid Compositions of Some Dietary Lipids* Lauric and Myristic

Palmitic

Stearic

Oleic

Linoleic

5

24–32

20–25

37–43

2–3

25

12

33

3

10

2

7

Corn

8–12

3–4

19–49

34–62

Olive

9

2

84

4

Palm

Source Beef Milk Coconut

74

39

4

40

8

Safflower

6

3

13

78

Soybean

9

6

20

52

Sunflower

6

1

21

66

Data from Merck Index, 10th ed. Rahway, NJ: Merck and Co.; and Wilson, E. D., et al., 1979, Principles of Nutrition, 4th ed. New York: Wiley. *Values are percentages of total fatty acids.

is not found in plants, can be synthesized by mammals only from linoleic acid. At least one function of the essential fatty acids is to serve as a precursor for the synthesis of eicosanoids, such as prostaglandins, a class of compounds that exert hormone-like effects in many physiological processes (discussed in Chapter 9). Fats in the modern human diet vary widely in their fatty acid composition (Table 8.2 above). The incidence of cardiovascular disease is correlated with diets high in saturated fatty acids. By contrast, a diet relatively higher in unsaturated fatty acids (especially polyunsaturated fatty acids) may reduce the risk of heart attacks and strokes. Although vegetable  oils usually contain a higher proportion of unsaturated fatty acids than do animal oils and fats, several plant oils are actually high in saturated fats. Palm oil is low in polyunsaturated fatty acids and particularly high in (saturated) palmitic acid. Coconut oil is particularly high in lauric and myristic acids (both saturated) and contains little unsaturated fatty acid. Canola oil has been promoted as a healthy dietary oil because it consists primarily of oleic acid (60%), linoleic acid (20%), and a-linolenic acid (9%), with very low saturated fat content (7%). Canola oil is actually rapeseed oil, from the seeds of the rape plant Brassica rapa (from the Latin rapa, meaning “turnip”), a close relative of mustard, kale, cabbage, and broccoli. Asians and Europeans used rapeseed oil in lamps for hundreds of years, but it was not usually considered edible because of its high content of erucic acid, a 22:1!13 monounsaturated fatty acid (often 20% to 60%). In the first half of the 20th century, it was used as a steam engine lubricant (especially in World War II). Conventional breeding techniques have reduced the erucic acid content to less than 1%, producing the “canola oil” (the name is derived from Canadian oil, low acid) now used so commonly for cooking and baking. Although most unsaturated fatty acids in nature are cis fatty acids, trans fatty acids are formed by some bacteria via double-bond migration and isomerization. These bacterial reactions produce trans fats in ruminant animals (which carry essential bacteria in their rumen), and butter, milk, cheese, and the meat of these animals contain modest quantities of trans fats (typically 2% to 8% by weight), such as those in Figure 8.2. Margarine and other processed fats, made by partial hydrogenation of polyunsaturated oils (e.g., corn, safflower, and sunflower), contain substantial levels of various trans fats, and clinical research has shown that chronic consumption of processed foods containing partially hydrogenated vegetable oils can contribute to cardiovascular disease. Diets high in trans fatty acids raise plasma low-density lipoprotein (LDL) cholesterol levels and triglyceride levels while lowering high-density lipoprotein (HDL) cholesterol levels. The effects of trans fatty acids on LDL, HDL, and cholesterol levels are similar to those of saturated fatty acids. Diets aimed at reducing the risk of coronary heart disease should be low in both trans and saturated fatty acids.

H

O C OH

H Elaidic acid trans double bond at 9,10 position

H

O C H

OH

Vaccenic acid trans double bond at 11,12 position

FIGURE 8.2 Structures of elaidic acid and vaccenic acid, 2 trans fatty acids.

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232

Part 1 Building Blocks of the Cell

HUMAN BIOCHEMISTRY

Fast Foods in Canada

Africa Studio/Shutterstock

In industrialized nations such as Canada, the meals that fast food chains provide are well known for their characteristic fried nature, inherently containing high amounts of trans and saturated fatty acids.* The evidence supporting links to cardiovascular disease, diabetes, obesity, and stroke due to the increased dietary intake of these foods is overwhelming.** In 2005, attempts were made to reduce trans and saturated fatty acid consumption in Canada due in part to new regulations mandating their labelling on packaged foods such as donuts, cookies, muffins, and french fries.† Results from a 2009 national assessment of trans fatty acid content in select Canadian foods demonstrated that three-quarters of the grocery and restaurant products assessed had undergone reformulation, leading to an average reduction to less than 2% trans fatty acid content.† These reformulations consisted of a change to increased cis, unsaturated fat content† [see graphs in the Ratnayake et al. (2009) reference]. Clearly, the overall health benefits arising from the transition to lower trans and saturated fatty acid levels in foods, in addition to heightened public awareness of the consequences associated with the continual consumption of these “bad” fats, are undisputable.

8.2

*Martin, C. A., Milinak, M.C., et al., 2007. Trans fatty acid-forming processes in foods: A review. Anais da Academia Brasileira de Ciencias 79:1–16. **Mozaffarian, D., Aro, A., et al., 2009. Health effects of trans-fatty acids: experimental and observational evidence. European Journal of Clinical Nutrition 63:S5–S21. †Ratnayake W. M. N., L’Abbe, M. R., et al. 2009. Nationwide product reformulations to reduce trans fatty acids in Canada: When trans fat goes out, what goes in? European Journal of Clinical Nutrition 63:808–811.

What Are the Structures and Chemistry of Triacylglycerols?

A significant number of the fatty acids in plants and animals exist in the form of triacylglycerols (also called triglycerides). Triacylglycerols are a major energy reserve and the principal neutral derivatives of glycerol found in animals. These molecules consist of a glycerol esterified with 3 fatty acids (Figure 8.3). If all 3 fatty acid groups are the same, the molecule is called a simple triacylglycerol. Examples include tristearoylglycerol (common name tristearin) and trioleoylglycerol (triolein). Mixed triacylglycerols contain 2 or 3 different fatty acids. Triacylglycerols in animals are found primarily in the adipose tissue (body fat), which serves as a depot or storage site for lipids. Monoacylglycerols and diacylglycerols also exist, but they are far less common than the triacylglycerols. Most natural plant and animal fat is composed of mixtures of simple and mixed triacylglycerols. Acylglycerols can be hydrolyzed by heating with acid or base or by treatment with lipases. Hydrolysis with alkali is called saponification and yields salts of free fatty acids and glycerol. This is how our ancestors made soap (a metal salt of an acid derived from fat). One method used potassium hydroxide (potash) leached from wood ashes to hydrolyze animal fat (mostly triacylglycerols). (The tendency of such soaps to be precipitated by Mg2+ and Ca2+ ions in hard water makes them less useful than modern detergents.) When the fatty acids esterified at the first and third carbons of glycerol are different, the second carbon is asymmetric. The various acylglycerols are normally soluble in benzene, chloroform, ether, and hot ethanol. Although triacylglycerols are insoluble in water, monoacylglycerols and diacylglycerols readily form organized structures in water (see Chapter 10), owing to the polarity of their free hydroxyl groups. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 8

233

Lipids

A DEEPER LOOK

Polar Bears Prefer Non-polar Food

H2C

CH

CH2

H2C

CH

HO

OH

OH

O

O

O

C

C

O C

Glycerol

O

© John Shaw/Photo Researchers, Inc.

The polar bear is magnificently adapted to thrive in its harsh Arctic environment. Research by Malcolm Ramsay (at the University of Saskatchewan) and others has shown that polar bears eat only during a few weeks of the year and then fast for periods of 8 months or more, consuming no food or water during that time. Eating mainly in the winter, the adult polar bear feeds almost exclusively on seal blubber (largely composed of triacylglycerols), thus building up its own triacylglycerol reserves. Through the Arctic summer, the polar bear maintains normal physical activity, roaming over long distances, but relies entirely on its body fat for sustenance, burning as much as 1–1.5 kg of fat per day. It neither urinates nor defecates for extended periods. All the water needed to sustain life is provided from the metabolism of triacylglycerols (because oxidation of fatty acids yields carbon dioxide and water). The word Arctic comes from the ancient Greeks, who understood that the northernmost part of Earth lay under the stars of the constellation Ursa Major, the Great Bear. Although unaware of the polar bear, they called this region Arktikós, which means “the country of the great bear.”

CH2

H2C

O

O

CH

O

O

O

C

C

O C

Myristic Tristearin (a simple triacylglycerol)

CH2

O

Palmitoleic

Stearic A mixed triacylglycerol

FIGURE 8.3 Triacylglycerols are formed from glycerol and fatty acids.

Triacylglycerols are rich in highly reduced carbons and thus yield large amounts of energy in the oxidative reactions of metabolism. Complete oxidation of 1 g of triacylglycerols yields about 38 kJ of energy, whereas proteins and carbohydrates yield only about 17 kJ/g. Also, their hydrophobic nature allows them to aggregate in highly anhydrous forms, whereas polysaccharides and proteins are highly hydrated. For these reasons, triacylglycerols are the molecules of choice for energy storage in animals. Body fat (mainly triacylglycerols) also provides good insulation. Whales and Arctic mammals rely on body fat for both insulation and energy reserves.

8.3

What Are the Structures and Chemistry of Glycerophospholipids?

A 1,2-diacylglycerol that has a phosphate group esterified at carbon atom 3 of the glycerol backbone is a glycerophospholipid, also known as a phosphoglyceride or a glycerol phosphatide (Figure 8.4). These lipids form one of the largest and most important classes of NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

234

Part 1 Building Blocks of the Cell O C O

O

C

O

CH2 C

H

CH2

O O

P

O–

O– FIGURE 8.4 Phosphatidic acid, the parent compound for glycerophospholipids.

natural lipids. They are essential components of cell membranes and are found in small concentrations in other parts of the cell. It should be noted that all glycerophospholipids are members of the broader class of lipids known as phospholipids. The numbering and nomenclature of glycerophospholipids present a dilemma in that the number 2 carbon of the glycerol backbone of a phospholipid is asymmetric. It is possible to name these molecules as either D- or L-isomers. Thus, glycerol phosphate itself can be referred to either as D-glycerol-1-phosphate or as L-glycerol-3-phosphate (Figure 8.5). Instead of naming the glycerol phosphatides in this way, biochemists have adopted the stereospecific numbering or sn- system. The stereospecific numbering system is based on the concept of prochirality. If a tetrahedral centre in a molecule has 2 identical substituents, it is referred to as prochiral because if either of the like substituents is converted to a different group, the tetrahedral centre then becomes chiral. Consider glycerol (Figure 8.5): The central carbon of glycerol is prochiral because replacing either of the —CH2OH groups would make the central carbon chiral. Nomenclature for prochiral centres is based on the (R,S ) system (see Chapter 4). To name the otherwise identical substituents of a prochiral centre, imagine slightly increasing the priority of one of them (by substituting a deuterium for a hydrogen, e.g.), as shown in Figure 8.5. The resulting molecule has an (S ) configuration about the (now chiral) central carbon atom. The group that contains the deuterium is thus referred to as the pro-S group. As a useful exercise, you should confirm that labelling the other —CH2OH group with a deuterium produces the (R) configuration at the central carbon so that this latter —CH2OH group is the pro-R substituent. Now consider the 2 presentations of glycerol phosphate in Figure 8.5. In the stereospecific numbering system, the pro-S position of a prochiral atom is denoted as the 1-position, the prochiral atom as the 2-position, and so on. When this scheme is used, the prefix sn- precedes the molecule name (glycerol phosphate in this case) and distinguishes this nomenclature from other approaches. In this way, the glycerol phosphate in natural phosphoglycerides is named sn-glycerol-3-phosphate. FIGURE 8.5 (a) The 2 identical i CH2OH groups on the central carbon of glycerol may be distinguished by imagining a slight increase in priority for one of them (by replacing an H by a D) as shown. (b) The absolute configuration of sn-glycerol-3-phosphate is shown. The pro-R and pro-S positions of the parent glycerol are also indicated.

D (a)

HOH2C

HOH23C

CH2OH C

H

1CHOH 2C

H

OH

OH

1-d, 2(S)-Glycerol (S-configuration at C-2)

Glycerol

(b) pro-S position HO pro-R position

CH2OPO32–

CH2 OH C

H

H

CH2OPO32– L-Glycerol-3-phosphate

C

OH

CH2 OH D-Glycerol-1-phosphate

sn-Glycerol-3-phosphate NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Glycerophospholipids Are the Most Common Phospholipids Phosphatidic acid, the parent compound for the glycerol-based phospholipids (Figure 8.4), consists of sn-glycerol-3-phosphate, with fatty acids esterified at the 1- and 2-positions. Phosphatidic acid is found in small amounts in most natural systems and is an important intermediate in the biosynthesis of the more common glycerophospholipids (Figure 8.6). In these compounds, a variety of polar groups are esterified to the phosphoric acid moiety of the molecule. The phosphate, together with such esterified entities, is referred to as a “head” group. Phosphatides with choline and ethanolamine are referred to as phosphatidylcholine (known commonly as lecithin) and phosphatidylethanolamine, respectively. These phosphatides are 2 of the most common constituents of biological membranes. Other common head groups found in phosphatides are glycerol, serine, and inositol (Figure 8.6). Another kind of glycerol phosphatide found in many tissues is diphosphatidylglycerol. First O C

O CH2

O C

O

C

H

CH2

CH3

O O

P O–

Phosphatidylcholine

O

CH2CH2

N + CH3 CH3

GLYCEROLIPIDS WITH OTHER HEAD GROUPS: O O

P

O O

CH2CH2

+ NH3

O

O–

O–

Phosphatidylethanolamine O

P

O

CH2

O–

O

CH2

H

C

O

CH2

P O–

CH + NH3

Diphosphatidylglycerol (Cardiolipin) H

Phosphatidylserine H O O

P O–

O O

CH2

CH

CH2

OH

OH

Phosphatidylglycerol

OH

O

COO–

O O

P

O

P

HO O

OH

OH H H HO

H OH

H

O– Phosphatidylinositol

FIGURE 8.6 Structures of several glycerophospholipids and space-filling models of phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

236

Part 1 Building Blocks of the Cell

FIGURE 8.7 A space-filling model of 1-stearoyl-2-oleoyl-phosphatidylcholine.

observed in heart tissue, it is also called cardiolipin. In cardiolipin, a phosphatidylglycerol is esterified through the C-1 hydroxyl group of the glycerol moiety of the head group to the phosphoryl group of another phosphatidic acid molecule. Phosphatides exist in many different varieties, depending on the fatty acids esterified to the glycerol group. As we shall see, the nature of the fatty acids can greatly affect the chemical and physical properties of the phosphatides and the membranes that contain them. In most cases, glycerol phosphatides have a saturated fatty acid at position 1 and an unsaturated fatty acid at position 2 of the glycerol. Thus, 1-stearoyl-2-oleoyl-phosphatidylcholine (Figure 8.7) is a common constituent in natural membranes, but 1-linoleoyl-2palmitoylphosphatidylcholine is not. Both structural and functional strategies govern the natural design of the many different kinds of glycerophospholipid head groups and fatty acids. The structural roles of these different glycerophospholipid classes are described in Chapter 10. Certain phospholipids, including phosphatidylinositol and phosphatidylcholine, participate in complex cellular signalling events. These roles are described in Section 8.8 and Chapter 11.

Ether Glycerophospholipids Include PAF and Plasmalogens Ether glycerophospholipids possess an ether linkage instead of an acyl group at the C-1 position of glycerol (Figure 8.8a). One of the most versatile biochemical signal molecules found in mammals is platelet-activating factor, or PAF, a unique ether glycerophospholipid (Figure 8.8b). The alkyl group at C-1 of PAF is typically a 16-carbon chain, but the acyl group at C-2 is a 2-carbon acetate unit. By virtue of this acetate group, PAF is much more water-soluble than other lipids, allowing PAF to function as a soluble messenger in signal transduction. (a)

O –O

P

O

CH2

CH2

+ NH3

O H2C Ether linkage

CH

O

O

R1

C

CH2 Ester linkage O

R2 (b)

O –O

P

O

CH2

CH2

O H2C O

CH

CH3 + N CH3 CH3

CH2

O C CH3

O

Plateletactivating factor

FIGURE 8.8 (a) A 1-alkyl 2-acylphosphatidylethanolamine (an ether glycerophospholipid). (b) The structure of 1-alkyl 2-acetyl-phosphatidylcholine, also known as plateletactivating factor or PAF. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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237

HUMAN BIOCHEMISTRY

Platelet-Activating Factor: A Potent Glyceroether Mediator Platelet-activating factor (PAF) was first identified by its ability (at  low levels) to cause platelet aggregation and dilation of blood vessels, but it is now known to be a potent mediator in inflammation, allergic responses, and shock. PAF effects are observed at tissue concentrations as low as 10-12M. PAF causes a dramatic inflammation of air passages and induces asthmalike symptoms in laboratory animals. Toxic shock syndrome occurs when fragments of destroyed bacteria act as toxins and induce the synthesis of PAF. PAF causes

CH3

O –O Choline plasmalogen

H C C

CH2

CH

O

O C

P O CH2

a drop in blood pressure and a reduced volume of blood pumped by the heart, which leads to shock and, in severe cases, death. Beneficial effects have also been attributed to PAF. In reproduction, PAF secreted by the fertilized egg is instrumental in the implantation of the egg in the uterine wall. PAF is produced in significant quantities in the lungs of the fetus late in pregnancy and may stimulate the production of fetal lung surfactant, a protein–lipid complex that prevents collapse of the lungs in a newborn infant.

O

CH2CH2

N + CH3

FIGURE 8.9 The structure and a space-filling model of a choline plasmalogen.

CH3 The ethanolamine plasmalogens have ethanolamine in place of choline.

O

H

Plasmalogens are ether glycerophospholipids in which the alkyl moiety is cis-a, b-unsaturated (Figure 8.9). Common plasmalogen head groups are choline, ethanolamine, and serine. These lipids are referred to as phosphatidal choline, phosphatidal ethanolamine, and phosphatidal serine.

8.4

What Are Sphingolipids, and How Are They Important for Higher Animals?

Sphingolipids represent another class of lipids frequently found in biological membranes. Rather than glycerol, an 18-carbon amino alcohol, sphingosine (Figure 8.10a), forms the backbone of these lipids. Typically, a fatty acid is joined to a sphingosine via an amide linkage to form a ceramide (Figure 8.10b). Sphingomyelins represent a phosphoruscontaining subclass of sphingolipids and are especially important in the nervous tissue of higher animals. A sphingomyelin is formed by the esterification of a phosphorylcholine or a phosphorylethanolamine to the 1-hydroxy group of a ceramide (Figure 8.10c). There is another class of ceramide-based lipids that, like the sphingomyelins, are important components of muscle and nerve membranes in animals. These are the glycosphingolipids, and they consist of a ceramide with 1 or more sugar residues in a b-glycosidic linkage at the 1-hydroxyl moiety. The neutral glycosphingolipids contain only neutral (uncharged) sugar residues. When a single glucose or galactose is bound in this manner, the molecule is a cerebroside (Figure 8.10d). Another class of lipids is formed when a sulfate is NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

238

Part 1 Building Blocks of the Cell (a)

OH

H

C

H C

C

(b)

OH H2O

CH2

H

+NH 3

H

OH

C R COOH Fatty acid

C H

C

C

H

NH

C

C

H

(c)

OH

H

O P

OH

H

O

C

C

CH2

CH2

O

H

H C

R

CH3

–O

O

CH2CH2

N + CH3 CH3

NH

C

C

O

H

Sphingosine

Ceramide

Choline sphingomyelin with stearic acid

(d)

(e)

GM1 GM2

"-D-Galactose

HO H

CH2OH O H OH H H

HO H OH

OH

H

H

C

C

H C

O

C H

C R

D-Galactose

D-Galactose

CH2OH O H OH H

CH2OH O H H

CH2OH O H H

H

OH

HO H

H O

H

O CH3

O H

H

NH

O

CH2

NH

GM3

N-AcetylD-galactosamine

C

H

D-Glucose

CH2OH O H OH H

H O H

OH

H

C O

CH3 H H N

O CHOH

COO–

CHOH

O H

CH2OH H H OH

H

H C

H

OH OH

H

O

C

C

CH2

NH

C H

C

O

Ganglioside GM1

R

H

N-Acetylneuraminidate (a sialic acid)

A cerebroside

Gangliosides GM1, GM2, and GM3

FIGURE 8.10 Sphingolipids are based on the structure of sphingosine. A ceramide with a phosphocholine head group is a choline sphingomyelin. A ceramide with a single sugar is a cerebroside. Gangliosides are ceramides with 3 or more sugars esterified, 1 of which is a sialic acid.

esterified at the 3-position of the galactose to make a sulfatide. Gangliosides (Figure 8.10e) are more complex glycosphingolipids that consist of a ceramide backbone with 3 or more sugars esterified, 1 of these being a sialic acid such as N-acetylneuraminic acid. These latter compounds are referred to as acidic glycosphingolipids, and they have a net negative charge at neutral pH. The glycosphingolipids have a number of important cellular functions, despite the fact that they are present only in small amounts in most membranes. Glycosphingolipids at cell surfaces appear to determine, at least in part, certain elements of tissue and organ specificity. Cell–cell recognition and tissue immunity depend on specific glycosphingolipids. Gangliosides NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 8

Lipids

239

A DEEPER LOOK

Moby Dick and Spermaceti: A Valuable Wax from Whale Oil When oil from the head of the sperm whale is cooled, spermaceti, a translucent wax with a white, pearly lustre, crystallizes from the mixture. Spermaceti, which makes up 11% of whale oil, is composed mainly of the wax cetyl palmitate:

In the literary classic Moby Dick, Herman Melville describes Ishmael’s impressions of spermaceti, when he muses that the waxes “discharged all their opulence, like fully ripe grapes their wine; as I snuffed that uncontaminated aroma—literally and truly, like the smell of spring violets.”*

CH3(CH2)14 i COO i (CH2)15CH3 as well as smaller amounts of cetyl alcohol:

*Melville, H., 1984. Moby Dick. London: Octopus Books, p. 205. (Adapted from Waddell, T. G., and Sanderlin, R. R., 1986. Chemistry in Moby Dick. Journal of Chemical Education 63:1019–1020.)

HO i (CH2)15CH3 Spermaceti and cetyl palmitate have been widely used in the making of cosmetics, fragrant soaps, and candles.

are present in nerve endings and are important in nerve impulse transmission. A number of genetically transmitted diseases involve the accumulation of specific glycosphingolipids due to an absence of the enzymes needed for their degradation. Such is the case for ganglioside GM2 in the brains of people with Tay–Sachs disease, a rare but fatal childhood disease characterized by a red spot on the retina, gradual blindness, and self-mutilation.

8.5

What Are Waxes, and How Are They Used?

Waxes are esters of long-chain alcohols with long-chain fatty acids. The resulting molecule can be viewed (in analogy to the glycerolipids) as having a weakly polar head group (the ester moiety itself) and a long, non-polar tail (the hydrocarbon chains) (Figure 8.11). Fatty acids found in waxes are usually saturated. The alcohols found in waxes may be saturated or unsaturated and may include sterols, such as cholesterol (see Section 8.7). Waxes are water-insoluble due to their mostly hydrocarbon composition. As a result, this class of molecules confers water-repellent character to animal skin, to the leaves of certain plants, and to bird feathers. The glossy surface of a polished apple results from a wax coating. Carnauba wax, obtained from the fronds of a species of palm tree in Brazil, is a particularly hard wax used for high-gloss finishes, such as in automobile wax, boat wax, floor wax, and shoe polish. Lanolin,1 a wool wax, is used as a base for pharmaceutical and cosmetic O O

C

O CH3(CH2)14

C

O

CH2

(CH2)16

CH3

O Oleoyl alcohol

Stearic acid

R1

C

O

R2

General forumula of a wax O CH3(CH2)14

C

O

CH2

(CH2)28

CH3

Triacontanol palmitate Oleoyl stearate

FIGURE 8.11 Waxes consist of long-chain alcohols esterified to long-chain fatty acids. Triacontanol palmitate is the principal component of beeswax. Waxes are components of the waxy coating on the leaves of plants, such as jade plants (shown here). Such species typically contain dozens of different waxy esters. 1

Lanolin is a complex mixture of waxes with 33 different alcohols esterified to 36 different fatty acids.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

© Steven Lunetta Photography, 2012

Stearyl palmitate

Part 1 Building Blocks of the Cell

products because it is rapidly assimilated by human skin. The brand name Oil of Olay was coined by Graham Wulff, a South African chemist who developed it. The name refers to lanolin, a key ingredient.

8.6

What Are Terpenes, and What Is Their Relevance to Biological Systems?

The terpenes are a class of lipids formed from combinations of 2 or more molecules of 2-methyl-1,3-butadiene, better known as isoprene (a 5-carbon unit that is abbreviated C5). A monoterpene (C10) consists of 2 isoprene units, a sesquiterpene (C15) consists of 3 isoprene units, a diterpene (C20) has 4 isoprene units, and so on. Isoprene units can be linked in terpenes to form straight-chain or cyclic molecules, and the usual method of linking isoprene units is head to tail (Figure 8.12). Monoterpenes occur in all higher plants, whereas sesquiterpenes and diterpenes are less widely known. Several examples of these classes of terpenes are shown in Figure 8.13. The triterpenes are C30 terpenes and include OH CH2

H

Head-to-tail linkage

R

C

Tail-to-tail linkage

C H2C

R

CH3 Geraniol

Isoprene

FIGURE 8.12 The structure of isoprene (2-methyl-1,3-butadiene) and the structure of head-to-tail and tail-to-tail linkages. Isoprene itself can be formed by distillation of natural rubber, a linear head-to-tail polymer of isoprene units. SESQUITERPENES

DITERPENES

OH

...

MONOTERPENES

H CHO

Limonene

OH

Citronellal

Menthol

...

CH2OH

O

......

240

HO

Bisabolene

Phytol

C

H CH3

COOH

O Gibberellic acid HO Camphene

CHO

Eudesmol

!-Pinene

TRITERPENES

All-trans-retinal

TETRATERPENES

HO Squalene

H Lanosterol

Lycopene

FIGURE 8.13 Many monoterpenes are readily recognized by their characteristic flavours or odours (limonene in lemons; citronellal in roses, geraniums, and some perfumes; and menthol from peppermint, used in cough drops and nasal inhalers). The diterpenes, which are C20 terpenes, include retinal (the essential light-absorbing pigment in rhodopsin, the photoreceptor protein of the eye) and phytol (a constituent of chlorophyll). The triterpene lanosterol is a constituent of wool fat. Lycopene is a carotenoid found in ripe fruit, especially tomatoes. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 8

241

Lipids

A DEEPER LOOK

Why Do Plants Emit Isoprene?

jo Crebbin/Shutterstock

The Blue Ridge Mountains of Virginia are so named for the misty blue vapour or haze that hangs over them through much of the summer season. This haze is composed in part of isoprene that is produced and emitted by the plants and trees of the mountains. Global emission of isoprene from vegetation is estimated at 3 * 1014 g/yr. Plants frequently emit as much as 15% of the carbon fixed in photosynthesis as isoprene, and Thomas Sharkey, a botanist at the University of Wisconsin, has shown that the kudzu plant can emit as much as 67% of its fixed carbon as isoprene as a result of water stress. Why should plants and trees emit large amounts of isoprene and other hydrocarbons? Sharkey has shown that an isoprene atmosphere or “blanket” can protect leaves from irreversible damage induced by high (summerlike) temperatures. He hypothesizes that isoprene in the air around plants dissolves into leaf-cell membranes, altering the lipid bilayer and/or lipid–protein and protein–protein interactions within the membrane to increase thermal tolerance.

# Blue Ridge Mountains

squalene and lanosterol, 2 of the precursors of cholesterol and other steroids (discussed in Section 8.7). Tetraterpenes (C40) are less common but include the carotenoids, a class of colourful photosynthetic pigments. b-carotene is the precursor of vitamin A, whereas lycopene, similar to b-carotene, is a pigment found in tomatoes. Long-chain polyisoprenoid molecules with a terminal alcohol moiety are called polyprenols. The dolichols, one class of polyprenols (Figure 8.14), consist of 16 to 22 isoprene units and, in the form of dolichyl phosphates, function to carry carbohydrate units in the biosynthesis of glycoproteins in animals. Polyprenyl groups serve to anchor certain proteins to biological membranes (discussed in Chapter 10).

The Membranes of Archaea Are Rich in Isoprene-Based Lipids Archaea are found primarily in harsh environments. Some thrive in the high temperatures of geysers and ocean steam vents, whereas others are found in extremely acidic, cold, or salty environments. Archaea also live in extremes of pH in the digestive tracts of cows, termites,

CH3 H

CH2

C

O

CH3 CH

CH2

CH2

CH

CH2

CH2

O

13 – 23

P

O–

O–

Dolichol phosphate

CH3 H3C

C

CH3 H C

CH2

CH2

C

CH3 CH

CH2

CH2

C

CH

CH2OH

9

Undecaprenyl alcohol (bactoprenol) FIGURE 8.14 Dolichol phosphate is an initiation point for the synthesis of carbohydrate polymers in animals. The analogous alcohol in bacterial systems, undecaprenol, also known as bactoprenol, consists of 11 isoprene units. Undecaprenyl phosphate delivers sugars from the cytoplasm for the synthesis of cell wall components such as peptidoglycans, lipopolysaccharides, and glycoproteins. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

242

Part 1 Building Blocks of the Cell CH2OH

H

H2C

O

C

O

O

C

O

CH2

H

Glycerol

HOCH2 Isoprene units

Glycerol

Caldarchaeol FIGURE 8.15 The structure of caldarchaeol, an isoprene-based lipid found in archaea.

and humans. Archaea are ideally adapted to their harsh environments, and one such adaptation is found in their cell membranes, which contain isoprene-based lipids (Figure 8.15). These isoprene chains are linked at both ends by ether bonds to glycerols. Ether bonds are more stable to hydrolysis than the ester linkages of glycerophospholipids (Figure 8.6). With a length twice that of typical glycerophospholipids, these molecules can completely span a cell membrane, providing additional stability. Interestingly, the glycerols in archaeal lipids are in the (R) configuration, whereas glycerolipids of animals, plants, and eubacteria are almost always in the (S) configuration.

HUMAN BIOCHEMISTRY

Coumadin or Warfarin—Agent of Life or Death The isoprene-derived molecule whose structure is shown here is known both as Coumadin and as warfarin. By the former name, it is a widely prescribed anticoagulant. By the latter name, it is a component of rodent poisons. How can the same chemical species be used for such disparate purposes? The key to both uses lies in its ability to act as an antagonist of vitamin K in the body. Vitamin K is necessary for the carboxylation of glutamate residues on certain proteins, including some proteins in the bloodclotting cascade (including prothrombin, factor VII, factor IX, and factor X, which undergo a Ca2+-dependent conformational change in the course of their biological activity, as well as protein C and protein S, 2 regulatory proteins in coagulation). Carboxylation of these coagulation factors is catalyzed by a carboxylase that requires the reduced form of vitamin K (vitamin KH2), molecular oxygen, and carbon dioxide. KH2 is oxidized to vitamin K epoxide, which is recycled to KH2 by the enzymes vitamin K epoxide reductase (1) and vitamin K reductase (2, 3). Coumadin/warfarin exerts its anticoagulant effect by inhibiting vitamin K epoxide reductase and possibly also vitamin K reductase. This inhibition depletes vitamin KH2 and reduces the activity of the carboxylase. Coumadin/warfarin, given at a typical dosage of 4–5 mg/day, prevents the deleterious formation in the bloodstream of small blood clots and thus reduces the risk of heart attacks and strokes for individuals whose arteries contain sclerotic plaques. Taken in much larger doses, as for example in rodent poisons, Coumadin/warfarin can cause massive hemorrhages and death.

O

O

O

CH

CH2

C

CH3

HO Warfarin (Coumadin) Glu

#-carboxy-Glu O

CH

H

CH2 O

3

C

CH

C

H O-

-O

O

CH2 O

C

CH

C

O-

CO2

Warfarin resistant K

C

N

H2C

O N

KH2

KO

1

2 K

Warfarin inhibits

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Chapter 8

8.7

Lipids

What Are Steroids, and What Are Their Cellular Functions?

Cholesterol A large and important class of terpene-based lipids is the steroids. This molecular family, whose members affect an amazing array of cellular functions, is based on a common structural motif of three 6-membered rings and one 5-membered ring all fused together. Cholesterol (Figure 8.16) is the most common steroid in animals and the precursor for all other animal steroids. The numbering system for cholesterol applies to all such molecules. Many steroids contain methyl groups at positions 10 and 13 and an 8- to 10-carbon alkyl side chain at position 17. The polyprenyl nature of this compound is particularly evident in the side chain. Many steroids contain an oxygen at C-3, either a hydroxyl group in sterols or a carbonyl group in other steroids. Significantly, the carbons at positions 10 and 13 and the alkyl group at position 17 are nearly always oriented on the same side of the steroid nucleus, the b-orientation. Alkyl groups that extend from the other side of the steroid backbone are in an a-orientation. Cholesterol is a principal component of animal cell plasma membranes, and smaller amounts of cholesterol are found in the membranes of intracellular organelles. The relatively rigid fused ring system of cholesterol and the weakly polar alcohol group at the C-3 position have important consequences for the properties of plasma membranes. Cholesterol is also a component of lipoprotein complexes in the blood, and it is one of the constituents of plaques that form on arterial walls in atherosclerosis.

Steroid Hormones Are Derived from Cholesterol Steroids derived from cholesterol in animals include 5 families of hormones (the androgens, estrogens, progestins, glucocorticoids, and mineralocorticoids) and bile acids (Figure 8.17). Androgens, such as testosterone, and estrogens, such as estradiol, mediate the development of sexual characteristics and sexual function in animals. The progestins, such as progesterone, participate in control of the menstrual cycle and pregnancy. Glucocorticoids (e.g., cortisol) participate in the control of carbohydrate, protein, and lipid metabolism, whereas the mineralocorticoids regulate salt (Na+, K+, and Cl–) balances in tissues. The bile acids (including cholic and deoxycholic acid) are detergent molecules secreted in bile from the gallbladder that assist in the absorption of dietary lipids in the intestine.

26

CH3 27

25 HC 24 23 22

H3C 11

H3C 2 3

HO

1 19

A 4

5

10

9

B 6

12

C

CH2 CH2 CH2

HC

18 13 14

CH3

20 21

17

D

CH3

16 15

8 7

Cholesterol FIGURE 8.16 The structure of cholesterol, shown with steroid ring designations and carbon numbering.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

243

244

Part 1 Building Blocks of the Cell CH2OH C HO

CH3

O OH

OH

C

O

O

HO

O

Cortisol

Testosterone

Estradiol

Progesterone

HO

HO COOH

HO

OH

O

COOH

OH

HO

Cholic acid

Deoxycholic acid FIGURE 8.17 The structures of several important sterols derived from cholesterol.

8.8

How Do Lipids and Their Metabolites Act as Biological Signals?

Glycerophospholipids and sphingolipids play important structural roles as the principal components of biological membranes (see Chapter 10). However, their modification and breakdown also produce an eclectic assortment of substances that act as powerful chemical signals (Figures 8.18 and 8.19). In contrast to the steroid hormones (Figure 8.17), which travel from tissue to tissue in the blood to exert their effects, these lipid metabolites act locally, either within the cell in which they are made or on nearby cells. Signal molecules typically initiate a cascade of reactions with multiple possible effects, and the lifetimes of

(a)

Phospholipase D

O

CH2

CH2

O Phospholipase C

+

N

CH3

CH3

Phospholipase A 2 CH2 CH

O O

C O

CH2

O

Diamondback rattlesnake

Joe McDonald/CORBIS

P

Tom Bean/CORBIS

–O

(b)

CH3

O

C Phospholipase A 1 Indian cobra FIGURE 8.18 (a) Phospholipases A1 and A2 cleave fatty acids from a glycerophospholipid, producing lysophospholipids. Phospholipases C and D hydrolyze on either side of the phosphate in the polar head group. (b) Phospholipases are components of the venoms of many poisonous snakes. The pain and physiological consequences of a snake bite partly result from breakdown of cell membranes by phospholipases.

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Chapter 8

Lipids

245

A DEEPER LOOK

Glycerophospholipid Degradation: One of the Effects of Snake Venom The venoms of poisonous snakes contain (among other things) a class of enzymes known as phospholipases, enzymes that cause the breakdown of phospholipids. For example, the venoms of the eastern diamondback rattlesnake (Crotalus adamanteus) and the Indian cobra (Naja naja) both contain phospholipase A2, which catalyzes

the hydrolysis of fatty acids at the C-2 position of glycerophospholipids (Figure 8.18). The phospholipid breakdown product of this reaction, lysolecithin, acts as a detergent and dissolves the membranes of red blood cells, causing them to rupture. Indian cobras kill several thousand people each year.

these powerful signals in or near a cell are usually very short. Thus, the creation and breakdown of signal molecules is almost always carefully timed and regulated. Enzymes known as phospholipases hydrolyze the ester bonds of glycerophospholipids as shown in Figure 8.18. Phospholipases A1 and A2 remove fatty acid chains from the 1- and 2-positions, respectively, of glycerophospholipids. Phospholipases C and D attack the polar head group of a glycerophospholipid. Hydrolysis of inositol phospholipids by phospholipase C produces a diacylglycerol and inositol-1,4,5-trisphosphate (IP3) (Figure 8.19), 2 signal molecules whose combined actions trigger signalling cascades that regulate many cell processes (see Chapter 11). Action of phospholipase A2 on a phosphatidic acid releases a fatty acid and a lysophosphatidic acid (LPA; Figure 8.19). If the fatty acid is arachidonic acid, further chemical modifications can produce a family of 20-carbon compounds, that is, eicosanoids. The eicosanoids are local hormones produced as a response to injury and inflammation. They exert their effects on cells near their sites of synthesis (see Chapter 9). LPA produced outside the cell is a signal that can bind to receptor proteins on nearby cells, thereby regulating a host of processes, including brain development, cell proliferation and survival, and olfaction (the sense of smell).

Phosphatidic acid

Phosphatidylinositol 2 ATP

PLA2

Arachidonic acid

+

2 ADP

Lysophosphatidic acid (LPA) (extracellular)

Phosphatidylinositol-4,5-bisphosphate (PIP2) PLC

Prostaglandins Thromboxanes Leukotrienes

Effects

Diacylglycerol

Inositol-1,4,5-trisphosphate (IP3)

Activation of protein kinase C

Increase in cellular Ca2+

Phosphorylation in signalling pathways

Binding and regulation

Effects

FIGURE 8.19 Modification and breakdown of glycerophospholipids produce a variety of signals and regulatory effects. Phospholipase A2 cleaves a fatty acid from phosphatidic acid to produce lysophosphatidic acid (LPA), which can act as an extracellular signal. If the fatty acid released is arachidonic acid, it can be the substrate for synthesis of prostaglandins, thromboxanes, and leukotrienes. Phospholipase C action on phosphatidylinositol-4,5-bisphosphate produces diacylglycerol and inositol-1,4,5-trisphosphate, 2 signal molecules that work together to activate protein kinases—enzymes that phosphorylate other proteins in signalling pathways.

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246

Part 1 Building Blocks of the Cell OH C H

H

C

H

O

C

CH2

PO32–

+NH 3

C H

Sphingosine-1-phosphate (S1P)

HOOC

O

HOOC CH3

O

CH3 OH

NH2

CH3

CH3

HOOC

OH

O

HOOC

OH

O Fumonisin B1

FIGURE 8.20 Structures of sphingosine-1-phosphate (S1P) and fumonisin B1.

Sphingolipids can also be modified or broken down to produce chemical signals. Sphingosine itself can be phosphorylated to produce sphingosine-1-phosphate (S1P) inside cells (Figure 8.20). S1P may either exert a variety of intracellular effects or be excreted from the cell, where it can bind to membrane receptor proteins, either on adjacent cells or on the cell from which the S1P was released. Excreted S1P binds to many different receptor proteins and provokes many different cell and tissue effects, among them inflammation in allergic reactions, heart rate, and movement and migration of certain cells. Sphingolipid signal molecules are carefully balanced and regulated in organisms, and chemical agents that disturb this balance can be highly toxic. For example, fumonisin is a common fungal contaminant of corn and corn-based products that inhibits sphingolipid biosynthesis (Figure 8.20; see also Chapter 9). Fumonisin can trigger esophageal cancer in humans and leucoencephalomalacia, a fatal neurological disease in horses. Cellular lipidomics provides a framework for understanding the myriad roles of lipids, which include (but are not limited to) membrane transport (see Chapter 10); cell signalling (see Chapter 11); and metabolic regulation (see Chapters 9, 17–26, and 33). For example, 6 different classes of lipids have been shown to modulate systems important in the regulation of pain responses. Each of these classes of lipids exerts its action by interacting with 1 or more receptor proteins. True understanding of the molecular basis for diseases and metabolic and physiologic conditions may require comprehensive and simultaneous analyses of many lipid species and their respective receptors.

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Chapter 8

Lipids

247

HUMAN BIOCHEMISTRY

Plant Sterols and Stanols—Natural Cholesterol Fighters Dietary guidelines for optimal health call for reducing cholesterol intake. One strategy involves eating plant sterols and stanols in place of cholesterol-containing fats such as butter (see figure). Despite their structural similarity to cholesterol, minor isomeric differences and the presence of methyl and ethyl groups in the side chains of these substances result in their poor absorption by intestinal mucosal cells. Interestingly, stanols are even less well absorbed than their sterol counterparts. Both sterols and stanols bind to cholesterol receptors on intestinal cells and block H3C

the absorption of cholesterol itself. Stanols esterified with longchain fatty acids form micelles (see Chapter 10, page 302) that are more effectively distributed in the fat phase of food digestion and provide the most effective blockage of cholesterol uptake. (Stanols are fully reduced sterols.) Raisio Group, a Finnish company, has developed Benecol, a stanol ester spread that can lower LDL cholesterol by up to 14% if consumed daily (see graph). McNeil Nutritionals has partnered with Raisio Group to market Benecol in the United States. H3C

CH3

H3C

H3C

CH3 CH2CH3

H 3C

CH3 CH2CH3

H3C

HO

HO Stigmasterol

H

CH3

Stigmastanol

CH3 H3C H3C

CH3 H3C

CH3

H3C

CH3

H3C

CH3 CH3

H3C

HO

HO " - Sitosterol

H " - Sitostanol

250

Cholesterol (mg/dL)

240

230

" Serum cholesterol levels before and after the

220

210 Sitostanol-ester margarine 200 "2

0

2

4 6 8 Study period (mo)

10

12

14

consumption of margarine with and without sitostanol ester for 12 months. Green circles: 0 g/day. Red squares: 2.6 g/day. Blue triangles: 1.8 g/day. Note: The y-axis begins at 200 mg cholesterol/dL. (Adapted from Miettinen, T. A., et al., 1995. Reduction of serum cholesterol with sitostanol-ester margarine in a mildly hypercholesterolemic population. New England Journal of Medicine 333:1308–1312.)

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Part 1 Building Blocks of the Cell

HUMAN BIOCHEMISTRY

17b-Hydroxysteroid Dehydrogenase 3 Deficiency Testosterone, the principal male sex steroid hormone, is synthesized in 5 steps from cholesterol, as shown in the following figure. In the last step, 5 isozymes catalyze the 17b-hydroxysteroid dehydrogenase reaction that interconverts 4-androstenedione and testosterone. Defects in the synthesis or action of testosterone can impair the development of the male phenotype during embryogenesis and cause the disorders of human sexuality termed “male pseudohermaphroditism.” Specifically, mutations in isozyme 3 of the 17b-hydroxysteroid dehydrogenase in the fetal testes impair

the formation of testosterone and give rise to genetic males with female external genitalia and blind-ending vaginas. Such individuals are typically raised as females but virilize at puberty, due to an increase in serum testosterone, and develop male hair growth patterns. Fourteen different mutations of 17b-hydroxysteroid dehydrogenase 3 have been identified in 17 affected families in the United States, the Middle East, Brazil, and western Europe. These families account for about 45% of the patients with this disorder reported in scientific literature. O

H3C

O

H3C Desmolase (Mitochondria)

H3C

HO

H3C

H3C

H3C (Endoplasmic reticulum)

HO

O

O Cholesterol

C

Progesterone

Pregnenolone

H

17!-Hydroxylase

Isocaproic aldehyde

H3C H3C

O

OH

H3C 17"-Hydroxysteroid dehydrogenase

H3C

H3C

OH

H3C

17,20-Lyase (Gonads)

O Testosterone

O

O

O 4-Androstenedione

17!-Hydroxyprogesterone

SUMMARY Lipids are a class of biological molecules defined by low solubility in water and high solubility in non-polar solvents. As molecules that are largely hydrocarbon in nature, lipids represent highly reduced forms of carbon and, upon oxidation in metabolism, yield large amounts of energy. Lipids are thus the molecules of choice for metabolic energy storage. The lipids found in biological systems are either hydrophobic (containing only non-polar groups) or amphipathic (containing both polar and non-polar groups). The hydrophobic nature of lipid molecules allows membranes to act as effective barriers to more polar molecules. 8.1 What Are the Structures and Chemistry of Fatty Acids? A fatty acid is composed of a long hydrocarbon chain (“tail”) and a terminal carboxyl group (“head”). The carboxyl group is normally ionized under physiological conditions. Fatty acids occur in large amounts in biological systems but only rarely in the free, uncomplexed state. They are typically esterified to glycerol or other backbone structures. 8.2 What Are the Structures and Chemistry of Triacylglycerols? A significant number of the fatty acids in plants and animals exist in the form of triacylglycerols (also called triglycerides). Triacylglycerols are a major energy reserve and the principal neutral derivatives of glycerol

found in animals. These molecules consist of a glycerol esterified with 3 fatty acids. Triacylglycerols in animals are found primarily in the adipose tissue (body fat), which serves as a depot or storage site for lipids. Monoacylglycerols and diacylglycerols also exist, but they are far less common than the triacylglycerols. 8.3 What Are the Structures and Chemistry of Glycerophospholipids? A 1,2-diacylglycerol that has a phosphate group esterified at carbon atom 3 of the glycerol backbone is a glycerophospholipid, also known as a phosphoglyceride or a glycerol phosphatide. These lipids form one of the largest and most important classes of natural lipids. They are essential components of cell membranes and are found in small concentrations in other parts of the cell. All glycerophospholipids are members of the broader class of lipids known as phospholipids. 8.4 What Are Sphingolipids, and How Are They Important for Higher Animals? Sphingolipids represent another class of lipids in biological membranes. An 18-carbon amino alcohol, sphingosine, forms the backbone of these lipids rather than glycerol. Typically, a fatty acid is joined to a sphingosine via an amide linkage to form a NEL

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Chapter 8

ceramide. Sphingomyelins are a phosphorus-containing subclass of sphingolipids especially important in the nervous tissue of higher animals. A sphingomyelin is formed by the esterification of a phosphorylcholine or a phosphorylethanolamine to the 1-hydroxy group of a ceramide. Glycosphingolipids are another class of ceramide-based lipids that, like the sphingomyelins, are important components of muscle and nerve membranes in animals. Glycosphingolipids consist of a ceramide with 1 or more sugar residues in a b-glycosidic linkage at the 1-hydroxyl moiety. 8.5 What Are Waxes, and How Are They Used? Waxes are esters of long-chain alcohols with long-chain fatty acids. The resulting molecule can be viewed (in analogy to the glycerolipids) as having a weakly polar head group (the ester moiety itself) and a long, nonpolar tail (the hydrocarbon chains). Fatty acids found in waxes are usually saturated. The alcohols found in waxes may be saturated or unsaturated and may include sterols, such as cholesterol. Waxes are water-insoluble due to their predominantly hydrocarbon nature. 8.6 What Are Terpenes, and What Is Their Relevance to Biological Systems? The terpenes are a class of lipids formed from combinations of 2 or more molecules of 2-methyl-1,3-butadiene, better known as isoprene (a 5-carbon unit abbreviated C5). A monoterpene (C10) consists of 2 isoprene units, a sesquiterpene (C15) consists of 3 isoprene units, a diterpene (C20) has 4 isoprene units, and so on. Isoprene units can be linked in terpenes to form straight-chain or cyclic molecules, and the usual method of linking isoprene units is head to tail. Monoterpenes occur in all higher plants, whereas sesquiterpenes and diterpenes are less widely known.

Lipids

249

8.7 What Are Steroids, and What Are Their Cellular Functions? A large and important class of terpene-based lipids is the steroids. This molecular family, whose members affect an amazing array of cellular functions, is based on a common structural motif of three 6-membered rings and one 5-membered ring all fused together. Cholesterol is the most common steroid in animals and the precursor for all other animal steroids. The numbering system for cholesterol applies to all such molecules. The polyprenyl nature of this compound is particularly evident in the side chain. Many steroids contain an oxygen at C-3, either a hydroxyl group in sterols or a carbonyl group in other steroids. The methyl groups at positions 10 and 13 and the alkyl group at position 17 are usually oriented on the same side of the steroid nucleus, the b-orientation. Alkyl groups that extend from the other side of the steroid backbone are in an a-orientation. Cholesterol is a principal component of animal cell plasma membranes. Steroids derived from cholesterol in animals include 5 families of hormones (the androgens, estrogens, progestins, glucocorticoids, and mineralocorticoids) and bile acids. 8.8 How Do Lipids and Their Metabolites Act as Biological Signals? Modification and breakdown of cellular lipids produce an eclectic assortment of substances that act as powerful chemical signals. Signal molecules typically initiate a cascade of reactions with multiple possible effects. The creation and breakdown of signal molecules is almost always carefully timed and regulated. Phospholipases initiate the production of a variety of lipid signals, including arachidonic acid (the precursor to eicosanoids), lysophosphatidic acid, inositol-1,4, 5-trisphosphate, and diacylglycerol.

PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. Draw the structures of (a) all the possible triacylglycerols that can be formed from glycerol with stearic and arachidonic acid and (b) all the phosphatidylserine isomers that can be formed from palmitic and linolenic acids. 2. Describe in your own words the structural features of a. a ceramide and how it differs from a cerebroside. b. a phosphatidylethanolamine and how it differs from a phosphatidylcholine. c. an ether glycerophospholipid and how it differs from a plasmalogen. d. a ganglioside and how it differs from a cerebroside. e. testosterone and how it differs from estradiol. 3. From your memory of the structures, name a. the glycerophospholipids that carry a net positive charge. b. the glycerophospholipids that carry a net negative charge. c. the glycerophospholipids that have zero net charge.

6. In a departure from his usual and highly popular westerns, author Louis L’Amour wrote a novel in 1987, Last of the Breed (Bantam Press), in which a military pilot of Native American ancestry is shot down over the former Soviet Union and is forced to use the survival skills of his ancestral culture to escape his enemies. On the rare occasions when he is able to trap and kill an animal for food, he selectively eats the fat, not the meat. Based on your reading of this chapter, what is his reasoning for doing so? 7. As you read Section 8.7, you might have noticed that phospholipase A2, the enzyme found in rattlesnake venom, is also the enzyme that produces essential and beneficial lipid signals in most organisms. Explain the differing actions of phospholipase A2 in these processes. 8. Visit a grocery store near you, stop by the rodent poison section, and examine a container of warfarin or a related product. From what you can glean from the packaging, how much warfarin would a typical dog (20 kg) have to consume to risk hemorrhages and/or death? 9. Refer to Figure 8.13 and draw each of the structures shown and try to identify the isoprene units in each of the molecules. (Note that there may be more than one correct answer for some of these molecules, unless you have the time and facilities to carry out 14C labelling studies with suitable organisms.)

4. Compare and contrast 2 individuals, one whose diet consists largely of meats containing high levels of cholesterol and the other whose diet is rich in plant sterols. Are their risks of cardiovascular disease likely to be similar or different? Explain your reasoning.

10. (Integrates with Chapter 3.) As noted in the Deeper Look box on polar bears, a polar bear may burn as much as 1.5 kg of fat resources per day. What mass of seal blubber would you have to ingest if you were to obtain all your calories from this energy source?

5. In the United States, James G. Watt, Secretary of the Interior (1981–1983) in Ronald Reagan’s first term as president, provoked substantial controversy by stating publicly that trees cause significant amounts of air pollution. Based on your reading of this chapter, evaluate Watt’s remarks.

11. If you are still at the grocery store working on Problem 8, stop by the cookie shelves and choose your 3 favourite cookies from the shelves. Estimate how many calories of fat, and how many other calories from other sources, are contained in 100 g of each of these cookies. Survey the ingredients listed on each package, and describe the contents of

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250

Part 1 Building Blocks of the Cell

the package in terms of (a) saturated fat, (b) cholesterol, and (c) trans fatty acids. (Note that food makers are required to list ingredients in order of decreasing amounts in each package.) 12. Describe all of the structural differences between cholesterol and stigmasterol. 13. Describe in your own words the functions of androgens, glucocorticoids, and mineralocorticoids. 14. Look through your refrigerator, your medicine cabinet, and your cleaning solutions shelf or cabinet, and find at least 3 commercial products that contain fragrant monoterpenes. Identify each one by its scent and then draw its structure. 15. Our ancestors kept clean with homemade soap (page 232), often called “lye soap.” Go to http://www.wikihow.com/Make-Your-OwnSoap and read the procedure for making lye soap from vegetable oils and lye (sodium hydroxide). What chemical process occurs in the making of lye soap? Draw reactions to explain. How does this soap work as a cleaner? 16. Mayonnaise is mostly vegetable oil and vinegar. So what is the essential difference between oil and vinegar salad dressing and mayonnaise? Learn for yourself: Combine a half cup of pure vegetable oil (olive oil will work) with 2 tablespoons of vinegar in a bottle, cap it securely, and shake the mixture vigorously. What do you see? Now let the mixture sit undisturbed for an hour. What do you see now? Add 1 egg yolk to the mixture, and shake vigorously again. Let the mixture stand as before. What do you see after an hour? After 2 hours? Egg yolk is rich in phosphatidylcholine. Explain why the egg yolk caused the effect you observed.

18. Statins are cholesterol-lowering drugs that block cholesterol synthesis in the human liver (see Chapter 9). Would you expect the beneficial effects of stanol esters and statins to be duplicative or additive? Explain. 19. If most plant-derived food products contain plant sterols and stanols, would it be as effective (for cholesterol-lowering purposes) to simply incorporate plant fats in one’s diet as to use a sterol- or stanol-fortified spread like Benecol? Consult a suitable reference (e.g., http://lpi.oregonstate.edu/infocenter/phytochemicals/ sterols/#sources at the Linus Pauling Institute) to compose your answer. 20. Tetrahydrogestrinone is an anabolic steroid. It was banned by the U.S. Food and Drug Administration in 2003, and is also banned in Canada, but it has been used illegally since then by athletes to increase muscle mass and strength. Nicknamed “The Clear,” it has received considerable attention in high-profile steroid abuse cases among athletes such as baseball player Barry Bonds and track star Marion Jones. Use your favourite Web search engine to learn more about this illicit drug. How is it synthesized? Who is “the father of prohormones”—the man who first synthesized it? Why did so many prominent athletes use The Clear (and its relative, “The Cream”) when less expensive and more commonly available anabolic steroids are in common use? (Hint: There are at least 2 answers to this last question.) OH H

17. The cholesterol-lowering benefit of stanol-ester margarine is only achieved after months of consumption of stanol esters (see graph, page 247). Suggest why this might be so. Suppose dietary sources represent approximately 25% of total serum cholesterol. Based on the data in the graph, how effective are stanol esters at preventing uptake of dietary cholesterol?

H O Tetrahydrogestrinone

FURTHER READING General

Isoprenes and Prenyl Derivatives

Robertson, R. N., 1983. The Lively Membranes. Cambridge: Cambridge University Press.

Dowd, P., Ham, S.-W., et al., 1995. The mechanism of action of vitamin K. Annual Review of Nutrition 15:419–440.

Seachrist, L., 1996. A fragrance for cancer treatment and prevention. The Journal of NIH Research 8:43.

Hirsh, J., Dalen, J. E., et al., 1995. Oral anticoagulants: Mechanism of action, clinical effectiveness, and optimal therapeutic range. Chest 108:231S–246S.

Vance, D. E., and Vance, J. E. (eds.), 1985. Biochemistry of Lipids and Membranes. Menlo Park, CA: Benjamin/Cummings. Sterols Anderson, S., Russell, D. W., et al., 1996. 17b-Hydroxysteroid dehydrogenase 3 deficiency. Trends in Endocrinology and Metabolism 7:121–126.

Sharkey, T. D., 1995. Why plants emit isoprene. Nature 374:769. Sharkey, T. D., 1996. Emission of low molecular-mass hydrocarbons from plants. Trends in Plant Science 1:78–82. Eicosanoids

DeLuca, H. F., and Schneos, H. K., 1983. Vitamin D: Recent advances. Annual Review of Biochemistry 52:411–439.

Chakrin, L. W., and Bailey, D. M., 1984. The Leukotrienes—Chemistry and Biology. Orlando: Academic Press.

Denke, M. A., 1995. Lack of efficacy of low-dose sitostanol therapy as an adjunct to a cholesterol-lowering diet in men with moderate hypercholesterolemia. American Journal of Clinical Nutrition 61:392–396.

Keuhl, F. A., and Egan, R. W., 1980. Prostaglandins, arachidonic acid and inflammation. Science 210:978–984.

Thompson, G., and Grundy, S., 2005. History and development of plant sterol and stanol esters for cholesterol-lowering purposes. American Journal of Cardiology 96:3D–9D.

Hakamori, S., 1986. Glycosphingolipids. Scientific American 254:44–53.

Vanhanen, H. T., Blomqvist, S., et al., 1993. Serum cholesterol, cholesterol precursors, and plant sterols in hypercholesterolemic subjects with different apoE phenotypes during dietary sitostanol ester treatment. Journal of Lipid Research 34:1535–1544.

Sphingolipids

Trans Fatty Acids Katan, M. B., Zock, P. L., et al., 1995. Trans fatty acids and their effects on lipoproteins in humans. Annual Review of Biochemistry 15:473–493.

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Chapter 8

Lipids

251

Lipids of Archaea

Lipids as Signalling Molecules

Hanford, M., and Peebles, T., 2002. Archaeal tetraetherlipids: Unique structures and applications. Applied Biochemistry and Biotechnology 97:45–62.

Eyster, K., 2007. The membrane and lipids as integral participants in signal transduction: Lipid signal transduction for the non-lipid biochemist. Advances in Physiology Education 31:5–16.

Lipid Alterations in Disease States

Fernandis, A., and Wenk, M., 2007. Membrane lipids as signaling molecules. Current Opinion in Lipidology 18:121–128.

Malan, T. P., and Porreca, F., 2005. Lipid mediators regulating pain sensitivity. Prostaglandins and Other Lipid Mediators 77:123–130. Smith, L. E. H., and Connor, K. M., 2005. A radically twisted lipid regulates vascular death. Nature Medicine 11:1275–1276.

Rosen, H., and Goetzl, E., 2005. Sphingosine-1-phosphate and its receptors: An autocrine and paracrine network. Nature Reviews Immunology 5:560–570.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

9

Lipid Biosynthesis

! Walruses basking on the beach.

To everything there is a season, and a time for every purpose under heaven…A time to break down and a time to build up. Ecclesiastes 3:1–3

9.1

How Are Fatty Acids Synthesized?

9.2

How Are Complex Lipids Synthesized?

9.3

How Are Eicosanoids Synthesized, and What Are Their Functions?

9.4

How Is Cholesterol Synthesized?

9.5

How Are Lipids Transported throughout the Body?

9.6

How Are Bile Acids Biosynthesized?

9.7

How Are Steroid Hormones Synthesized and Utilized?

© Alissa Crandall/CORBIS

KEY QUESTIONS

ESSENTIAL QUESTION We turn now to the biosynthesis of lipid structures. We begin with a discussion of the biosynthesis of fatty acids, stressing the basic pathways, additional means of elongation, mechanisms for the introduction of double bonds, and regulation of fatty acid synthesis. Sections then follow on the biosynthesis of glycerophospholipids, sphingolipids, eicosanoids, and cholesterol. The transport of lipids through the body in lipoprotein complexes is described, and the chapter closes with discussions of the biosynthesis of bile salts and steroid hormones. What are the pathways of lipid synthesis in biological systems?

WHY IT MATTERS?

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

Before we begin our discussion of lipid biosynthesis, we pose the question, “Why does lipid biosynthesis matter?” As we will see in the upcoming chapter, lipids are fundamental molecules used for energy and the production of hormones, and are the major constituents of cellular membranes. Lipid biosynthesis is vital to a cell since alterations in one biosynthetic pathway versus another can have catastrophic effects on the wide variety of biological functions that a lipid must carry out. In line with this, we have certainly been bombarded with declarations from health professionals and worldwide media outlets to reduce excessive dietary consumption of the lipid cholesterol. The reason for this? It is well known that increased blood levels of this steroid have long been associated with narrowing of blood vessels (or, colloquially speaking, “hardening of the arteries”) and cardiovascular disease. We have already seen several cases in which the synthesis of a class of biomolecules is conducted differently from degradation (e.g, glycolysis versus gluconeogenesis, and glycogen or starch breakdown versus polysaccharide synthesis). Likewise, the synthesis of fatty acids and other lipid components is different from their degradation. Fatty acid

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Chapter 9 Normal artery

Heart

Plaque

Lipid Biosynthesis

" Cholesterol buildup. (Nucleus Medical Art/ Visuals Unlimited, Inc.)

Blocked artery

synthesis involves a set of reactions that follow a strategy different in several ways from the corresponding degradative process: 1. Intermediates in fatty acid synthesis are linked covalently to the sulfhydryl groups of special proteins, the acyl carrier proteins (ACPs). In contrast, fatty acid breakdown intermediates are bound to the—SH group of coenzyme A. 2. Fatty acid synthesis occurs in the cytosol, whereas fatty acid degradation takes place in mitochondria. 3. In animals, the enzymes of fatty acid synthesis are components of one long polypeptide chain, the fatty acid synthase, whereas no similar association exists for the degradative enzymes. (Plants and bacteria employ separate enzymes to carry out the biosynthetic reactions.) 4. The coenzyme for the oxidation–reduction reactions of fatty acid synthesis is NADP+/ NADPH, whereas degradation involves the NAD+/NADH couple.

9.1

253

How Are Fatty Acids Synthesized?

Formation of Malonyl-CoA Activates Acetate Units for Fatty Acid Synthesis The design strategy for fatty acid synthesis is this: 1. Fatty acid chains are constructed by the addition of 2-carbon units derived from acetyl-CoA. 2. The acetate units are activated by formation of malonyl-CoA (at the expense of ATP). 3. The addition of 2-carbon units to the growing chain is driven by decarboxylation of malonyl-CoA. 4. The elongation reactions are repeated until the growing chain reaches 16 carbons in length (palmitic acid). 5. Other enzymes then add double bonds and additional carbon units to the chain.

Fatty Acid Biosynthesis Depends on the Reductive Power of NADPH The net reaction for the formation of palmitate from acetyl-CoA is acetyl-CoA + 7 malonyl-CoA- + 14 NADPH + 13 H+ + H2O 4 palmitate- + 7 HCO3- + 8 CoASH + 14 NADP+ (Levels of free fatty acids are very low in the typical cell. The palmitate made in this process is rapidly converted to CoA esters in preparation for the formation of triacylglycerols and phospholipids.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

Cells Must Provide Cytosolic Acetyl-CoA and Reducing Power for Fatty Acid Synthesis Eukaryotic cells face a dilemma in providing suitable amounts of substrate for fatty acid synthesis. Sufficient quantities of acetyl-CoA, malonyl-CoA, and NADPH must be generated in the cytosol for fatty acid synthesis. Malonyl-CoA is made by carboxylation of acetyl-CoA, so the problem reduces to generating sufficient acetyl-CoA and NADPH. There are 3 principal sources of acetyl-CoA (Figure 9.1): 1. Amino acid degradation produces cytosolic acetyl-CoA. 2. Fatty acid oxidation produces mitochondrial acetyl-CoA. 3. Glycolysis yields cytosolic pyruvate, which (after transport into the mitochondria) is converted to acetyl-CoA by pyruvate dehydrogenase. The acetyl-CoA derived from amino acid degradation is normally insufficient for fatty acid biosynthesis, and the acetyl-CoA produced by pyruvate dehydrogenase and by fatty  acid oxidation cannot cross the mitochondrial membrane to participate directly in  fatty acid synthesis. Instead, acetyl-CoA is linked with oxaloacetate to form citrate, which is transported from the mitochondrial matrix to the cytosol (Figure 9.1). Here it can be converted back into acetyl-CoA and oxaloacetate by ATP–citrate lyase. In this manner, Inner mitochondrial membrane

Mitochondrial matrix

Fatty acylFatty acid carnitine oxidation

Cytosol

Fatty acylcarnitine

Acetyl-CoA Oxaloacetate

Amino acid catabolism Citrate

Citrate synthase

TCA cycle

Citrate

Malate dehydrogenase

NAD+

ADP

+

NADP+

Pi

Malic enzyme

Pyruvate carboxylase ATP

+

Fatty acids

Malate

Malate

Pyruvate dehydrogenase NAD+

Acetyl-CoA

ATP-citrate lyase

NADH

NAD+

NADH

Amino acids

Oxaloacetate

NADH Malate dehydrogenase

CO2

Fatty acids

Fatty acyl-CoA

CO2

NADPH

CO2

Pyruvate

Pyruvate NADH Glycolysis NAD+ Glucose FIGURE 9.1 The citrate–malate–pyruvate shuttle provides cytosolic acetate units and some reducing equivalents (electrons) for fatty acid synthesis. The shuttle collects carbon substrates, primarily from glycolysis but also from fatty acid oxidation and amino acid catabolism. Pathways that provide carbon for fatty acid synthesis are shown in blue; pathways that supply electrons for fatty acid synthesis are shown in red. NEL

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Chapter 9

Lipid Biosynthesis

255

mitochondrial acetyl-CoA becomes the substrate for cytosolic fatty acid synthesis. (Oxaloacetate returns to the mitochondria in the form of either pyruvate or malate, which is then reconverted to acetyl-CoA or oxaloacetate respectively.) NADPH can be produced in the pentose phosphate pathway as well as by malic enzyme (Figure 9.1). Reducing equivalents (electrons) derived from glycolysis in the form of NADH can be transformed into NADPH by the combined action of malate dehydrogenase and malic enzyme: oxaloacetate + NADH + H+ 4 malate + NAD+ malate + NADP+ 4 pyruvate + CO2 + NADPH + H+ How many of the 14 NADPH needed to form 1 palmitate (see equation on page 253) can be made in this way? The answer depends on the status of malate. Every citrate entering the cytosol produces 1 acetyl-CoA and 1 malate (Figure 9.1). Every malate oxidized by malic enzyme produces 1 NADPH, at the expense of a decarboxylation to pyruvate. Thus, when malate is oxidized, 1 NADPH is produced for every acetyl-CoA. Conversion of 8 acetyl-CoA units to 1 palmitate would then be accompanied by production of 8 NADPH. (The other 6 NADPH required, as shown in the equation on page 253, would be provided by the pentose phosphate pathway.) On the other hand, for every malate returned to the mitochondria, 1 NADPH fewer is produced.

Acetate Units Are Committed to Fatty Acid Synthesis by Formation of Malonyl-CoA Rittenberg and Bloch showed in the late 1940s that acetate units are the building blocks of fatty acids. Their work, together with the discovery by Salih Wakil that bicarbonate is required for fatty acid biosynthesis, eventually made clear that this pathway involves synthesis of malonyl-CoA. The carboxylation of acetyl-CoA to form malonyl-CoA is essentially irreversible and is the committed step in the synthesis of fatty acids (Figure 9.2). The reaction is catalyzed by acetyl-CoA carboxylase, which contains a biotin prosthetic group. This carboxylase is the only enzyme of fatty acid synthesis in animals that is not part of the multi-enzyme complex called fatty acid synthase.

Acetyl-CoA Carboxylase Is Biotin-Dependent and Displays Ping-Pong Kinetics The biotin prosthetic group of acetyl-CoA carboxylase is covalently linked to the !-amino group of an active-site lysine in a manner similar to pyruvate carboxylase (see Figure 21.2). The reaction mechanism is also analogous to that of pyruvate carboxylase (see Figure 21.3): ATP-driven carboxylation of biotin is followed by transfer of the activated CO2 to acetylCoA to form malonyl-CoA. The enzyme from Escherichia coli has 3 subunits: (1) a biotin carboxyl carrier protein (a dimer of 22.5 kD subunits); (2) biotin carboxylase (a dimer of 51 kD subunits), which adds CO2 to the prosthetic group; and (3) carboxyl transferase (an "2 b2 tetramer with 30 and 35 kD subunits), which transfers the activated CO2 unit to acetyl-CoA. The long, flexible biotin–lysine chain (biocytin) enables the activated carboxyl group to be carried between the biotin carboxylase and the carboxyl transferase (Figure 9.3 on page 257).

Acetyl-CoA Carboxylase in Animals Is a Multifunctional Protein In animals, acetyl-CoA carboxylase (ACC) is a filamentous polymer (4 to 8 × 106 D) composed of 265 kD protomers. Each of these subunits contains the biotin carboxyl carrier moiety, biotin carboxylase, and carboxyl transferase activities, as well as allosteric regulatory sites. Animal ACC is thus a multifunctional protein. The polymeric form is active, but the 265 kD protomers are inactive. The activity of ACC is thus dependent upon the position of the equilibrium between these 2 forms: inactive protomers ∆ active polymer.

Biotin carboxylase domain of human acetyl-CoA carboxylase 2 (pdb id = 2HJW)

The biotin carboxylase domain from human acetyl-CoA carboxylase 2, with the A-subdomain in blue, the B-subdomain in red, the A–B linker in green, and the C-subdomain in yellow.

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Part 1 Building Blocks of the Cell

FIGURE 9.2 (a) The acetyl-CoA carboxylase reaction produces malonyl-CoA for fatty acid synthesis. (b) A mechanism for the acetyl-CoA carboxylase reaction. Bicarbonate is activated for carboxylation reactions by formation of N-carboxybiotin. ATP drives the reaction forward, with transient formation of a carbonyl phosphate intermediate (Step 1). In a typical biotindependent reaction, nucleophilic attack by the acetylCoA carbanion on the carboxyl carbon of N-carboxybiotin—a transcarboxylation—yields the carboxylated product (Step 2).

(a) O CH3

C

S

CoA

+

+

ATP

– HCO3

O

O C

CH2

C

S

CoA

+

ADP

+

+

Pi

H+

–O

(b) Step 1

The carboxylation of biotin ADP

+

ATP

HCO3–

O –O

C

Pi

O O

P

O

O–

–O

O

C N

O–

NH

O S HN

Lys

NH

Biotin S Step 2

Lys

The transcarboxylation reaction O H2C

–

–O

C

SCoA

O

O

N

O

O

C NH

H2C

C

SCoA

+

HN

NH

COO– S

Carboxyltransferase domain of yeast acetyl-CoA carboxylase (pdb id = 1OD2)

The carboxyl transferase domain dimer of acetyl-CoA carboxylase-1 from Saccharomyces cerevisiae. The Nand C-subdomains of one monomer are cyan and yellow, whereas those of the other monomer are purple and green. CoA is shown as a ball-and-stick model in 1 subunit.

Lys

S

Lys

Because this enzyme catalyzes the committed step in fatty acid biosynthesis, it is carefully regulated. Palmitoyl-CoA, the final product of fatty acid biosynthesis, shifts the equilibrium toward the inactive protomers, whereas citrate, an important allosteric activator of this enzyme, shifts the equilibrium toward the active polymeric form of the enzyme. Acetyl-CoA carboxylase shows the kinetic behaviour of a Monod–Wyman–Changeux, V-system, allosteric enzyme in which allosteric effectors shift the T/R equilibrium between active R conformers and inactive T conformers.

Phosphorylation of ACC Modulates Activation by Citrate and Inhibition by Palmitoyl-CoA The regulatory effects of citrate and palmitoyl-CoA are dependent on the phosphorylation state of acetyl-CoA carboxylase. The animal enzyme is phosphorylated at 8–10 sites on each enzyme subunit (Figure 9.4). Some of these sites are regulatory, whereas others are NEL

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Chapter 9

Lipid Biosynthesis

257

Biotin carboxyl carrier protein Carboxyltransferase

23 25

P

29

P

P

SC oA

O S

C C O

N

–O

N

CH

–

2

NH

S

S

O

“silent” and have no effect on enzyme activity. Unphosphorylated acetyl-CoA carboxylase binds citrate with high affinity and thus is active at very low citrate concentrations (Figure 9.5). Phosphorylation of the regulatory sites decreases the affinity of the enzyme for citrate, and in this case, high levels of citrate are required to activate the carboxylase. The inhibition by fatty acyl-CoAs operates in a similar but opposite manner. Thus, low levels of fatty acyl-CoA inhibit the phosphorylated carboxylase, but the dephosphoenzyme is inhibited only by high levels of fatty acyl-CoA. Specific phosphatases act to dephosphorylate ACC, thereby increasing the sensitivity to citrate.

–O

C O

–O

O

2– 3 PO

C

O

– O

O

O

N

C

P

Residue number

C

CO H N

N

NH

S

NH

O

3

CH

S

C

–O

HN

C HN O

O NH

C

N

O

SC oA

O

NH

O

C

C O

H

O

N

N

H

Biotin carboxylase

FIGURE 9.3 In the acetyl-CoA carboxylase reaction, the biotin ring, on its flexible tether, acquires carboxyl groups from carbonyl phosphate on the biotin carboxylase subunit and transfers them to acyl-CoA molecules on the carboxyl transferase subunits. Colours of the domains correspond to those in Figure 9.4.

Calmodulin-dependent protein kinase Casein kinase II

1 83 Biotin carboxylase

BCCP

621 661 821

1200

P

76 77

P P

95

P

cAMP-dependent protein kinase (PKA), protein kinase C (PKC) AMP-dependent protein kinase (AMPK) Protein kinase C (PKC)

cAMP-dependent protein kinase (PKA), AMP-dependent protein kinase (AMPK)

1574

Carboxyltransferase

2346

FIGURE 9.4 Schematic of the acetyl-CoA carboxylase polypeptide, with domains and phosphorylation sites indicated, along with the protein kinases responsible. Phosphorylation at Ser1200 is primarily responsible for decreasing the affinity for citrate.

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Part 1 Building Blocks of the Cell

Acyl Carrier Proteins Carry the Intermediates in Fatty Acid Synthesis

Dephospho-acetyl-CoA carboxylase (Low [citrate] activates, high [fatty acyl-CoA] inhibits)

ATP

The basic building blocks of fatty acid synthesis are acetyl and malonyl groups, but they are not transferred directly from CoA to the growing fatty acid chain. Rather, they are first passed to ACP. This protein consists (in E. coli) of a single polypeptide chain of 77 residues to which is attached (on a serine residue) a phosphopantetheine group, the same group that forms the “business end” of coenzyme A. Thus, ACP is a somewhat larger version of coenzyme A, specialized for use in fatty acid biosynthesis (Figure 9.6).

Pi

Kinases

Phosphatases

ADP

H2O

In Some Organisms, Fatty Acid Synthesis Takes Place in Multi-enzyme Complexes

Phospho-acetyl-CoA carboxylase (High [citrate] activates, low [fatty acyl-CoA] inhibits) P

P

P

P

P

P

P

P

P

FIGURE 9.5 The activity of acetyl-CoA carboxylase is modulated by phosphorylation and dephosphorylation. The dephospho form of the enzyme is activated by low [citrate] and inhibited only by high levels of fatty acylCoA. In contrast, the phosphorylated form of the enzyme is activated only by high levels of citrate but is very sensitive to inhibition by fatty acyl-CoA.

The enzymes that catalyze the formation of acetyl-ACP and malonyl-ACP and the subsequent reactions of fatty acid synthesis are organized differently in different organisms. Fatty acid synthesis in mammals occurs on homodimeric fatty acyl synthase I (FAS I), each 270 kD polypeptide of which contains all reaction centres required to produce a fatty acid. In lower eukaryotes, such as yeast and fungi, the enzymatic activities of FAS are distributed on 2 multifunctional peptide chains, which form 2.6 megadalton !6 "6 complexes. In plants, most bacteria, and parasites, the enzymes of fatty acid synthesis are separated and independent, and this collection of enzymes is referred to as fatty acyl synthase II (FAS II). The individual steps in the elongation of the fatty acid chain are similar across all organisms. The mammalian pathway (Figure 9.7) is a cycle of elongation that involves 6 enzyme activities. The elongation cycle is initiated by transfer of the acyl moiety of acetylCoA to the acyl carrier protein by the malonyl-CoA–acetyl-CoA-ACP transacylase (MAT), which also transfers the malonyl group of malonyl-CoA to ACP.

H HS

CH2

CH2

H C

N

CH2

CH2

N

O

HO

CH3

C

C

C

O

H

CH3

O CH2

O

P

O O

O–

P

HS

CH2

CH2

N

H C

CH2

CH2

O

N

HO C

C

O

H

CH3

O

H

Adenine H

H

2–O

H

3PO

CH3

C

CH2

O–

Phosphopantetheine group of coenzyme A

H

O

OH

O CH2

O

P

O

CH2

Ser

Acyl carrier protein

O–

Phosphopantetheine prosthetic group of ACP FIGURE 9.6 Fatty acids are conjugated to both coenzyme A and acyl carrier protein through the sulfhydryl of phosphopantetheine prosthetic groups.

A DEEPER LOOK

Choosing the Best Organism for the Experiment The selection of a suitable and relevant organism is an important part of any biochemical investigation. The studies that revealed the secrets of fatty acid synthesis are a good case in point. The paradigm for fatty acid synthesis in plants has been the avocado, which has 1 of the highest fatty acid contents in the plant

kingdom. Early animal studies centred primarily on pigeons, which are easily bred and handled and which possess high levels of fats in their tissues. Other animals, richer in fatty tissues, might be even more attractive but more challenging to maintain. Grizzly bears, for example, carry very large fat reserves but are difficult to work with in the lab!

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Chapter 9 FIGURE 9.7 The pathway of palmitate synthesis from acetyl-CoA and malonyl-CoA. Acetyl and malonyl building blocks are introduced as ACP conjugates. Decarboxylation drives the "-ketoacyl-ACP synthase and results in the addition of 2-carbon units to the growing chain. The first turn of the cycle begins at Step 1 and goes to butyryl-ACP; subsequent turns of the cycle are indicated as Steps 2 through 6.

Lipid Biosynthesis

CH3 O

C

S-CoA Acetyl-CoA MAT

CH3 O

C KS

COO– Palmitate

S-ACP

COO– CH2 C

C

S-CoA

TE Thioesterase 7

CH3

ACP-SH

O MAT

Malonyl-CoA

S-KSase

COO– CH2

CoASH

C

O

1

CO2

!-Ketoacyl-ACP NADPH + Acyl (Cn+2)-ACP

CH3

CH2

CH2

C

Butyryl-ACP

S

ACP NADP+ NADPH + H+

H+

C

CH2

C

S

NADPH + H+

KR !-Ketoacyl-ACP reductase

!-Hydroxyacyl-ACP

ACP

Acetoacetyl-ACP

NADP+

2 3 4 5 6

NADP+

DH !-Enoyl-ACP

O

OH H2O

NADP+

CH3

KR

ER

O

O

KS

O

KS

!-Ketoacyl synthase

S-ACP

CO2

O

CH3

C

CH2

C

S

H

!-Enoyl-ACP ER reductase

D-!-Hydroxybutyryl-ACP

DH

NADPH + H+

!-HydroxyacylACP dehydratase H CH3

C H

C

C

S

ACP

H2O

O Crotonyl-ACP

Decarboxylation Drives the Condensation of Acetyl-CoA and Malonyl-CoA The !-ketoacyl-ACP synthase (KS) catalyzes the decarboxylative condensation of the acyl group with malonyl-ACP to produce a "-ketoacyl-ACP intermediate (acetoacetyl-ACP in the first cycle). The mechanism (Figure 9.8) begins with acetyl group transfer to MAT, followed with attack by the ACP thiol sulfur to form an acetyl-ACP. The acetyl group is NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

ACP

259

260

Part 1 Building Blocks of the Cell HS MAT

MAT

OH O H3C

C

O SCoA

H3C

KS

O

MAT

KS

MAT

S

O

C

C

CH3

CH2

O

C

OH

C

S

O

CH3

ACP

SH ACP

–OOC

CH2C

O SCoA

SH

O

COO–

ACP KS

O

O–

O

S

C

C

CH2

CH3

C

KS

CH3

SH

C

O

CH2 O

CO2

C

S

S

ACP

ACP

O

FIGURE 9.8 A mechanism for mammalian ketoacyl synthase. An acetyl group is transferred from CoA to MAT, then to the acyl carrier protein, and then to ketoacyl synthase. Next, a malonyl group is transferred to MAT and then to the acyl carrier protein. Decarboxylation of the malonyl group creates a transient carbanion on the acyl group of ACP, which attacks the KS acetyl group to form a ketoacyl-ACP. A cycle (see Figure 9.7) of keto group reduction (by KR), water removal (by DH), and double-bond reduction (by ER; see next section) will finally produce an acyl group increased in length by 2 carbons.

transferred to a cysteine sulfur on KS, freeing the ACP thiol to acquire the malonyl group. In the condensation reaction that follows, decarboxylation of the malonyl group creates a transient, highly nucleophilic carbanion that can attack the acetate group. The net reaction for each turn of this cycle (see Figure 9.7) is addition of a 2-carbon unit to the acyl group. Why is the 3-carbon malonyl group used here as a 2-carbon donor? The answer is that this is yet another example of a decarboxylation driving a desired but otherwise thermodynamically unfavourable reaction. The decarboxylation that accompanies the reaction with malonyl-ACP drives the synthesis of acetoacetyl-ACP. Note that hydrolysis of ATP drove the carboxylation of acetyl-CoA to form malonyl-ACP, so, indirectly, ATP is responsible for the condensation reaction to form acetoacetylACP. Malonyl-CoA can be viewed as a form of stored energy for driving fatty acid synthesis. It is also worth noting that the carbon of the carboxyl group that was added to drive this reaction is the one removed by the condensing enzyme. Thus, all the carbons of acetoacetyl-ACP (and of the fatty acids to be made) are derived from acetate units of acetyl-CoA.

Reduction of the !-Carbonyl Group Follows a Now Familiar Route The next 3 steps—reduction of the "-carbonyl group by !-ketoacyl-ACP reductase (KR) to form a "-alcohol, then dehydration by !-hydroxyacyl-ACP dehydratase (DH), and reduction by 2,3-trans-enoyl-ACP reductase (ER) to saturate the chain (see Figure 9.7)—look very similar to the fatty acid degradation pathway in reverse. However, there are 2 crucial differences between fatty acid biosynthesis and fatty acid oxidation (besides the fact that different enzymes are involved): First, the alcohol formed in biosynthesis has the D-configuration rather than the L-form seen in catabolism; second, the reducing coenzyme is NADPH, whereas NAD# and FAD are the oxidants in the catabolic pathway. The net result of the first turn of the biosynthetic cycle is the synthesis of a 4-carbon unit, a butyryl group, from 2 smaller building blocks. In the next cycle of the process, this butyryl-ACP condenses with another malonyl-ACP to make a 6-carbon "-ketoacyl-ACP and CO2. Subsequent reduction to a "-alcohol, dehydration, and another reduction yield a 6-carbon saturated acyl-ACP. This cycle continues with the net addition of a 2-carbon unit in each turn until the chain is 16 carbons long (see Figure 9.7). The KS cannot accommodate larger substrates, so the reaction cycle ends with a 16-carbon chain. Hydrolysis of the C16-acyl-ACP yields a palmitic acid and the free ACP. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 9

Lipid Biosynthesis

In the end, 7 malonyl-CoA molecules and 1 acetyl-CoA yield a palmitate (shown here as palmitoyl-CoA): acetyl-CoA + 7 malonyl-CoA$ + 14 NADPH + 14 H# 4 palmitoyl-CoA + 7 HCO3$ + 14 NADP# + 7 CoASH The formation of 7 malonyl-CoA molecules requires 7 acetyl-CoA + 7 HCO3$ + 7 ATP4$ 4 7 malonyl-CoA$ + 7 ADP3$ + 7 Pi2$ + 7 H# Thus, the overall reaction of acetyl-CoA to yield palmitic acid is 8 acetyl-CoA + 7 ATP 4$ + 14 NADPH + 7 H# 4 palmitoyl-CoA + 14 NADP# + 7 CoASH + 7 ADP3$ + 7Pi2$ Note: These equations are stoichiometric and are charge balanced. See Problem 1 at the end of the chapter for practice in balancing these equations.

Eukaryotes Build Fatty Acids on Megasynthase Complexes The multiple enzyme domains of eukaryotic fatty acyl synthases are arrayed on large protein structures termed megasynthases. The individual enzyme domains of these structures in all eukaryotes are homologous to the corresponding small, discrete enzymes of bacterial FAS pathways. Remarkably, however, lower eukaryotes such as fungi and higher eukaryotes such as mammals have evolved entirely different megasynthase architectures for fatty acid synthesis. Mammalian homodimeric FAS has a flattened X-shape, whereas the fungal dodecameric FAS is a large, closed barrel, with 2 reaction chambers separated by equatorial stabilizing struts (Figure 9.9). In the fungal structure, the 6 "-subunits form a central ring that is a “trimer of dimers” (Figure 9.10a, b). Each "-subunit contributes an extended "-helical segment to the centre of the structure. Pairs of these helices form 3 coiled-coil

Fungal fatty acid synthase Reaction chamber

AT: Acetyl transferase MPT: Malonyl/palmitoyl transferase MAT: Malonyl-CoA–acetyl-CoA-ACP DH transacylase TE: Thioesterase ACP: Acyl carrier protein MPT PPT: Phosphopantetheinyl transferase KR: !-Ketoacyl reductase KR2 KS: !-Ketoacyl synthase ER: !-Enoyl reductase DH: Dehydratase

AT

% Active sites

DH

ER

ER2 KR

KR

MPT

DH KS2

MPT

Reaction chamber KS2

MAT

DH

ER

DH

Reaction chamber

KR2

MPT DH

Mammalian fatty acid synthase

MAT

AT Reaction chamber

" !

A C P

KR

KS

PPT "

AT

ER

DH

KS

MPT

MAT DH

ER

KR

A C P

TE

FIGURE 9.9 Organization of enzyme functions on 2 eukaryotic fatty acid synthases. (left) Fungal FAS is a closed barrel 260 Å high and 230 Å wide. (right) Mammalian (pig) FAS is an asymmetric X-shape 210 Å high, 180 Å wide, and 90 Å deep. The arrangement of functional domains along the FAS polypeptides is shown at the bottom of the figure. KS domains form dimers in both structures. KR domains form dimers in the fungal enzyme, whereas ER and DH domains form dimers in the mammalian complex. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

261

262

Part 1 Building Blocks of the Cell (a)

(b)

Central wheel KR dimer 90° Interchamber opening

Upper reaction chamber

Lower reaction chamber

KS dimer (c)

Tilted side view

L1

L2

PPT

Central anchor

Peripheral anchor

ACP

FIGURE 9.10 (a) FAS from S. cerevisiae possesses 2 trimeric reaction chambers separated by equatorial stabilizing struts. ACP domains are located at the equatorial base of each reaction chamber, close to the catalytic site of KS. (b) The equatorial base of this structure is an "6 trimer of dimers, with alternating KS and KR domains. The central stabilizing struts are "-helical extensions of the functional domains arranged around the outside of the ring. A central 6-helix bundle stabilizes the structure. (c) The ACP domains (red) are tethered on flexible linkers (yellow) so that they can move from one active site to the next in the catalytic cycle. Comparison of the ACP domains of S. cerevisiae and E. coli reveals that the ACP domains probably extend the acyl-phosphopantetheine group to active sites but then retract the acyl group into a hydrophobic cleft while moving from one site to the next [pdb = 2 UV8; images courtesy of Marc Leibundgut and Nenad Ban, ETH (Zurich)].

struts anchored by a 6-helix bundle in the centre of the barrel. Each "-subunit contains KR and KS domains. Three KR and 3 KS active sites are oriented toward the upper reaction chamber, and 3 of each face the lower chamber. The "-subunit trimers form rounded caps over the upper and lower reaction chambers. Each chamber contains 3 pores that allow substrates (acetyl-CoA and malonyl-CoA) to diffuse in and palmitoyl-CoA to exit. On each end of the structure, the active sites of the 4 "-subunit enzyme domains (see Figure 9.9) are oriented toward the interior of the reaction chamber. Three ACP domains in each chamber shuttle growing acyl chains from site to site during the catalytic cycle. Each ACP NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 9

Lipid Biosynthesis

ER2

KR

KR

DH

DH

MAT

KS2

MAT

FIGURE 9.11 Structural studies reveal that mammalian FAS homodimer is X-shaped. The ACP domains are probably located adjacent to the KR domains at the ends of the arms [pdb id % 2VZ8; Marc Leibundgut and Nenad Ban, ETH (Zurich)

is tethered by 2 flexible linker peptides, which facilitates its site-to-site movement (Figure 9.10c). The phosphopantetheine arm on each ACP can extend outward to reach into active sites or may retract to insert its acyl chain in a protective hydrophobic cavity during intersite transport. The homodimeric mammalian FAS contains all 6 functional enzyme domains on each subunit (Figures 9.9 and 9.11). In the X-shaped dimer, 3 of the domains—including KS, ER, and DH—form dimeric structures, whereas the KR and MAT domains are separated and lie near the ends of the extended “arms.” The arms form reaction chambers on either side of the structure. The flexible ACP domains do not appear in this structure (probably because they are not fixed in any one position in the crystals used for the structural studies). However, since it follows the KR domain in the polypeptide sequence, the ACP domain probably lies at the end of each KR arm, where it can rotate to interact with the adjacent active sites. In both the fungal and the mammalian FAS structures, the close association of enzymatic domains within one large complex permits efficient transfer of intermediates from one active site to the next. In addition, the presence of all these enzyme domains on 1 or 2 polypeptides allows the cell to coordinate synthesis of all enzymes needed for fatty acid synthesis.

C16 Fatty Acids May Undergo Elongation and Unsaturation Additional Elongation As seen already, palmitate is the primary product of the fatty acid synthase. Cells synthesize many other fatty acids. Shorter chains are easily made if the chain is released before reaching 16 carbons in length. Longer chains are made through special elongation reactions, which occur both in the mitochondria and at the surface of NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

263

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Part 1 Building Blocks of the Cell

FIGURE 9.12 (Step 1) Elongation of fatty acids in mitochondria is initiated by the thiolase reaction. The "-ketoacyl intermediate thus formed undergoes the same 3 reactions (in reverse order) that are the basis of "-oxidation of fatty acids. (Step 2) Reduction of the "-keto group is followed by (Step 3) dehydration to form a double bond. (Step 4) Reduction of the double bond yields a fatty acyl-CoA that is elongated by 2 carbons. Note that the reducing coenzyme for the second step is NADH, whereas the reductant for the fourth step is NADPH.

O CH3

CH2

C

S CoA HSCoA

O R

C

O R

SCoA Thiolase 1

Acyl-CoA

CH2

C

O CH2

C

SCoA

!-Ketoacyl-CoA NADH +

H+

L-!-hydroxyacyl-CoA

dehydrogenase

2 NAD+ O

H O

H R

CH2

H 2O

C

C

C

SCoA

Enoyl-CoA hydratase 3

H

R

CH2

C

CH2

C

SCoA

OH L-!-Hydroxyacyl-CoA

", !-trans-Enoyl-CoA

NADPH + H+ 4 NADP+

Acetyl-ACP + 4 Malonyl-ACP

O R

Four rounds of fatty acyl synthase

CH2

C

CH2

C

S-ACP

H !-Hydroxydecanoyl–ACP !-Hydroxydecanoyl thioester dehydrase

H2O

O H CH3(CH2)5 C #

H C !

CH2

CH2

C

SCoA

Acyl-CoA (2 carbons longer)

O

OH CH3(CH2)5

CH2

CH2

C

S-ACP

Three rounds of fatty acyl synthase Palmitoleoyl-ACP (16:1∆9–ACP) Elongation at ER cis-Vaccenoyl-ACP 18:1∆11–ACP FIGURE 9.13 Double bonds are introduced into the growing fatty acid chain in E. coli by specific dehydrases. Palmitoleoyl-ACP is synthesized by a sequence of reactions involving 4 rounds of chain elongation, followed by double-bond insertion by "-hydroxydecanoyl thioester dehydrase and 3 additional elongation steps. Another elongation cycle produces cis-vaccenic acid.

the endoplasmic reticulum (ER). The ER reactions are similar to those we have just discussed: addition of 2-carbon units at the carboxyl end of the chain by means of oxidative decarboxylations involving malonyl-CoA. As was the case for the fatty acid synthase, this decarboxylation provides the thermodynamic driving force for the condensation reaction. The mitochondrial reactions involve addition (and subsequent reduction) of acetyl units. These reactions (Figure 9.12 above) are essentially a reversal of fatty acid oxidation, with the exception that NADPH is utilized in the saturation of the double bond, instead of FADH2. Introduction of a Single cis Double Bond Both prokaryotes and eukaryotes are capable of introducing a single cis double bond in a newly synthesized fatty acid. Bacteria such as E. coli carry out this process in an O2-independent pathway, whereas eukaryotes have adopted an O2-dependent pathway. There is a fundamental chemical difference between the two. The O2-dependent reaction can occur anywhere in the fatty acid chain, with no (additional) need to activate the desired bond toward dehydrogenation. However, in the absence of O2, some other means must be found to activate the bond in question. Thus, in the bacterial reaction, dehydrogenation occurs while the bond of interest is still near the "-carbonyl or "-hydroxy group and the thioester group at the end of the chain. In E. coli, the biosynthesis of a monounsaturated fatty acid begins with 4 normal cycles of elongation to form a 10-carbon intermediate, "-hydroxydecanoyl-ACP (Figure 9.13). At this point, !-hydroxydecanoyl thioester dehydrase forms a double bond ",# to the thioester and in the cis configuration. This is followed by 3 rounds of the normal elongation reactions to form palmitoleoyl-ACP. Elongation may terminate at this point or may be followed by additional biosynthetic events. The principal unsaturated fatty acid in E. coli, cis-vaccenic acid, is formed by an additional elongation step, using palmitoleoyl-ACP as a substrate. NEL

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Chapter 9

Lipid Biosynthesis

Unsaturation Reactions Occur in Eukaryotes in the Middle of an Aliphatic Chain The addition of double bonds to fatty acids in eukaryotes does not occur until the fatty acyl chain has reached its full length (usually 16–18 carbons). Dehydrogenation of stearoyl-CoA occurs in the middle of the chain, despite the absence of any useful functional group on the chain to facilitate activation: CH3 i (CH2)16CO i SCoA h CH3 i (CH2)7CH “ CH(CH2)7CO i SCoA This impressive reaction is catalyzed by stearoyl-CoA desaturase, a 53 kD enzyme containing a non-heme iron centre. NADH and O2 are required, as are 2 other proteins: cytochrome b5 reductase (a 43 kD flavoprotein) and cytochrome b5 (16.7 kD). All 3 proteins are associated with the ER membrane. Cytochrome b5 reductase transfers a pair of electrons from NADH through FAD to cytochrome b5 (Figure 9.14). Oxidation of reduced cytochrome b5 is coupled to reduction of non-heme Fe3+ to Fe2+ in the desaturase. The Fe3+ accepts a pair of electrons (1 at a time in a cycle) from cytochrome b5 and creates a cis double bond at the 9,10-position of the stearoyl-CoA substrate. O2 is the terminal electron acceptor in this fatty acyl desaturation cycle. Note that 2 water molecules are made, which means that 4 electrons are transferred overall: 2 come through the reaction sequence from NADH, and 2 come from the fatty acyl substrate that is being dehydrogenated.

The Unsaturation Reaction May Be Followed by Chain Elongation Additional chain elongation can occur following this single desaturation reaction. The oleoyl-CoA produced can be elongated by 2 carbons to form a 20:1 cis-∆11 fatty acyl-CoA. If the starting fatty acid is palmitate, reactions similar to the preceding scheme yield palmitoleoyl-CoA (16:1 cis-∆9), which subsequently can be elongated to yield cis-vaccenic acid (18:1 cis-∆11). Similarly, C16 and C18 fatty acids can be elongated to yield C22 and C24 fatty acids, such as are often found in sphingolipids.

Mammals Cannot Synthesize Most Polyunsaturated Fatty Acids Organisms differ with respect to formation, processing, and utilization of polyunsaturated fatty acids. E. coli, for example, does not have any polyunsaturated fatty acids. Eukaryotes do synthesize a variety of polyunsaturated fatty acids, certain organisms more than others. For example, plants manufacture double bonds between the ∆9 and the methyl end of the chain, but mammals cannot. Plants readily desaturate oleic acid at the 12-position (to give linoleic acid) or at both the 12- and 15-positions (producing linolenic acid). Mammals require polyunsaturated fatty acids but must acquire them in their diet. As such, these fatty acids are referred to as essential fatty acids. On the other hand, mammals can introduce

2 H2O

+

2 H+

H

NADH

+

H+

Cytochrome b5 reductase (FAD)

2 Cytochrome b5 (Fe2+) (Reduced)

Desaturase (Fe3+)

CH3

(CH2)7

O

H

C C (CH2)7C Oleoyl-CoA

S CoA

O NAD+

Cytochrome b5 reductase (FADH2)

2 Cytochrome b5 (Fe3+) (Oxidized)

Desaturase (Fe2+)

CH3

(CH2)16

C

Stearoyl-CoA

S CoA

+

O2

+

2 H+

FIGURE 9.14 The conversion of stearoyl-CoA to oleoyl-CoA in eukaryotes is catalyzed by stearoyl-CoA desaturase in a reaction sequence that also involves cytochrome b5 and cytochrome b5 reductase. Two electrons are passed from NADH through the chain of reactions as shown, and 2 electrons are derived from the fatty acyl substrate. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 1 Building Blocks of the Cell

double bonds between the double bond at the 8- or 9-position and the carboxyl group. Enzyme complexes in the ER desaturate the 5-position, provided a double bond exists at the 8-position, and form a double bond at the 6-position if one already exists at the 9-position. Thus, oleate can be unsaturated at the 6,7-position to give an 18:2 cis-∆6,∆9 fatty acid.

Arachidonic Acid Is Synthesized from Linoleic Acid by Mammals Mammals can add additional double bonds to unsaturated fatty acids in their diets. Their ability to make arachidonic acid from linoleic acid is one example (Figure 9.15). This fatty acid is the precursor for prostaglandins and other biologically active derivatives such as leukotrienes. Synthesis involves formation of a linoleoyl ester of CoA from dietary linoleic acid, followed by introduction of a double bond at the 6-position. The triply unsaturated product is then elongated (by malonyl-CoA with a decarboxylation step) to yield a 20-carbon fatty acid with double bonds at the 8-, 11-, and 14-positions. A second desaturation reaction at the 5-position, followed by a reverse acyl-CoA synthetase reaction (see Chapter 22), liberates the product, a 20-carbon fatty acid with double bonds at the 5-, 8-, 11-, and 14-positions.

Regulatory Control of Fatty Acid Metabolism Is an Interplay of Allosteric Modifiers and Phosphorylation–Dephosphorylation Cycles The control and regulation of fatty acid synthesis is intimately related to regulation of fatty acid breakdown, glycolysis, and the TCA cycle. Acetyl-CoA is an important intermediate metabolite in all these processes. In these terms, it is easy to appreciate the interlocking relationships in Figure 9.16. Malonyl-CoA can act to prevent fatty acyl-CoA derivatives 12

COO–

9

Linoleic acid (18:2∆9,12) CoA + ATP Acyl-CoA AMP + P P synthetase

O C

CoA

S

Linoleoyl-CoA (18:2∆9,12–CoA) Desaturation 2H 12

9

6

C

Linolenoyl-CoA (18:3∆6,9,12–CoA)

CoA

S

O Malonyl-CoA CO2 + CoA 14

11

Elongation

8

C

(20:3∆8,11,14–CoA)

S

CoA

O Desaturation 2H 14

11

8

O 5

C

CoA

S

Arachidonoyl-CoA (20:4∆5,8,11,14–CoA) H2O FIGURE 9.15 Arachidonic acid is synthesized from linoleic acid in eukaryotes. This is the means by which animals synthesize fatty acids with double bonds at positions other than C-9.

CoA 14

11

8

O 5

C

_

O

Arachidonic acid NEL

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Chapter 9

Lipid Biosynthesis

267

HUMAN BIOCHEMISTRY

V3 and V6: Essential Fatty Acids with Many Functions Linoleic acid and "-linolenic acid are termed essential fatty acids because animals cannot synthesize them and must acquire them in their diet. Linoleic acid is the precursor of arachidonic acid, and both are referred to as V6 fatty acids because, counting from the end (omega; v) carbon of the chain, the first double bond is at the sixth position (see figure). Similarly, "-linolenic acid is the precursor of eicosapentaenoic acid and docosahexaenoic acid (DHA), and these 3 are termed V3 fatty acids. Vegetable oils are rich in linoleic acid and thus satisfy the body’s v6 dietary requirements, whereas fish oils (e.g., cod, herring, menhaden, and salmon) are rich in v3 fatty acids. The v6 fatty acids are precursors of prostaglandins, thromboxanes, and leukotrienes (see Section 9.3). The v3 fatty acids have

beneficial effects in a variety of organs and biological processes, including growth regulation, platelet activation, and lipoprotein metabolism. The v3 fats are generally cardioprotective, anti-inflammatory, and anticarcinogenic. Interestingly, especially high levels of DHA have been found in rod cell membranes in animal retina and in neural tissue. DHA is approximately 22% of total fatty acids in animal retina and 35%–40% of the fatty acids in retinal phosphatidylethanolamine. DHA supports neural and visual development, in part because it is a precursor for eicosanoids that regulate numerous cell and organ functions. Infants can synthesize DHA and other polyunsaturated fatty acids, but the rates of synthesis are low. Strong evidence exists for the importance of these fatty acids in infant nutrition.

$6 Essential fatty acid COOH Linoleic acid (18:2 $6) COOH Arachidonic acid (20:4 $6) $3 Essential fatty acid COOH "-Linoleic acid (18:3 $3)

" The “v” numbering system, where the position of

COOH

the first double bond relative to the methyl (v) group is indicated (red box), is useful because the v doublebond position is retained during chain elongation and desaturation.

Eicosapentaenoic acid (EPA; 20:5 $3) COOH Docosahexaenoic acid (DHA; 22:6 $3) Glucose

Malate

2 Pyruvate

Oxaloacetate Citrate

Acetyl-CoA

O

xa

!-Oxidation

lo

ac

et

e

at

at

tr Ci

Acetyl-CoA

e Citric acid cycle

Acetyl-CoA carboxylase

Malonyl-CoA

Fatty acyl-CoA

Carnitine acyltransferase-1

+

Fatty acyl-CoA

Fatty acid

Carnitine

Triacylglycerol

FIGURE 9.16 Regulation of fatty acid synthesis and fatty acid oxidation are coupled as shown. Malonyl-CoA, produced during fatty acid synthesis, inhibits the uptake of fatty acyl carnitine (and thus fatty acid oxidation) by mitochondria. When fatty acyl-CoA levels rise, fatty acid synthesis is inhibited and fatty acid oxidation activity increases. Rising citrate levels (which reflect an abundance of acetyl-CoA) similarly signal the initiation of fatty acid synthesis.

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268

Part 1 Building Blocks of the Cell

from entering the mitochondria by inhibiting the carnitine acyl transferase of the outer mitochondrial membrane that initiates this transport. In this way, when fatty acid synthesis is turned on (as signalled by higher levels of malonyl-CoA), "-oxidation is inhibited. As we pointed out earlier, citrate is an important allosteric activator of acetyl-CoA carboxylase, and fatty acyl-CoAs are inhibitors. The degree of inhibition is proportional to the chain length of the fatty acyl-CoA; longer chains show a higher affinity for the allosteric inhibition site on acetyl-CoA carboxylase. Palmitoyl-CoA, stearoyl-CoA, and arachidyl-CoA are the most potent inhibitors of the carboxylase.

Hormonal Signals Regulate ACC and Fatty Acid Biosynthesis As described earlier, citrate activation and palmitoyl-CoA inhibition of acetyl-CoA carboxylase are strongly dependent on the phosphorylation state of the enzyme. This provides a crucial connection to hormonal regulation. Many of the enzymes that act to phosphorylate acetyl-CoA carboxylase (see Figure 9.4) are controlled by hormonal signals. Glucagon is a good example (Figure 9.17). As noted in Chapter 21, glucagon binding to membrane receptors activates an intracellular cascade involving activation of adenylyl cyclase. Cyclic AMP produced by the cyclase activates a protein kinase, which then phosphorylates acetylCoA carboxylase. Unless citrate levels are high, phosphorylation causes inhibition of fatty acid biosynthesis. The carboxylase (and fatty acid synthesis) can be reactivated by

Glucagon

Insulin

Glucagon receptor

Insulin receptor

Adenylyl cyclase

G protein Phosphodiesterase

ATP

AMP

cAMP Protein kinase (inactive)

Protein kinase (active)

Triacylglycerol lipase (inactive)

Dephospho-acetyl-CoA carboxylase (Active at low [citrate])

_

HPO42

_

ATP

ATP

Phosphatases

Phosphatases

H 2O

P

ADP

ADP

Phospho-acetyl-CoA carboxylase (Active only at high [citrate]) P

P

HPO42

P

P

P

P

H2O

Triacylglycerol lipase (active)

P

Triacylglycerols FIGURE 9.17 Hormonal signals regulate fatty acid synthesis, primarily through actions on acetyl-CoA carboxylase. Availability of fatty acids also depends upon hormonal activation of triacylglycerol lipase.

Fatty acids and glycerol

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Chapter 9

Lipid Biosynthesis

a specific phosphatase, which dephosphorylates the carboxylase. Also indicated in Figure 9.17 is the simultaneous activation by glucagon of triacylglycerol lipases, which hydrolyze triacylglycerols, releasing fatty acids for "-oxidation. Both the inactivation of acetyl-CoA carboxylase and the activation of triacylglycerol lipase are counteracted by insulin, whose receptor acts to stimulate a phosphodiesterase that converts cAMP to AMP.

9.2

How Are Complex Lipids Synthesized?

Complex lipids consist of backbone structures to which fatty acids are covalently bound. Principal classes include the glycerolipids, for which glycerol is the backbone, and sphingolipids, which are built on a sphingosine backbone. The 2 major classes of glycerolipids are glycerophospholipids and triacylglycerols. The phospholipids, which include both glycerophospholipids and sphingomyelins, are crucial components of membrane structure. They are also precursors of hormones such as the eicosanoids (e.g., prostaglandins) and signal molecules (such as the breakdown products of phosphatidylinositol). Different organisms possess greatly different complements of lipids and therefore invoke somewhat different lipid biosynthetic pathways. For example, sphingolipids and triacylglycerols are produced only in eukaryotes. In contrast, bacteria usually have simple lipid compositions. Phosphatidylethanolamine accounts for at least 75% of the phospholipids in E. coli, with phosphatidylglycerol and cardiolipin accounting for most of the rest. E. coli membranes possess no phosphatidylcholine, phosphatidylinositol, sphingolipids, or cholesterol. On the other hand, some bacteria (such as Pseudomonas) can synthesize phosphatidylcholine, for example. In this section and the one following, we consider some of the pathways for the synthesis of glycerolipids, sphingolipids, and the eicosanoids, which are derived from phospholipids.

Glycerolipids Are Synthesized by Phosphorylation and Acylation of Glycerol A common pathway operates in nearly all organisms for the synthesis of phosphatidic acid, the precursor to other glycerolipids. Glycerokinase catalyzes the phosphorylation of glycerol to form glycerol-3-phosphate, which is then acylated at both the 1- and the 2-positions to yield phosphatidic acid (Figure 9.18). The first acylation, at position 1, is catalyzed by glycerol-3-phosphate acyl transferase, an enzyme that in most organisms is specific for saturated fatty acyl groups. Eukaryotic systems can also utilize dihydroxyacetone phosphate as a starting point for synthesis of phosphatidic acid (Figure 9.18). Again a specific acyl transferase adds the first acyl chain, followed by reduction of the backbone keto group by acyl dihydroxyacetone phosphate reductase, using NADPH as the reductant. Alternatively, dihydroxyacetone phosphate can be reduced to glycerol-3-phosphate by glycerol-3phosphate dehydrogenase.

Eukaryotes Synthesize Glycerolipids from CDP-Diacylglycerol or Diacylglycerol In eukaryotes, phosphatidic acid is converted directly either to diacylglycerol or to cytidine diphosphate diacylglycerol (or simply CDP-diacylglycerol; Figure 9.19 on page 271). From these 2 precursors, all other glycerophospholipids in eukaryotes are derived. Diacylglycerol is a precursor for synthesis of triacylglycerol, phosphatidylethanolamine, and phosphatidylcholine. Triacylglycerol is synthesized mainly in adipose tissue, liver, and intestines and serves as the principal energy storage molecule in eukaryotes. Triacylglycerol biosynthesis in liver and adipose tissue occurs via diacylglycerol acyl transferase, an enzyme bound to the cytoplasmic face of the ER. A different route is used, however, in intestines. As shown in Figure 22.3, triacylglycerols from the diet are broken down to 2-monoacylglycerols by

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269

270

Part 1 Building Blocks of the Cell CH2OH HO

C

H

CH2OH Glycerol ATP Glycerokinase ADP CH2OH C

O

O

CH2

NADH

O

P

O–

+ H+

CH2OH

NAD+

HO

C CH2

Glycerol-3-P dehydrogenase

O– Dihydroxyacetone-P

O

H O

O–

P

O– Glycerol-3-P

O R1

Dihydroxyacetone-P acyltransferase

C

O SCoA

R1

Glycerol-3-P acyltransferase

O R1

C

C

CoA

CoA

O SCoA or R1

C

S-ACP

or ACP

O O

CH2 C CH2

NADPH

O

O O

P

O–

O– 1-Acyldihydroxyacetone-P

+

H+

NADP+

R1

C

O HO

CH2 C CH2

Acyldihydroxyacetone-P reductase

O

H O

O–

P

O– 1-Acylglycerol-3-P O 1-Acylglycerol-3-P acyltransferase

R2

C

CoASH

O SCoA or

R2

C

S-ACP

or ACP-SH

O R1

C

O

CH2

O

C

O R2

C

CH2 FIGURE 9.18 Synthesis of glycerolipids in eukaryotes begins with the formation of phosphatidic acid, which may be formed from dihydroxyacetone phosphate or glycerol as shown.

O

H O

P

O–

O– Phosphatidic acid

specific lipases (see Figure 22.3). Acyl transferases then acylate 2-monoacylglycerol to produce new triacylglycerols (Figure 9.20 on page 272).

Phosphatidylethanolamine Is Synthesized from Diacylglycerol and CDP-Ethanolamine Phosphatidylethanolamine synthesis begins with phosphorylation of ethanolamine to form phosphoethanolamine (see Figure 9.19). The next reaction involves transfer of a cytidylyl group from CTP to form CDP-ethanolamine and pyrophosphate. As always, PPi NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 9

Lipid Biosynthesis

O R1

C

O

CH2

R2

C

O

CH CH2

O

O O

O

P

O

P

CDP-diacylglycerol O–

+ HOCH2CH2NH3 Ethanolamine

Phosphatidate cytidylyltransferase

ATP

O

Cytidine

O–

P P

+ HOCH2CH2N(CH3)3

CTP

Choline

Ethanolamine kinase ADP O –O

P

+ OCH2CH2NH3

–O

Choline kinase

R1

C

O

CH2

R2

C

O

CH

O

Phosphoethanolamine CTP: Phosphoethanolamine cytidylyltransferase

ATP

O

CH2

O

P O–

O

CTP

P P ADP

P

+ OCH2CH2NH3

R1

C

O

CH2

R2

C

O

CH

O

R2

C O

O O

CH2 CH

R1

CH2

R3

O O

P

O– Phosphatidylethanolamine

O–

P

+ OCH2CH2N(CH3)3

O– CDP-choline

R2

C

SCoA

C

O

CH2

C

O

CH

O

Diacylglycerol acyltransferase

+ OCH2CH2NH3

O

O

CH2OH

O C

P

O

CMP

Diacylglycerol

O

O

CDP-choline: 1,2-diacylglycerol phosphocholine transferase

O

CMP

O

P

O–

CDP-ethanolamine: 1,2diacylglycerol phosphoethanolamine transferase

Cytidine

Phosphatidic acid phosphatase

ATP

CTP: Phosphocholine cytidylyltransferase

P P

H2O

Diacylglycerol kinase

+ OCH2CH2N(CH3)3

P

–O Phosphocholine

Phosphatidic acid

CDP-ethanolamine

R1

–O

O–

P

CTP

O O

O

O

O–

Cytidine O

ADP

O

CH2

O

P

+ OCH2CH2N(CH3)3

O– Phosphatidylcholine

CoASH O R1

C

O

CH2

R2

C

O

CH

O

CH2

O O

Triacylglycerol

C

R3

FIGURE 9.19 Diacylglycerol and CDP-diacylglycerol are the principal precursors of glycerolipids in eukaryotes. Phosphatidylethanolamine and phosphatidylcholine are formed by reaction of diacylglycerol with CDP-ethanolamine or CDP-choline respectively.

hydrolysis drives this reaction forward. A specific phosphoethanolamine transferase then links phosphoethanolamine to the diacylglycerol backbone. Biosynthesis of phosphatidylcholine is entirely analogous because animals can synthesize it directly. All the choline utilized in this pathway must be acquired from the diet. On the other hand, yeast, certain bacteria, and animal liver can convert phosphatidylethanolamine to phosphatidylcholine by methylation reactions involving S-adenosylmethionine (see Chapter 23). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

271

272

Part 1 Building Blocks of the Cell

Exchange of Ethanolamine for Serine Converts Phosphatidylethanolamine to Phosphatidylserine

Dietary triacylglycerols

Mammals synthesize phosphatidylserine (PS) in a calcium ion–dependent reaction involving amino alcohol exchange (Figure 9.21). The enzyme catalyzing this reaction is associated with the ER and will accept phosphatidylethanolamine (PE) and other phospholipid substrates. A mitochondrial PS decarboxylase can subsequently convert PS to PE. No other pathway converting serine to ethanolamine has been found.

Lipases

O R2

C

CH2OH O

C

H

CH2 OH

Eukaryotes Synthesize Other Phospholipids via CDP-Diacylglycerol

2-Monoacylglycerol O R1

C

S CoA

Monoacylglycerol acyltransferase CoASH O R1

C

Eukaryotes also use CDP-diacylglycerol, derived from phosphatidic acid, as a precursor for several other important phospholipids, including phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (Figure 9.22). PI accounts for only about 2%–8% of the lipids in most animal membranes, but breakdown products of PI, including inositol-1,4,5trisphosphate and diacylglycerol, are second messengers in a vast array of cellular signalling processes.

O

CH2

O

CH

Dihydroxyacetone Phosphate Is a Precursor to the Plasmalogens

CH2 OH

Certain glycerophospholipids possess alkyl or alkenyl ether groups at the 1-position in place of an acyl ester group. These glyceryl ether phospholipids are synthesized from dihydroxyacetone phosphate (Figure 9.23 on page 274). Acylation of dihydroxyacetone phosphate (DHAP) is followed by an exchange reaction in which the acyl group is removed as a carboxylic acid and a long-chain alcohol adds to the 1-position. This long-chain alcohol is derived from the corresponding acyl-CoA by means of an acyl-CoA reductase reaction involving oxidation of 2 molecules of NADH. The 2-keto group of the DHAP backbone is then reduced to an alcohol, followed by acylation. The resulting 1-alkyl-2-acylglycerol-3phosphate can react in a manner similar to phosphatidic acid to produce ether analogs of

O R2

C

Diacylglycerol O R3

C

SCoA

Diacylglycerol acyltransferase CoASH O R1 R2 R3

C O

O

CH2

C O

O

CH

C

O

CH2

O R1

C

O

CH2

R2

C

O

CH

Triacylglycerol

FIGURE 9.20 Triacylglycerols are formed primarily by the action of acyl transferases on monoacylglycerol and diacylglycerol.

CH2

O CO2

O O

P

O

CH2

+ NH3

CH2

O– Phosphatidylethanolamine Serine

Serine

Phosphatidylserine decarboxylase (mitochondria)

Base exchange enzyme (endoplasmic reticulum)

Ethanolamine

Ethanolamine

O R1

C

O

CH2

R2

C

O

CH

O

CH2

Serine O O

P

H O

O–

CH2

C

+ NH3

COO–

Phosphatidylserine FIGURE 9.21 The interconversion of phosphatidylethanolamine and phosphatidylserine in mammals. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 9

Lipid Biosynthesis

O R1

C

O

NH2

CH2 N

O R2

C

O

C CH2

O

O

H O

P

O

P

O O

O

O–

O–

N

CH2

OH OH CDP-diacylglycerol Inositol

Glycerol-3-P Glycerophosphate phosphatidyltransferase CMP

Phosphatidylinositol synthase CMP O R1

C

O O

CH2

R1

C

O R2

C

O

CH2

O O

C

O

H

CH2

O

P

R2

Phosphoglycerol

C

O

O–

C

H

CH2

O

HO

H H OH H H

O

P

CH2

O

O CH2

C

O–

OH OH H

H

O

O

O–

P O–

OH

Phosphatidylglycerol-P

H

Phosphatidylglycerol-P phosphatase

OH

Pi

Phosphatidylinositol O R1

C

O

CH2

O R2

Glycerol

C

O

C

O

H

CH2

O

H O

P

CH2

C

O–

CH2OH

OH

Phosphatidylglycerol CDP-diacylglycerol Cardiolipin synthase CMP O

O R1

C

O

CH2

CH2

O R2

C

O

C CH2

O

H O

P O–

H O

CH2

C

CH2

OH

R1&

O

Glycerol O

C

O

O

H

C

P

O

CH2

O

C

R2&

O–

Cardiolipin FIGURE 9.22 CDP-diacylglycerol is a precursor of phosphatidylinositol, phosphatidylglycerol, and cardiolipin in eukaryotes.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

273

274

Part 1 Building Blocks of the Cell CH2 OH C

O

CH2

O

O

O–

P

O– Dihydroxyacetone phosphate Dihydroxyacetone phosphate 1 acyltransferase

O

S

CoA

C

R R1

CoASH

C

H C

R2 O

O

CH2

O

CH

O

O R

H C

C

CH2 C

O

CH2 O

CH2

O

P

O

O

O– O–

P

Plasmalogen

O– 1-Acyldihydroxyacetone phosphate HO 1-Acyldihydroxyacetone phosphate synthase 2

+ CH2CH2NH3

O

1-Alkyl-2-acylglycerophosphoethanolamine desaturase 6

CH2CH2R1

R

NAD+

+

NADH

+

2 H2O O2

+

H+

O–

C

R1CH2CH2

O

O

CH2

O

C

O R1CH2CH2 O

R2

CH2 C CH2

O O

P

R1CH2CH2

O

NADPH

O

+

P

O

+ CH2CH2NH3

O–

O–

1-Alkyl-2-acylglycero-3-phosphoethanolamine CDP

CDP-ethanolamine transferase 5

H+

CDP- ethanolamine

NADP+ R1CH2CH2

O

CH2

HOCH CH2

O

H

CH2

O

O– 1-Alkyldihydroxyacetone phosphate

1-Alkyldihydroxyacetone phosphate 3 oxidoreductase

C

R2

O O

P O–

C

CH2

O

C

O S

CoA

CoASH

O–

1-Alkylglycero-3-phosphate

O

1-Alkylglycerophosphate acyltransferase 4

R2

C

CH2

H

O O

P

O–

O– 1-Alkyl-2-acylglycero-3-phosphate

FIGURE 9.23 Biosynthesis of plasmalogens in animals. (Step 1) Acylation at C-1 is followed by (Step 2) exchange of the acyl group for a long-chain alcohol. (Step 3) Reduction of the keto group at C-2 is followed by (Steps 4 and 5) transferase reactions, which add an acyl group at C-2 and a polar head-group moiety (as shown here for phosphoethanolamine), and a (Step 6) desaturase reaction that forms a double bond in the alkyl chain. The first 2 enzymes are of cytoplasmic origin, and the last transferase is located at the endoplasmic reticulum.

phosphatidylcholine, phosphatidylethanolamine, and so forth (Figure 9.23). In addition, specific desaturase enzymes associated with the ER can desaturate the alkyl ether chains of these lipids as shown. The products, which contain a,b-unsaturated ether-linked chains at the C-1 position, are plasmalogens; they are abundant in cardiac tissue and in the central nervous system. The desaturases catalyzing these reactions are distinct from but similar to those that introduce unsaturations in fatty acyl-CoAs. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 9 RCH2CH2

O HO

Lipid Biosynthesis

275

CH2 C

H

CH2

O

O O

P

+ CH2CH2N(CH3)3

O– 1-Alkyl-2-lysophosphatidylcholine O O CH3C O– CH3C SCoA Acetyl-CoA: 1-alkyl-2-lysoglycerophosphocholine transferase CoASH

Acetylhydrolase H2O RCH2CH2

O

CH2

CH3C

O

C

O

CH2

H O

O P

O

+ CH2CH2N(CH3)3

O– 1-Alkyl-2-acetylglycerophosphocholine (platelet-activating factor, PAF)

FIGURE 9.24 Platelet-activating factor, formed from 1-alkyl-2-lysophosphatidylcholine by acetylation at C-2, is degraded by the action of acetyl hydrolase.

Platelet-Activating Factor Is Formed by Acetylation of 1-Alkyl-2-Lysophosphatidylcholine A particularly interesting ether phospholipid with unusual physiological properties, 1-alkyl-2-acetylglycerophosphocholine, also known as platelet-activating factor, possesses an alkyl ether at C-1 and an acetyl group at C-2 (Figure 9.24). The very short chain at C-2 makes this molecule much more water soluble than typical glycerolipids. Platelet-activating factor displays a dramatic ability to dilate blood vessels (and thus reduce blood pressure in hypertensive animals) and to aggregate platelets.

Sphingolipid Biosynthesis Begins with Condensation of Serine and Palmitoyl-CoA Sphingolipids, ubiquitous components of eukaryotic cell membranes, are present at high levels in neural tissues. The myelin sheath that insulates nerve axons is particularly rich in sphingomyelin and other related lipids. Prokaryotic organisms normally do not contain sphingolipids. Sphingolipids are built upon sphingosine backbones rather than glycerol. The initial reaction, which involves condensation of serine and palmitoyl-CoA with release of bicarbonate, is catalyzed by 3-ketosphinganine synthase, a PLP-dependent enzyme (Figure 9.25). Reduction of the ketone product to form sphinganine is catalyzed by 3-ketosphinganine reductase, with NADPH as a reactant. In the next step, sphinganine is acylated to form N-acyl sphinganine, which is then desaturated to form ceramide. Sphingosine itself does not appear to be an intermediate in this pathway in mammals.

Ceramide Is the Precursor for Other Sphingolipids and Cerebrosides Ceramide is the building block for all other sphingolipids. Sphingomyelin, for example, is produced by transfer of phosphocholine from phosphatidylcholine (Figure 9.26 on page 277). Glycosylation of ceramide by sugar nucleotides yields cerebrosides, such as galactosyl ceramide, which makes up about 15% of the lipids of myelin sheath structures. Cerebrosides that contain 1 or more sialic acid (N-acetylneuraminic acid) moieties are called gangliosides. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

276

Part 1 Building Blocks of the Cell H –OOC

O CH3(CH2)14

C

S

CH2OH

C +NH

CoA

3

Palmitoyl-CoA

Serine

H2O

3-Ketosphinganine synthase

HCO3–

CoASH

CH3(CH2)14

O

H

C

C

CH2OH

+NH

3

2S-3-Ketosphinganine NADPH

+ H+

3-Ketosphinganine reductase NADP+ OH H CH3(CH2)14

CH2OH

C

C

H

+NH

3

2S,3R-Sphinganine Acyl- SCoA

CoASH N-acyl-sphinganine X

XH2 OH H

H CH3(CH2)12

C

C

C

C

H

H

NH C

CH2OH

O

(CH2)n Ceramide

CH3

FIGURE 9.25 Biosynthesis of sphingolipids in animals begins with the 3-ketosphinganine synthase reaction, a PLPdependent condensation of palmitoyl-CoA and serine. Subsequent reduction of the keto group, acylation, and desaturation (via reduction of an electron acceptor, X) form ceramide, the precursor of other sphingolipids.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 9

CH3

C H C

(CH2)12 R1

H C H N

277

CHOH CH

O

CH2

H C H N

C H C

(CH2)12

UDP-Gal

R1

O

O

CH3

CH3

CH2OH HO

Lipid Biosynthesis

UDP

Ceramide

!-D-Galactosylceramide

R1

UDP- Glu

–O

HO H

H

H OH

H

H

OH

P

O

O CH2

CH2OH

O

C

O

Sphingomyelin

UDP

CH2OH

CHOH

CH2

1,2-Diacylglycerol

UDP- Gal

UDP

H C H N

C H C O

CH2OH

UDP- galactosyltransferase

OH

(CH2)12

CH

O

OH

Phosphatidylcholine

CHOH

O H

O

H OH

H

H

OH

O

HC

H

CH2

O

CH2

C N H OH

HC

R1

H3C

+ N CH3 CH3

R2

!-D-Galactosyl-(1

4)-!-D-glucosylceramide

UDP- N-Acetylgalactosamine N-Acetylgalactosaminyltransferase UDP CH2OH HO

H OH

H

H CH3

CH2OH

O H

H

NH C

CH2OH

O

H O

H

O

O

H OH

H

H

OH

H

O

H OH

H

H

OH

O

HC

H

O

CH2

HC

C N H OH

R1

R2

!-D-N-Acetylgalactosamine-(1 4)!-D-galactosyl-(1 4)-!-D-glucosylceramide CH3 O

C HN H

CH2OH CMP- sialic acid

H HCOH O COH H H H OH

CMP

H

CH2OH

CH2OH HO H

H

H

NH

CH3

CH2OH

O

H OH

C

Sialytransferase

COO–

O H

O

H H

H

O

O

H

H

OH

O H

O

H OH

H

H

OH

O H

CH2 HC HC

O C N H OH

R1

R2 Ganglioside GM2

FIGURE 9.26 Glycosyl ceramides (such as galactosylceramide), gangliosides, and sphingomyelins are synthesized from ceramide in animals.

Several dozen gangliosides have been characterized, and the general form of the biosynthetic pathway is illustrated for the case of ganglioside GM2 (Figure 9.26). Sugar units are added to the developing ganglioside from nucleotide derivatives, including UDP–Nacetylglucosamine, UDP–galactose, and UDP–glucose. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

278

Part 1 Building Blocks of the Cell

9.3

How Are Eicosanoids Synthesized, and What Are Their Functions?

Eicosanoids, so named because all of them are derived from 20-carbon fatty acids, are ubiquitous breakdown products of phospholipids. In response to appropriate stimuli, cells activate the breakdown of selected phospholipids (Figure 9.27). Phospholipase A2 (see Chapter 8) selectively cleaves fatty acids from the C-2 position of phospholipids. Often these are unsaturated fatty acids, among which is arachidonic acid. Arachidonic acid may also be released from phospholipids by the combined actions of phospholipase C (which yields diacylglycerols) and diacylglycerol lipase (which releases fatty acids).

Eicosanoids Are Local Hormones Animal cells can modify arachidonic acid and other polyunsaturated fatty acids, in processes often involving cyclization and oxygenation, to produce so-called local hormones that (1) exert their effects at very low concentrations and (2) usually act near their sites of synthesis. These substances include the prostaglandins (PG) (Figure 9.27) as well as thromboxanes (Tx), leukotrienes, and other hydroxy eicosanoic acids. Thromboxanes, discovered in blood platelets (thrombocytes), are cyclic ethers (TxB2 is actually a hemiacetal; see Figure 9.27) with a hydroxyl group at C-15.

Prostaglandins Are Formed from Arachidonate by Oxidation and Cyclization All prostaglandins are cyclopentanoic acids derived from arachidonic acid. The biosynthesis of prostaglandins is initiated by an enzyme associated with the ER, called prostaglandin endoperoxide H synthase (PGHS), also known as cyclooxygenase (COX). The enzyme catalyzes simultaneous oxidation and cyclization of arachidonic acid. The enzyme is viewed as having 2 distinct activities, COX and peroxidase (POX), as shown in Figure 9.28 on page 280.

A Variety of Stimuli Trigger Arachidonate Release and Eicosanoid Synthesis The release of arachidonate and the synthesis or interconversion of eicosanoids can be initiated by a variety of stimuli, including histamine, hormones such as epinephrine and  bradykinin, proteases such as thrombin, and even serum albumin. An important mechanism of arachidonate release and eicosanoid synthesis involves tissue injury and

A DEEPER LOOK

The Discovery of Prostaglandins The name prostaglandin was given to this class of compounds by Ulf von Euler, their discoverer, in Sweden in the 1930s. He extracted fluids containing these components from human semen. Because he thought they originated in the prostate gland, he named them prostaglandins. Actually, they were synthesized in the seminal vesicles, and it is now known that similar substances are synthesized in most animal tissues (both male and female). von Euler observed that injection of these substances into animals caused smooth muscle contraction and dramatic lowering of blood pressure. von Euler (and others) soon found that it is difficult to analyze and characterize these obviously interesting compounds because they are present at extremely low levels. Prostaglandin E2a, or PGE2a, is present in human serum at a level of less than 10-14 M! In addition,

they often have half-lives of only 30 seconds to a few minutes, not lasting long enough to be easily identified. Moreover, upon dissection and homogenization, most animal tissues rapidly synthesize and degrade a variety of these substances, so the amounts obtained in isolation procedures are extremely sensitive to the methods used and are highly variable, even when procedures are carefully controlled. Sune Bergström, Bengt Samuelsson, and their colleagues described the first structural determinations of prostaglandins in the late 1950s. In the early 1960s, dramatic advances in laboratory techniques, such as NMR spectroscopy and mass spectrometry, made further characterization possible. von Euler received the Nobel Prize for Physiology or Medicine in 1970, and Bergström, Samuelsson, and John Vane shared the Nobel Prize for Physiology or Medicine in 1982.

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Chapter 9 Hormone (or other stimulus)

Lipid Biosynthesis

Receptor

Activation of PLC and diacylglycerol lipase

Activation of PLA2 Phospholipids

Arachidonate 2 O2 O COO–

H

HO

O

COO–

OH PGH2

H

S

CH2 CH

Leukotriene C

HO

C O

OH

N H

CH2CH2 CH H+ COO– 3N

H

PGD2

COO–

O COO–

HO

CH2COO–

O NH

COO– O

C

HO

O OH TxB2

–OOC

OH PGE2

O

OH COO– HO

OH

HO

PGF2a

OH PGI2

FIGURE 9.27 Arachidonic acid, derived from breakdown of phospholipids (PL), is the precursor of prostaglandins, thromboxanes, and leukotrienes. The letters used to name the prostaglandins are assigned on the basis of similarities in structure and physical properties. The class denoted PGE, for example, consists of "-hydroxy ketones that are soluble in ether, whereas PGF denotes 1,3-diols that are soluble in phosphate buffer. PGA denotes prostaglandins possessing a, b-unsaturated ketones. The number following the letters refers to the number of carbon–carbon double bonds. Thus, PGE2 contains 2 double bonds.

inflammation. When tissue damage or injury occurs, special inflammatory cells, monocytes and neutrophils, invade the injured tissue and interact with the resident cells (such as smooth muscle cells and fibroblasts). This interaction typically leads to arachidonate release and eicosanoid synthesis. Examples of tissue injury in which eicosanoid synthesis has been characterized include heart attack (myocardial infarction), rheumatoid arthritis, and ulcerative colitis. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

279

280

Part 1 Building Blocks of the Cell 8

H

O 11

O

5

COOH

H 14

5, 8,11,14-Eicosatetraenoic acid (arachidonic acid)

. . ...

...

...

.

COOH

........

COX

O

...

O

O

H

O

Peroxide radical H

H

O

COOH

O

O

H FIGURE 9.28 Prostaglandin endoperoxide H synthase (PGHS), the enzyme that converts arachidonic acid to prostaglandin PGH2, possesses 2 distinct activities: cyclooxygenase (COX) and a glutathione-dependent hydroperoxidase (POX). The mechanism of the reaction begins with hydrogen atom abstraction by a tyrosine radical on the enzyme, followed by rearrangement to cyclize and incorporate 2 oxygen molecules. Reduction of the peroxide at C-15 completes the reaction. COX is the site of action of aspirin and other analgesic agents.

H

OH PGG2 H POX

O

H COOH

O H

HO

H

PGH2

A DEEPER LOOK

The Molecular Basis for the Action of Non-steroidal Anti-inflammatory Drugs Prostaglandins are potent mediators of inflammation. The first and committed step in the production of prostaglandins from arachidonic acid is the bis-oxygenation of arachidonate to prostaglandin PGG2. This is followed by reduction to PGH2 in a peroxidase reaction. Both these reactions are catalyzed by PGHS or COX. This enzyme is inhibited by the family of drugs known as non-steroidal anti-inflammatory drugs, or NSAIDs. Aspirin, ibuprofen, flurbiprofen, and acetaminophen (trade name Tylenol) are all NSAIDs. There are 2 isoforms of COX in animals: COX-1, which carries out normal, physiological production of prostaglandins; and COX-2, which is induced by cytokines, mitogens, and endotoxins in inflammatory cells and is responsible for the production of prostaglandins in inflammation. The enzyme structure shown in panel a is that of residues 33–583 of COX-1 from sheep, inactivated by ibuprofen (cyan). These 551 residues comprise 3 distinct domains. The first of these, residues 33–72, is a small, compact module that is similar to epidermal growth factor. The second domain, composed of residues 73–116, forms a righthanded spiral of 4 "-helical segments. These "-helical segments form a membrane-binding motif. The helical segments are amphipathic, with most of the hydrophobic residues facing away from the protein, where they can interact with a lipid bilayer. The third domain of the COX enzyme, the catalytic domain, is a globular structure that contains both the COX and the peroxidase active sites.

The COX active site lies at the end of a long, narrow, hydrophobic tunnel or channel. Three of the "-helices of the membranebinding domain lie at the entrance to this tunnel. The walls of the tunnel are defined by 4 "-helices, formed by residues 106–123, 325– 353, 379–384, and 520–535. The COX-1 structure shown in panel a has a molecule of ibuprofen bound in the tunnel. Deep in the tunnel, at the far end, lies Tyr385, a catalytically important residue. Heme-dependent peroxidase activity is implicated in the formation of a proposed Tyr385 radical, which is required for COX activity. Aspirin and other NSAIDs block the synthesis of prostaglandins by filling and blocking the tunnel, preventing the migration of arachidonic acid to Tyr385 in the active site at the back of the tunnel. Why do the new “COX-2 inhibitors” bind to (and inhibit) COX-2 but not COX-1? A single amino acid substitution makes all the difference. Panel b shows an overlay of COX-1 (1EQG) and COX-2 (1CX2) structures. COX-2 has a valine (blue) at position 523, which leaves room for binding of a Celebrex-like inhibitor (orange). On the other hand, COX-1 has bulkier isoleucine (red) at position 523, which prevents binding of the inhibitor. COX-2 inhibitors were introduced as pain medications in Canada in 1999, and by 2004 more than half the 14 million prescriptions written annually for NSAIDs in Canada were COX-2 inhibitors. However, several COX-2 inhibitors were taken off both the Canadian NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 9

(a) pdb id = 1EQG

(b) Superposition of pdb id = 1EQG and 1CX2

(c) NH2

CHF2

O S

F

N

O N COOH O

Bromoaspirin

N CF3

N

O CH3 O O Deramaxx for dogs*

COOH

S

CH2Br

H3N

CH3 Celebrex

281

alleviate pain without the risk of adverse gastrointestinal effects, less than 5% of patients that used COX-2 prescriptions at the peak of their popularity were at high risk for these adverse effects (see www .stacommunications.com/journals/cpm/2004/October/Pdf/39.pdf).

and U.S. market in late 2004 and early 2005, when their use was linked to heart attacks and strokes in a small percentage of users. Since that time, prescriptions for COX-2 inhibitors have dropped by 65%. Interestingly, although COX-2 inhibitors were originally intended to

O

Lipid Biosynthesis

Ibuprofen

* Abby Garrett took this.

“Take Two Aspirin and…” Inhibit Your Prostaglandin Synthesis In 1971, biochemist John Vane was the first to show that aspirin (acetylsalicylate; Figure 9.29) exerts most of its effects by inhibiting the biosynthesis of prostaglandins. Its site of action is PGHS. COX activity is destroyed when aspirin O-acetylates Ser530 on the enzyme. From this you may begin to infer something about how prostaglandins (and aspirin) function. Prostaglandins are known to enhance inflammation in animal tissues. Aspirin exerts its powerful anti-inflammatory effect by inhibiting this first step in their synthesis. Aspirin does not have any measurable effect on the peroxidase activity of the synthase. Other non-steroidal anti-inflammatory agents, such as ibuprofen (Figure 9.29) and phenylbutazone, inhibit COX by competing at the active site with arachidonate or with the peroxy-acid intermediate (PGG2, as in Figure 9.28). See A Deeper Look opposite. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

282

Part 1 Building Blocks of the Cell (a)

(b)

HO

NH

C

O

COO–

O

C

O CH3

CH3

Acetylsalicylate (aspirin) O

Acetaminophen Ser CH3

H3C

CH

H3C

CH2

CH

COOH

OH

Ser COO–

Active cyclooxygenase

O

C

CH3

Inactive cyclooxygenase

OH

Salicylate

Ibuprofen

FIGURE 9.29 (a) The structures of several common analgesic agents. Acetaminophen is marketed under the trade name Tylenol. Ibuprofen is sold as Motrin, Nuprin, and Advil. (b) Acetylsalicylate (aspirin) inhibits the COX activity of endoperoxide synthase via acetylation (covalent modification) of Ser530.

21

(a)

H

21

H3C H3C

19

11

CH3 2

HO

3

1

A 4

10 5

9

B 6

12

C

C

18

13 14

20

17 D 16 15

22

24 23

H C

H3C

(b) 27

26

3

8

CH3

2

HO

CH3

18

19

CH3

25

4

1

A 5

10 6

CH3

11 9

B

8 7

C

12

13

H C

20

16

22

24 23

H C

27

25

CH3

CH3

26

D 17 14

15

7

FIGURE 9.30 The structure of cholesterol, drawn (a) in the traditional planar motif and (b) in a form that more accurately describes the conformation of the ring system.

9.4

How Is Cholesterol Synthesized?

The most prevalent steroid in animal cells is cholesterol (Figure 9.30). Plants contain no cholesterol, but they do contain other steroids very similar in structure to cholesterol (see page 285). Cholesterol serves as a crucial component of cell membranes and as a precursor to bile acids (such as cholate, glycocholate, taurocholate) and steroid hormones (such as testosterone, estradiol, progesterone). Also, vitamin D3 is derived from 7-dehydrocholesterol, the immediate precursor of cholesterol. Liver is the primary site of cholesterol biosynthesis.

Mevalonate Is Synthesized from Acetyl-CoA via HMG-CoA Synthase The cholesterol biosynthetic pathway begins in the cytosol with the synthesis of mevalonate from acetyl-CoA (Figure 9.31). The first step is the !-ketothiolase–catalyzed Claisen condensation of 2 molecules of acetyl-CoA to form acetoacetyl-CoA. In the next reaction, acetylCoA and acetoacetyl-CoA join to form 3-hydroxy-3-methylglutaryl-CoA, which is abbreviated HMG-CoA. The reaction, a second Claisen condensation, is catalyzed by HMG-CoA synthase. The third step in the pathway is the rate-limiting step in cholesterol biosynthesis. Here, HMG-CoA undergoes 2 NADPH-dependent reductions to produce 3R-mevalonate (Figure 9.32). The reaction is catalyzed by HMG-CoA reductase, a 97 kD glycoprotein that spans the ER membrane, with its active site facing the cytosol. As the rate-limiting step, HMG-CoA reductase is the principal site of regulation in cholesterol synthesis. Three different regulatory mechanisms are involved: 1. Phosphorylation by cAMP-dependent protein kinases inactivates the reductase. This inactivation can be reversed by 2 specific phosphatases (Figure 9.33 on page 284). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 9

2. Degradation of HMG-CoA reductase. This enzyme has a half-life of only 3 hours, and the half-life itself depends on cholesterol levels: High [cholesterol] means a short half-life for HMG-CoA reductase. 3. Gene expression. Cholesterol levels control the amount of mRNA. If [cholesterol] is high, levels of mRNA coding for the reductase are reduced. If [cholesterol] is low, more mRNA is made. (Regulation of gene expression is discussed in Chapter 29.)

O CH3

CH3

C

SCoA

Acetyl-CoA

O C

SCoA

Acetyl-CoA

A Thiolase Brainteaser Asks Why Thiolase Cannot Be Used in Fatty Acid Synthesis If acetate units can be condensed by the thiolase reaction to yield acetoacetate in the first step of cholesterol synthesis, why couldn’t this same reaction also be used in fatty acid synthesis, avoiding all the complexity of the fatty acyl synthase? The answer is that the thiolase reaction is more or less reversible but slightly favours the cleavage reaction. In the cholesterol synthesis pathway, subsequent reactions, including HMG-CoA reductase and the following kinase reactions, pull the thiolase-catalyzed condensation forward. However, in the case of fatty acid synthesis, a succession of 8 thiolase condensations would be distinctly unfavourable from an energetic perspective. Given the necessity of repeated reactions in fatty acid synthesis, it makes better energetic sense to use a reaction that is favourable in the desired direction.

283

Lipid Biosynthesis

Thiolase

CoASH

O CH3

C

CH2

C

SCoA

Acetoacetyl-CoA

O CH3

O

C

SCoA

Acetyl-CoA

HMG-CoA synthase

CoASH

Squalene Is Synthesized from Mevalonate The biosynthesis of squalene involves conversion of mevalonate to 2 key 5-carbon intermediates, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, which join to yield farnesyl pyrophosphate and then squalene. A series of 4 reactions convert mevalonate to isopentenyl pyrophosphate and then to dimethylallyl pyrophosphate (Figure 9.34). The first 3 steps each consume an ATP, 2 for the purpose of forming a pyrophosphate at the (a)

–OOC

CH2

3-Hydroxy-3methylglutarylCoA (HMG-CoA)

C

CH2

C

OH

CoA

S H

H

+

CH2

HMG-CoA reductase

CH3 –OOC

CH2

C

H CH2

OH

CH2

Enzyme-bound intermediate

C

C

CoA

S

HMG-CoA reductase (pdb id = 1DQA)

H

OH

H

H

O C

N

NH2

Second NADPH

R CoASH CH3 –OOC

CH2

C

H CH2

OH 3R -Mevalonate

C H

OH

H

FIGURE 9.31 The biosynthesis of 3R-mevalonate from acetyl-CoA.

O CH2

C

3R -Mevalonate

H CH3

SCoA

2 H+

NH2

R

–OOC

C

CoASH

First NADPH

N

C

2 NADP+

O C

CH2

OH

2 NADPH

O

CH3

–OOC

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)

(b)

H+

O

CH3

OH

FIGURE 9.32 (a) A reaction mechanism for HMG-CoA reductase. Two successive NADPH-dependent reductions convert the thioester, HMG-CoA, to a primary alcohol. (b) HMG-CoA reductase structure.

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284

Part 1 Building Blocks of the Cell H3C –OOC

HMG-CoA reductase kinase (inactive)

ATP HMG-CoA reductase kinase kinase ADP

OH CH2OH

C

ATP

Mevalonate kinase

HPO24–

ADP

HMG-CoA reductase kinase phosphatase

HMG-CoA reductase kinase (active)

ATP

Phosphomevalonate kinase

H2O

H 3C –OOC

P

ADP OH

HMG-CoA reductase phosphatase ADP

ADP

+

H2O CO2

P P

CH2O CH2

C

+

P

Isopentenyl pyrophosphate

H2C Isopentenyl pyrophosphate isomerase

H2O

C

H3C

P

P P

CH2O

H3C C

HMG-CoA reductase (inactive)

+

ATP

Pyrophosphomevalonate decarboxylase

HPO24–

5-Pyrophosphomevalonate

CH2

H3C ATP

P P

CH2O

C

CH2

HMG-CoA reductase (active)

Mevalonate

CH2

CH2

Dimethylallyl pyrophosphate

H

Isopentenyl pyrophosphate P P

FIGURE 9.33 HMG-CoA reductase activity is modulated by a cycle of phosphorylation and dephosphorylation.

Isopentenyl pyrophosphate P P H3C H3C H3C

CH2 C

H3C

C

CH2 C

C

CH2

C

CH2

CH2O

P P

C H

H

H

Farnesyl pyrophosphate NADPH

+

H+

NADP+

+

2 P P

Squalene FIGURE 9.34 The conversion of mevalonate to squalene. In the last step, 2 farnesyl-PPs condense to form squalene.

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Chapter 9

Lipid Biosynthesis

285

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

The Long Search for the Route of Cholesterol Biosynthesis Heilbron, Kamm, and Owens suggested as early as 1926 that squalene is a precursor of cholesterol. That same year, H. J. Channon demonstrated that animals fed squalene from shark oil produced more cholesterol in their tissues. Bloch and Rittenberg showed in the 1940s that a significant amount of the carbon in the tetracyclic moiety and in the aliphatic side chain of cholesterol was derived from acetate. In 1934, Sir Robert Robinson suggested a scheme for the cyclization of squalene to form cholesterol before the biosynthetic link between acetate and squalene was understood. Squalene is actually a polymer of isoprene units, and Bonner and Arreguin suggested in 1949 that 3 acetate units could join to form 5-carbon isoprene units (see figure a). In 1952, Konrad Bloch and Robert Langdon showed conclusively that labelled squalene is synthesized rapidly from labelled acetate and also that cholesterol is derived from squalene. Langdon, a graduate student of Bloch’s, performed the critical experiments in Bloch’s laboratory at the University of Chicago while Bloch spent the summer in Bermuda attempting to demonstrate that radioactively labelled squalene would be converted to cholesterol in shark liver. As Bloch himself admitted, “All I was able to learn was that sharks of manageable length are very difficult to catch and their oily livers impossible to slice” (Bloch, 1987). In 1953, Bloch, together with the eminent organic chemist R. B. Woodward, proposed a new scheme (see figure b) for the cyclization of squalene. (Together with Fyodor Lynen, Bloch received the Nobel Prize in Medicine or Physiology in 1964 for his work.) The picture was nearly complete, but 1 crucial question remained: How could isoprene be the intermediate in the transformation of acetate into

squalene? In 1956, Karl Folkers and his colleagues at Merck Sharp & Dohme isolated mevalonic acid and also showed that mevalonate was the precursor of isoprene units. The search for the remaining details (described in the text) made the biosynthesis of cholesterol one of the most enduring and challenging bioorganic problems of the 1940s, 1950s, and 1960s. Even today, several of the enzyme mechanisms are poorly understood. (b)

Squalene

OH

(a)

Lanosterol CH3

CH2

C

C

CH2

(Many steps)

H Isoprene Cholesterol

# (a) An isoprene unit and a scheme for head-to-tail linking of isoprene units. (b) The cyclization of squalene to form lanosterol, as proposed by Bloch and Woodward.

5-position and the third to drive the decarboxylation and double-bond formation in the third step. Pyrophosphomevalonate decarboxylase phosphorylates the 3-hydroxyl group, and this is followed by trans elimination of the phosphate and carboxyl groups to form the double bond in isopentenyl pyrophosphate. Isomerization of the double bond yields the dimethylallyl pyrophosphate. Condensation of these two 5-carbon intermediates produces geranyl pyrophosphate; addition of another 5-carbon isopentenyl group gives farnesyl pyrophosphate. Both steps in the production of farnesyl pyrophosphate occur with release of pyrophosphate, hydrolysis of which drives these reactions forward. Note too that the linkage of isoprene units to form farnesyl pyrophosphate occurs in a head-to-tail fashion. This is the general rule in biosynthesis of molecules involving isoprene linkages. The next step—the joining of 2 farnesyl pyrophosphates to produce squalene—is a “tail-to-tail” condensation and represents an important exception to the general rule. Squalene monooxygenase, an enzyme bound to the ER, converts squalene to squalene2,3-epoxide (Figure 9.35 on page 287). This reaction employs FAD and NADPH as coenzymes and requires O2 as well as a cytosolic protein called soluble protein activator. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

286

Part 1 Building Blocks of the Cell

HUMAN BIOCHEMISTRY

Statins Lower Serum Cholesterol Levels Chemists and biochemists have long sought a means of reducing serum cholesterol levels to reduce the risk of heart attack and cardiovascular disease. Because HMG-CoA reductase is the rate-limiting step in cholesterol biosynthesis, this enzyme is a likely drug target. Mevinolin, also known as lovastatin (see accompanying figure), was isolated from a strain of Aspergillus terreus and developed at Merck Sharp & Dohme for this purpose. It is now a widely prescribed cholesterol-lowering drug. Dramatic reductions of serum cholesterol are observed at dosages of 20–80 mg per day. Lovastatin is administered as an inactive lactone. After oral ingestion, it is hydrolyzed to the active mevinolinic acid, a competitive (a)

O

HO

inhibitor of the reductase with a KI of 0.6 nM. Mevinolinic acid is thought to behave as a transition-state analog (see Chapter 13) of the tetrahedral intermediate formed in the HMG-CoA reductase reaction (see figure). Derivatives of lovastatin have been found to be even more potent in cholesterol-lowering trials. Synvinolin lowers serum cholesterol levels at much lower dosages than lovastatin. Lipitor, shown bound at the active site of HMG-CoA reductase, is the mostprescribed drug in the United States, with annual sales of $9 billion and over $1 billion in Canada.

HO

OH

O O CH3

R

O

H

CH3

O

CH3

H

COO–

CH3

CH3

R

CH3

HO H3C

H O

H

CH3

COO– OH

Mevalonate

HO H3C

COO– OH H SCoA

Tetrahedral intermediate in HMG-CoA reductase mechanism

CH3

1 R=H Mevinolin (Lovastatin, MEVACOR) 2 R=CH3 Synvinolin (Simvastatin, ZOCOR)

(b)

HO

COO–

Mevinolinic acid

(c)

(d)

OH H O NHC

CH3 CH3 CH

CH2

N

F Lipitor (Atorvastatin)

HMG-CoA reductase with NADP+ (magenta), HMG (blue), and CoA (green) (pdb id = 1DQA)

Lipitor (red) bound at the active site of HMG-CoA reductase (pdb id = 1HWK)

# The structures of (a) (inactive) lovastatin, (active) mevinolinic acid, mevalonate, and (b) Lipitor (atorvastatin). (c) HMG-CoA reductase with NADP#, HMG, and CoA. (d) Lipitor bound at the HMG-CoA reductase active site.

A second ER membrane enzyme, 2,3-oxidosqualene lanosterol cyclase, catalyzes the second reaction, which involves a succession of 1,2 shifts of hydride ions and methyl groups.

Conversion of Lanosterol to Cholesterol Requires Twenty Additional Steps Although lanosterol may appear similar to cholesterol in structure, another 20 steps are required to convert lanosterol to cholesterol (Figure 9.35). All the enzymes responsible for this are associated with the ER. The primary pathway involves 7-dehydrocholesterol as the NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 9

Lipid Biosynthesis

287

Squalene Squalene monooxygenase

Squalene-2,3-epoxide

O

H+

2,3-Oxidosqualene: lanosterol cyclase

H+

H3C H3C CH3 HO

Lanosterol H3C

CH3

Many steps (alternative route)

Many steps H3C H3C

H3C H3C HO

Desmosterol

7-Dehydrocholesterol

HO

H3C H3C

Cholesterol

HO O R

C

SCoA

Acyl-CoA cholesterol acyltransferase (ACAT) CoASH

H3C H3C

O R

C

O Cholesterol esters

FIGURE 9.35 Cholesterol is synthesized from squalene via lanosterol. The primary route from lanosterol involves 20 steps, the last of which converts 7-dehydrocholesterol to cholesterol. An alternative route produces desmosterol as the penultimate intermediate.

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288

Part 1 Building Blocks of the Cell

TABLE 9.1 Composition and Properties of Human Lipoproteins Composition (% dry weight) Lipoprotein Class

Density (g/mL)

Diameter (nm)

Protein

Cholesterol

Phospholipid

Triacylglycerol

HDL

1.063–1.21

5–15

33

30

29

8

LDL

1.019–1.063

18–28

25

50

21

4

IDL

1.006–1.019

25–50

18

29

22

31

VLDL

0.95–1.006

30–80

10

22

18

50

[phosphoenolpyruvate] when the pyruvate kinase reaction reaches equilibrium? NEL

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Chapter 17

14. (Integrates with Chapter 3) The standard free energy change (!G"#) for hydrolysis of fructose-1,6-bisphosphate (FBP) to fructose-6-phosphate (F-6-P) and Pi is -16.7 kJ/mol: FBP + H2O h fructose-6-P + Pi. The standard free energy change (!G"#) for ATP hydrolysis is -30.5 kJ/mol: ATP + H2O h ADP + Pi. a. What is the standard free energy change for the phosphofructokinase reaction: ATP + fructose-6-P h ADP + FBP b. What is the equilibrium constant for this reaction? c. Assuming the intracellular concentrations of [ATP] and [ADP] are maintained constant at 4 mM and 1.6 mM respectively in a rat liver cell, what will be the ratio of [FBP]>[fructose-6-P] when the phosphofructokinase reaction reaches equilibrium? 15. (Integrates with Chapter 3) The standard free energy change (!G"#) for hydrolysis of 1,3-bisphosphoglycerate (1,3-BPG) to 3-phosphoglycerate (3-PG) and Pi is -49.6 kJ/mol: 1,3-BPG + H2O h 3-PG + Pi. The standard free energy change (!G"#) for ATP hydrolysis is -30.5 kJ/mol: ATP + H2O h ADP + Pi. a. What is the standard free energy change for the phosphoglycerate kinase reaction: ADP + 1,3-BPG h ATP + 3-PG

Glycolysis

589

16. The standard-state free energy change, !G"#, for the hexokinase reaction is -16.7 kJ/mol. Use the values in Table 17.2 to calculate the value of !G for this reaction in the erythrocyte at 37°C. 17. Taking into consideration the equilibrium constant for the adenylate kinase reaction (page 570), calculate the change in concentration in AMP that would occur if 8% of the ATP in an erythrocyte (red blood cell) were suddenly hydrolyzed to ADP. In addition to the concentration values in Table 17.2, it may be useful to assume that the initial concentration of AMP in erythrocytes is 5 mM. 18. Fructose bisphosphate aldolase in animal muscle is a class I aldolase that forms a Schiff base intermediate between substrate (e.g., fructose-1,6-bisphosphate or dihydroxyacetone phosphate) and a lysine at the active site (see Figure 17.12). The chemical evidence for this intermediate comes from studies with aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of the enzyme with dihydroxyacetone phosphate and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate. Write a mechanism that explains these observations and provides evidence for the formation of a Schiff base intermediate in the aldolase reaction. 19. As noted on page 583, the galactose-1-phosphate uridylyl transferase reaction proceeds via a Ping-Pong mechanism. Consult Chapter 12, page 428, to refresh your knowledge of Ping-Pong mechanisms, and draw a diagram to show how a Ping-Pong mechanism would proceed for the uridylyl transferase. 20. Genetic defects in glycolytic enzymes can have serious consequences for humans. For example, defects in the gene for pyruvate kinase can result in a condition known as hemolytic anemia. Consult a reference to learn about hemolytic anemia, and discuss why such genetic defects lead to this condition.

b. What is the equilibrium constant for this reaction? c. If the steady-state concentrations of [1,3-BPG] and [3-PG] in an erythrocyte are 1 mM and 120 mM respectively, what will be the ratio of [ATP]>[ADP], assuming the phosphoglycerate kinase reaction is at equilibrium?

FURTHER READING General Fothergill-Gilmore, L., 1986. The evolution of the glycolytic pathway. Trends in Biochemical Sciences 11:47–51.

Jeffery, C. J., 2004. Molecular mechanisms for multitasking: Recent crystal structures of moonlighting proteins. Current Opinion in Structural Biology 14:663–668.

Kim, J-W., and Dang, C. V., 2006. Cancer’s molecular sweet tooth and the Warburg effect. Cancer Research 66:8927–8930.

Kim, J-W., and Dang, C. V, 2005. Multifaceted roles of glycolytic enzymes. Trends in Biochemical Sciences 30:142–150.

Sparks, S., 1997. The purpose of glycolysis. Science 277:459–460.

Lee, J. H., Chang, K. Z., et al., 2001. Crystal structure of rabbit phosphoglucose isomerase complexed with its substrate D-fructose 6-phosphate. Biochemistry 40:7799–7805.

Waddell, T. G., 1997. Optimization of glycolysis: A new look at the efficiency of energy coupling. Biochemical Education 25:204–205. Enzymes of Glycolysis Aleshin, A. E., Kirby, C., et al., 2000. Crystal structures of mutant monomeric hexokinase I reveal multiple ADP-binding sites and conformational changes relevant to allosteric regulation. Journal of Molecular Biology 296:1001–1015. Choi, K. H., Shi, J., et al., 2001. Snapshots of catalysis: The structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate. Biochemistry 40:13868–13875. Didierjean, C., Corbier, C., et al., 2003. Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate. Journal of Biological Chemistry 278:12968–12976. Jeffery, C. J., 1999. Moonlighting proteins. Trends in Biochemical Sciences 24:8–11.

Lolis, E., and Petsko, G., 1990. Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5 Å resolution: Implications for catalysis. Biochemistry 29:6619–6625. Schirmer, T., and Evans, P. R., 1999. Structural basis of the allosteric behaviour of phosphofructokinase. Nature 343:140–145. Valentini, G., Chiarelli, L., et al., 2000. The allosteric regulation of pyruvate kinase. Journal of Biological Chemistry 275:18145–18152. Wilson, J. E., 2003. Isozymes of mammalian hexokinase: Structure, subcellular localization and metabolic function. Journal of Experimental Biology 206:2049–2057. Zhang, E., Brewer, J. M., et al., 1997. Mechanism of enolase: The crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/ enolase-phosphoenolpyruvate at 2.0 Å resolution. Biochemistry 36: 12526–12534.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

590

Part 3 Metabolism and Its Regulation

Muscle Biochemistry Green, H. J., 1997. Mechanisms of muscle fatigue in intense exercise. Journal of Sports Sciences 15:247–256. HIF-1A and Glycolysis Cramer, T., Yamanishi, Y., et al., 2003. HIF-1a is essential for myeloid cell-mediated inflammation. Cell 112:645–657.

Melillo, G., 2006. Inhibiting hypoxia-inducible factor 1 for cancer therapy. Molecular Cancer Research 4:601–605. Melillo, G., 2007. Targeting hypoxia cell signaling for cancer therapy. Cancer and Metastasis Reviews 26:341–352. North, S., Moenner, M., et al., 2005. Recent developments in the regulation of the angiogenic switch by cellular stress factors in tumors. Cancer Letters 218:1–14.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

18

The Tricarboxylic Acid Cycle

! A time-lapse photograph of a Ferris wheel

at night. Aerobic cells use a metabolic wheel— the tricarboxylic acid cycle—to generate energy by acetyl-CoA oxidation.

Thus times do shift, each thing his turn does hold; New things succeed, as former things grow old. Robert Herrick

© Richard Cummins/Corbis

Hesperides (1648), “Ceremonies for Christmas Eve”

ESSENTIAL QUESTION The glycolytic pathway converts glucose to pyruvate and produces 2 molecules of ATP per glucose—only a small fraction of the potential energy available from glucose. Under anaerobic conditions, pyruvate is reduced to lactate in animals and to ethanol in yeast, and much of the potential energy of the glucose molecule remains untapped. In the presence of oxygen, however, a much more interesting and thermodynamically complete story unfolds. How is pyruvate oxidized under aerobic conditions, and what is the chemical logic that dictates how this process occurs?

WHY IT MATTERS Before we begin our discussion of the tricarboxylic acid (TCA) cycle, we pose the question: Why does this cycle matter? As we will see in the upcoming chapter, the TCA cycle is an important series of cyclic reactions employed by aerobic organisms to supply their metabolic needs. It will clearly become evident later in the chapter (see Figure 18.16, page 610) that the TCA cycle is a complex system that provides a juncture for numerous biosynthetic pathways, in addition to indirectly producing ATP as a result of NADH and FADH2 generation and their subsequent entry into the electron transport chain. The discovery of the TCA cycle by the German-born biochemist Sir Hans Krebs (1900– 1981) provided a greater understanding of metabolism in cellular organisms. Not only did Krebs receive the 1953 Nobel Prize in Physiology/Medicine for the TCA cycle, he was also instrumental in identifying and reporting the series of chemical reactions that accompany the urea cycle (see Chapter 23).* He was not only a physician, but also a biochemist, a researcher and a professor. Following his knighthood in 1958, Krebs received the Copley Medal from the Royal Society of London (1961), which recognizes outstanding accomplishments in the basic sciences. In his honour, the TCA cycle is often referred to today as the Krebs cycle. *Krebs, H. A., 1953. The citric acid cycle. The Nobel Lecture Transcript.

KEY QUESTIONS 18.1

What Is the Chemical Logic of the TCA Cycle?

18.2

How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA?

18.3

How Are 2 CO2 Molecules Produced from Acetyl-CoA?

18.4

How Is Oxaloacetate Regenerated to Complete the TCA Cycle?

18.5

What Are the Energetic Consequences of the TCA Cycle?

18.6

Can the TCA Cycle Provide Intermediates for Biosynthesis?

18.7

What Are the Anaplerotic, or “Filling-Up,” Reactions?

18.8

How Is the TCA Cycle Regulated?

18.9

Can Any Organisms Use Acetate as Their Sole Carbon Source?

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

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Part 3 Metabolism and Its Regulation

Under aerobic conditions, pyruvate from glycolysis is converted to acetyl-coenzyme A (acetyl-CoA) and oxidized to CO2 in the TCA cycle (also called the citric acid cycle). The electrons liberated by this oxidative process are passed via NADH and FADH2 through an elaborate, membrane-associated electron transport pathway to O2, the final electron acceptor. Electron transfer is coupled to creation of a proton gradient across the membrane. Such a gradient represents an energized state, and the energy stored in this gradient is used to drive the synthesis of many equivalents of ATP. ATP synthesis as a consequence of electron transport is termed oxidative phosphorylation; the complete process is diagrammed in Figure 18.1. Aerobic pathways permit the production of 30–38 molecules of ATP per glucose oxidized. Although 2 molecules of ATP come from glycolysis and 2 more directly out of the TCA cycle, most of the ATP arises from oxidative phosphorylation. Specifically, reducing equivalents released in the oxidative reactions of glycolysis, pyruvate decarboxylation, and the TCA cycle are captured in the form of NADH and enzyme-bound FADH2, and these reduced coenzymes fuel the electron transport pathway and oxidative phosphorylation. Complete oxidation of glucose to CO2 involves the removal of 24 electrons—that is, it is a 24-electron oxidation. In glycolysis, 4 electrons are removed as NADH, and 4 more exit as 2 more NADH in the decarboxylation of 2 molecules of pyruvate to 2 acetyl-CoA (Figure 18.1). For each acetyl-CoA oxidized in the TCA cycle, 8 more electrons are removed (as 3 NADH and 1 FADH2):

Science Source/Photo Researchers, Inc.

592

" Sir Hans Krebs

H3CCOO! " 2H2O " H" h 2CO2 " 8H

In the electron transport pathway, these 8 electrons combine with oxygen to form water: 8H " 2O2 h 4H2O

So the net reaction for the TCA cycle and electron transport pathway is H3CCOO! " 2O2 " H" h 2CO2 " 2H2O

Glycolysis

(a)

ADP

FIGURE 18.1 (a) Pyruvate produced in glycolysis is oxidized in (b) the tricarboxylic acid (TCA) cycle. (c) Electrons liberated in this oxidation flow through the electron-transport chain and drive the synthesis of ATP in oxidative phosphorylation. In eukaryotic cells, this overall process occurs in mitochondria.

Glucose

+ Pi

NAD+

ATP

NADH

Pyruvate NAD+ NADH

Acetyl-CoA

(c)

(b)

Oxidative phosphorylation

Electron transport

Intermembrane space

Malate

e

Proton gradient

ate

ate

et

ac

Citr

lo xa

O

H+

e

etog

e–

rate

NADH

+

H

AD

N

Pi

H+

H+ H+

e–

oA

GDP

H+

e–

e–

luta

yl-C

Succ inat

!-K

H+ H+

NADH

cin

GTP

H+

cit

Suc

m Fu

H+

e rat

Iso

Citric acid cycle

t ara

H+

[FADH2] NADH

H2

D FA

Mitochondrial matrix

O2 H2O

ADP

+

ATP

Pi

H+

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Chapter 18

The Tricarboxylic Acid Cycle

As Krebs showed in the 1930s, the 8-electron oxidation of acetate by the TCA cycle is accomplished with the help of oxaloacetate. Beginning with acetate, a series of 5 reactions produces 2 molecules of CO2, with 4 electrons extracted in the form of NADH and 4 electrons passed to oxaloacetate to produce a molecule of succinate. The pathway becomes a cycle by 3 additional reactions that accomplish a 4-electron oxidation of succinate back to oxaloacetate. This special trio of reactions is used repeatedly in metabolism: first, oxidation of a single bond to a double bond, then addition of the elements of water across the double bond, and finally oxidation of the resulting alcohol to a carbonyl. We will see it again in fatty acid oxidation (see Chapter 22) and in amino acid synthesis and breakdown (see Chapter 25). We saw it in reverse in fatty acid synthesis (see Chapter 9).

18.1

What Is the Chemical Logic of the TCA Cycle?

The entry of new carbon units into the cycle is through acetyl-CoA. This entry metabolite can be formed either from pyruvate (from glycolysis) or from oxidation of fatty acids (discussed in Chapter 22). Transfer of the 2-carbon acetyl group from acetyl-CoA to the 4-carbon oxaloacetate to yield 6-carbon citrate is catalyzed by citrate synthase. A dehydration–rehydration rearrangement of citrate yields isocitrate. Two successive decarboxylations produce !-ketoglutarate and then succinyl-CoA, a CoA conjugate of a 4-carbon unit. Several steps later, oxaloacetate is regenerated and can combine with another 2-carbon unit of acetyl-CoA. Thus, carbon enters the cycle as acetyl-CoA and exits as CO2. In the process, metabolic energy is captured in the form of ATP, NADH, and enzyme-bound FADH2 (symbolized as [FADH2]).

The TCA Cycle Provides a Chemically Feasible Way of Cleaving a 2-Carbon Compound The cycle shown in Figure 18.2 at first appears to be a complicated way to oxidize acetate units to CO2, but there is a chemical basis for the apparent complexity. Oxidation of an acetyl group to a pair of CO2 molecules requires C—C cleavage: CH3COO- h CO2 + CO2 In many instances, COC cleavage reactions in biological systems occur between carbon atoms ! and " and a carbonyl group:

O C

C#

C$

Cleavage

A good example of such a cleavage is the fructose bisphosphate aldolase reaction (see Figure 17.12). Another common type of COC cleavage is !-cleavage of an !-hydroxyketone:

O

OH

C

C#

Cleavage

(We see this type of cleavage in the transketolase reaction described in Chapter 21.) Neither of these cleavage strategies is suitable for acetate. It has no "-carbon, and the second method would require hydroxylation—not a favourable reaction for acetate. Instead, living things have evolved the clever chemistry of condensing acetate with oxaloacetate and then carrying out a "-cleavage. The TCA cycle combines this "-cleavage reaction with oxidation to form CO2, regenerate oxaloacetate, and capture the liberated metabolic energy in NADH and ATP. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

593

594

Part 3 Metabolism and Its Regulation O From glycolysis

H3C

O

C

C

O–

Pyruvate NAD+

CoASH

Pyruvate dehydrogenase NADH

+

H+

CO2 O

H3C

C

From "-oxidation of fatty acids

CoA

S

Acetyl-CoA O Malate dehydrogenase

C

COO–

H2C

COO–

8

Oxaloacetate

Citrate synthase

CoASH

1 H2O

HO C

COO–

H2C

COO–

H

NAD+

NADH

+ H+

H2C

COO–

C

COO–

H2C

COO–

HO

Malate Fumarase

7

Citrate 2

H2O

Aconitase

COO–

H C

TRICARBOXYLIC ACID CYCLE (citric acid cycle, Krebs cycle, TCA cycle)

C –OOC

H

Fumarate FADH2

Succinate dehydrogenase

H2C

COO–

Succinate

NADH

NADH

Succinyl-CoA synthetase 5

GTP Nucleoside ADP diphosphate kinase

+ H+

CoASH

NAD+

Pi

GDP

H2C

ATP

+

3 H+

H2C

COO–

HC

COO–

COO–

Isocitrate dehydrogenase

CO2

H2C

4

COO–

C

COO–

O

H2C C

HC

Isocitrate

NAD+

FAD COO–

COO–

OH

6

H2C

H2C

SCoA

O Succinyl-CoA

!-Ketoglutarate !-Ketoglutarate dehydrogenase CO2

FIGURE 18.2 The TCA cycle.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

18.2

The Tricarboxylic Acid Cycle

595

How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA?

Pyruvate produced by glycolysis is a significant source of acetyl-CoA for the TCA cycle. Because glycolysis occurs in the cytoplasm in eukaryotic cells, whereas the TCA cycle reactions and all subsequent steps of aerobic metabolism take place in the mitochondria, pyruvate must first enter the mitochondria to enter the TCA cycle. The oxidative decarboxylation of pyruvate to acetyl-CoA, Pyruvate + CoA + NAD + h acetyl-CoA + Co2 + NADH is the connecting link between glycolysis and the TCA cycle. The reaction is catalyzed by pyruvate dehydrogenase, a multienzyme complex. The pyruvate dehydrogenase complex (PDC) is formed from multiple copies of 3 enzymes: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). All are involved in the conversion of pyruvate to acetylCoA. The active sites of all 3 enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme, and so on, without diffusion of substrates and products through the solution. Eukaryotic PDC, one of the largest-known multienzyme complexes (with a diameter of approximately 500 Å) is a 9.5-megadalton assembly organized around an icosahedral 60-mer of E2 subunits, with 30 E1 heterotetramers and 12 homodimers of E3 (Figure 18.3). Eukaryotic PDC also contains an E3-binding protein (E3BP) that is required to bind E3 to the PDC. Trimeric units of E2 form the 20 vertices of the icosahedron, with E3BP bound in each of the 12 faces. The E2 subunits each carry a lipoic acid moiety covalently linked to a lysine residue. Flexible linker segments in E2 and E3BP impart the flexibility that allows the lipoic acid groups to visit all 3 active sites during catalysis. The pyruvate dehydrogenase reaction (Figure 18.4) is a tour de force of mechanistic chemistry, involving as it does a total of 3 enzymes and 5 different coenzymes. The first step of this reaction, decarboxylation of pyruvate and transfer of the acetyl group to lipoic acid, depends on accumulation of negative charge on the transferred 2-carbon fragment, as facilitated by the quaternary nitrogen on the thiazolium group of thiamine pyrophosphate (TPP). As shown in Figure 18.5, this cationic imine nitrogen plays 2 distinct roles in TPP-catalyzed reactions: 1. It provides electrostatic stabilization of the thiazole carbanion formed upon removal of the C-2 proton. (The sp2 hybridization and the availability of vacant d orbitals on the adjacent sulfur probably also facilitate proton removal at C-2.) 2. TPP attack on pyruvate leads to decarboxylation. The TPP cationic imine nitrogen can act as an effective electron sink to stabilize the negative charge that must develop on the carbon that has been attacked. This stabilization takes place by resonance interaction through the double bond to the nitrogen atom. This resonance-stabilized intermediate can be protonated to give hydroxyethyl-TPP. The reaction of hydroxyethyl-TPP with the oxidized form of lipoic acid yields the energy-rich acetyl-thiol ester of reduced lipoic acid through oxidation of the hydroxyl-carbon of the

FIGURE 18.3 Icosahedral model of PDC core structure (E3 not shown). E1 subunits (yellow) are joined to the E2 core (green) by linkers (blue). (Adapted from Zhou, Z. H., McCarthy, D. B., O’Connor, C. M., Reed, L. J., and Stoops, J. K., 2001. The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes. Proc Natl Acad Sci U S A 98:14802–14807. Copyright 2011 National Academy of Sciences, U.S.A.)

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596

Part 3 Metabolism and Its Regulation 1

Pyruvate loses CO2 and HETPP is formed

2

3

Hydroxyethyl group is transferred to lipoic acid and oxidized to form acetyl dihydrolipoate

O

O CH3

C

Acetyl group is transferred to CoA O CoASH CH3C SCoA

CH3

COO–

3

S

Thiamine pyrophosphate

Pyruvate

4

C

H S

SH

1

Lipoic acid is reoxidized NAD+

SH 4

2

[FAD]

Protein CO2

NADH

CH3 CH

+

H+

OH

TPP Hydroxyethyl TPP (HETPP)

S S Lipoic acid

Pyruvate dehydrogenase

Dihydrolipoyl transacetylase (dihydrolipoamide acetyltransferase)

Dihydrolipoyl dehydrogenase (dihydrolipoamide dehydrogenase)

FIGURE 18.4 The reaction mechanism of the pyruvate dehydrogenase complex. Decarboxylation of pyruvate occurs with formation of hydroxyethyl-TPP (step 1). Transfer of the 2-carbon unit to lipoic acid in step 2 is followed by formation of acetyl-CoA in step 3. Lipoic acid is reoxidized in step 4 of the reaction.

R R'

B

S + N

E

S

R

H

CH3

–

+ N

R'

R"

O ....H B

C

R E

R'

COO–

R"

CH3

S

C

+ N R"

Pyruvate

OH

C O–

O

CO2 CH3 O

C N

S

R

H S

S

H:B

C N

R'

R

OH R'

:B

H

O

S

SH

H

:B

–

N Lipoamide

CoA

:

N

H+

CH3

C

S

H

C

–

OH

CH3

S

C N

R'

OH

R"

Resonance-stabilized carbanion on substrate

Hydroxyethyl-TPP

CH3

+ N

R

R"

R" Lipoamide

CH3

S

:

–

H+

CH3

C

O

S

SH

H+

O CoA

S

C

CH3

+ Lipoamide

S

_

SH SH SH

Lipoamide Lipoamide

FIGURE 18.5 The mechanistic details of the first 3 steps of the pyruvate dehydrogenase complex reaction. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

597

The Tricarboxylic Acid Cycle

A DEEPER LOOK

The Coenzymes of the Pyruvate Dehydrogenase Complex Coenzymes are small molecules that bring unique chemistry to enzyme reactions. Five coenzymes are used in the pyruvate dehydrogenase reaction.

Thiamine Pyrophosphate TPP assists in the decarboxylation of !-keto acids (here) and in the formation and cleavage of !-hydroxy ketones (as in the transketolase reaction; see Chapter 21).

O– NH2 N H3C

N

H C H

H3C H C H

N +

S

H C H

OH

H3C

NH2

+

ATP

H C H

TPP Synthetase N

H

H3C

AMP

H C H

N +

H C H

O

O–

P

O

P

O

S

O–

O

H Acidic proton

N

Thiamine (vitamin B1)

Thiamine pyrophosphate (TPP)

The Nicotinamide Coenzymes NAD+/NADH and NADP+/NADPH carry out hydride (H:–) transfer reactions. All reactions involving these coenzymes are 2-electron transfers.

Nicotinamide (oxidized form)

Nicotinamide (reduced form) pro-R

O

H Nicotinamide adenine dinucleotide, NAD+

4

C

5 6

NH2

Hydride ion, H–

H

C

NH2

N+

2

N

...

–O

O

CH2

O

H

P

O

H

O

H H

OH OH O

NH2

N

N

P O

CH2 H

AMP

O H

3

1

–O

pro-S

O

N

N

H H

H OH OH

NADP+ contains a Pi on this 2%-hydroxyl (Continued)

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

598

Part 3 Metabolism and Its Regulation

A DEEPER LOOK

The Coenzymes of the Pyruvate Dehydrogenase Complex (Continued)

The Flavin Coenzymes—FAD/FADH2 Flavin coenzymes can exist in any of 3 redox states, allowing flavin coenzymes to participate in 1-electron transfer and 2-electron transfer reactions. Partly because of this, flavoproteins catalyze many reactions in biological systems and work with many electron acceptors and donors. Because the ribityl group is not a true pentose sugar (it is a sugar alcohol) and is not joined to riboflavin in a glycosidic bond, the molecule is not truly a “nucleotide,” and the terms flavin mononucleotide and dinucleotide are incorrect. Nonetheless, these designations are so deeply ingrained in common biochemical usage that the erroneous nomenclature persists.

(a)

N

H3C

N

H

N

Isoalloxazine

O

N

CH2

Riboflavin

Flavin mononucleotide, FMN

Flavin adenine dinucleotide, FAD

O H3C

HCOH HCOH D-Ribitol

HCOH CH2 O O

P

O– NH2

O O

P

N

O–

O

CH2 H

H

N

O H

N N

AMP

H

OH OH

(b) R Oxidized form λ max = 450 nm (yellow)

10

9

H3C

9a

N

1

10a

N

O

8

H3C

H– + H+

H3C

R

H

N

N

O

2

7

5a

6

N

NH

4a

5

3

4

H– + H+

O

H3C

FAD or FMN

H

O

FADH2 or FMNH2

H+, e–

H+, e–

H+, e–

N H

Reduced form (colourless)

N

H+, e– R H3C Semiquinone form λ max = 570 nm (blue)

N

R N

H3C

O

N

N

O Semiquinone anion

H3C

NH

N H

O

pKa ≅ 8.4

H3C

N –

λ max = 490 nm

N O

H

(red)

FADH or FMNH

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

Coenzyme A The two main functions of CoA are as follows:

599

The Tricarboxylic Acid Cycle

SH CH2

1. activation of acyl groups for transfer by nucleophilic attack 2. activation of the !-hydrogen of the acyl group for abstraction as a proton

β -Mercaptoethylamine

CH2 NH 4-Phosphopantetheine

The reactive sulfhydryl group on CoA mediates both of these functions. The sulfhydryl group forms thioester linkages with acyl groups. The two main functions of CoA are illustrated in the citrate synthase reaction (see Figure 18.6).

C

O

CH2 CH2 NH C

Pantothenic acid

O

HCOH H3C

C

CH3

CH2 O –O

P

O

O –O

P

NH2

O N

O

N

CH2

N N

O H

H 3'

O

H

H

OH

PO32– 3!,5!–ADP

Lipoic Acid Lipoic acid functions to couple acyl group transfer and electron transfer during oxidation and decarboxylation of !-keto acids. It is found in pyruvate dehydrogenase and !-ketoglutarate dehydrogenase. Lipoic acid is covalently bound to relevant enzymes through amide bond formation with the #-NH2 group of a lysine side chain.

(a)

(c)

(b) S

H2 C

S CHCH2CH2CH2CH2C CH2 Lipoic acid, oxidized form

O O–

HS H2 C

HS CHCH2CH2CH2CH2C CH2

O

S

H

S

HN

N

O–

CH O

Reduced form Lipoic acid

C Lysine

Lipoyllysine (lipoamide)

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

O

600

Part 3 Metabolism and Its Regulation

2-carbon substrate unit. Nucleophilic attack by CoA on the carbonyl carbon (a characteristic feature of CoA chemistry) results in transfer of the acetyl group from lipoic acid to CoA. The subsequent oxidation of lipoic acid is catalyzed by the FAD-dependent dihydrolipoyl dehydrogenase, and NAD+ is reduced.

18.3

How Are 2 CO2 Molecules Produced from Acetyl-CoA?

The Citrate Synthase Reaction Initiates the TCA Cycle The first reaction within the TCA cycle, the one by which carbon atoms are introduced, is the citrate synthase reaction (Figure 18.6). Here acetyl-CoA reacts with oxaloacetate in a Perkin condensation (a carbon–carbon condensation between a ketone or an aldehyde and an ester). The acyl group is activated in 2 ways in an acyl-CoA molecule: The carbonyl carbon is activated for attack by nucleophiles, and the C! carbon is more acidic and can be deprotonated to form a carbanion. The citrate synthase reaction depends upon the latter mode of activation. As shown in Figure 18.6, a general base on the enzyme accepts a proton from the methyl group of acetyl-CoA, producing a stabilized !-carbanion of acetylCoA. This strong nucleophile attacks the !-carbonyl of oxaloacetate, yielding citryl-CoA. This part of the reaction has an equilibrium constant near 1, but the overall reaction is driven to completion by the subsequent hydrolysis of the high-energy thioester to citrate and free CoA. The overall ∆G°% is −31.4 kJ/mol, and under standard conditions the reaction is essentially irreversible. Although the mitochondrial concentration of oxaloacetate is very low (much less than 1 μM—see example in Section 18.4), the strong negative ∆G°% drives the reaction forward. Citrate Synthase Is a Dimer Citrate synthase in mammals is a dimer of 49-kD subunits (Table 18.1). On each subunit, oxaloacetate and acetyl-CoA bind to the active site, which lies in a cleft between 2 domains and is surrounded mainly by !-helical segments (Figure 18.7). Binding of oxaloacetate induces a conformational change that facilitates the binding of acetyl-CoA and closes the active site so that the reactive carbanion of acetylCoA is protected from protonation by water. NADH Is an Allosteric Inhibitor of Citrate Synthase Citrate synthase is the first step in this metabolic pathway, and as stated the reaction has a large negative ∆G°%. As might be expected, it is a highly regulated enzyme. NADH, a product of the TCA cycle, is an allosteric inhibitor of citrate synthase, as is succinyl-CoA, the product of the fifth step in the cycle (and an acetyl-CoA analog). Citrate Is Isomerized by Aconitase to Form Isocitrate Citrate itself poses a problem: It is a poor candidate for further oxidation because it contains a tertiary alcohol, which could be oxidized only by breaking a carbon–carbon bond. An obvious solution to this problem is to isomerize the tertiary alcohol to a secondary alcohol, which the cycle proceeds to do in the next step.

H

H

C E E

B + B H

O C

O SCoA

H

H2C HO

O C H2C

COO– COO–

C

pro-S arm SCoA

C

COO–

H2C

COO–

Citryl-CoA

H2O

CoA HO

H2C

COO–

C

COO–

H2C

COO–

Citrate

pro-R arm

Oxaloacetate FIGURE 18.6 Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA. The mechanism involves nucleophilic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

The Tricarboxylic Acid Cycle

601

TABLE 18.1 The Enzymes and Reactions of the TCA Cycle Reaction

Enzyme

& G° % (kJ/mol)

&G (kJ/mol)

-31.4

-53.9

1. Acetyl-CoA + oxaloacetate + H2O m CoASH + citrate

Citrate synthase

2. Citrate m isocitrate

Aconitase

+6.7

+0.8

3. Isocitrate + NAD + m a-ketoglutarate + NADH + CO2

Isocitrate dehydrogenase

-8.4

-17.5

4. a-ketoglutarate + CoASH + NAD + m succinyl-CoA + NADH + CO2

!-ketoglutarate dehydrogenase complex

5. Succinyl-CoA + GDP + Pi m succinate + GTP + CoASH

Succinyl-CoA synthetase

-3.3

!0

6. Succinate + [FAD] m fumarate + [FADH2]

Succinate dehydrogenase

+0.4

!0

7. Fumarate + H2O m L-malate

Fumarase

-3.8

!0

+29.7

!0

8. L-malate +

NAD +

m oxaloacetate + NADH +

H+

malate dehydrogenase

-30

-43.9

&G values from Newsholme, E. A., and Leech, A. R., 1983. Biochemistry for the Medical Sciences. New York: Wiley.

Citrate is isomerized to isocitrate by aconitase in a 2-step process involving aconitate as an intermediate (Figure 18.8). In this reaction, the elements of water are first abstracted from citrate to yield aconitate, which is then rehydrated with H— and HO— adding back in opposite positions to produce isocitrate. The net effect is the conversion of a tertiary alcohol (citrate) to a secondary alcohol (isocitrate). Oxidation of the secondary alcohol of isocitrate involves breakage of a COH bond, a simpler matter than the C—C cleavage required for the direct oxidation of citrate. Inspection of the citrate structure shows a total of 4 chemically equivalent hydrogens, but only 1 of these—the pro-R H atom of the pro-R arm of citrate—is abstracted by aconitase, which is quite stereospecific. Formation of the double bond of aconitate following proton abstraction requires departure of hydroxide ion from the C-3 position. Hydroxide is a relatively poor leaving group, and its departure is facilitated in the aconitase reaction by coordination with an iron atom in an iron–sulfur cluster. Aconitase Utilizes an Iron–Sulfur Cluster Aconitase contains an iron–sulfur cluster consisting of 3 iron atoms and 4 sulfur atoms in a near-cubic arrangement (Figure 18.9).

CoASH

(a)

HO E

B

H2C

COO–

C

COO–

HR C

HS

COO–

pro-S arm

pro-R arm

Citrate

H2O

H2O

H2C

COO–

C

COO–

HC

COO–

H2O

cis-Aconitate

COO–

C

COO–

S

H H2O

H2C

HC R COO– OH Isocitrate

Aconitase removes the pro-R H of the pro-R arm of citrate (b)

FIGURE 18.7 Citrate synthase. In the monomer shown here, citrate is shown in blue, and CoA is red. (Top: pdb id = 1CTS; bottom: pdb id = 2CTS.) FIGURE 18.8 (a) The aconitase reaction converts citrate to cis-aconitate and then to isocitrate. Aconitase is stereospecific and removes the pro-R hydrogen from the pro-R arm of citrate. (b) The active site of aconitase. The iron–sulfur cluster (pink) is coordinated by cysteines (orange) and isocitrate (purple) (pdb id = 1B0J). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

602

Part 3 Metabolism and Its Regulation OH2 Cys

S

S Fe

O S – H O

Cys

Fe S

Cys

COO–

C

Fe S

Fe S S

O

O

H

H

O

S

S

Fe

Fe

Cys

S S

B

OH

–OOC

+

S S

Fe

C

C

Cys

CH2

C

COO–

OH2

Fe

–OOC

S

C C

CH2

H

Aconitate

Cys

Citrate FIGURE 18.9 The iron–sulfur cluster of aconitase. Binding of Fe2" to the vacant position of the cluster activates aconitase. The added iron atom coordinates the C-3 carboxyl and hydroxyl groups of citrate and acts as a Lewis acid, accepting an electron pair from the hydroxyl group and making it a better leaving group.

Cysteine residues from the enzyme coordinate the 3 iron atoms. In the inactive state of the enzyme, 1 corner of the cube is vacant. Binding of iron (as Fe2+) to this position activates aconitase. The iron atom in this position can coordinate the C-3 carboxyl and hydroxyl groups of citrate. This iron atom thus acts as a Lewis acid, accepting an unshared pair of electrons from the hydroxyl, making it a better leaving group. The equilibrium for the aconitase reaction favours citrate, and an equilibrium mixture typically contains about 90% citrate, 4% cis-aconitate, and 6% isocitrate. The ∆G°% is +6.7 kJ/mol. Fluoroacetate Blocks the TCA Cycle Fluoroacetate is an extremely poisonous agent that blocks the TCA cycle in vivo, although it has no apparent effect on any of the isolated enzymes. Its LD50, the lethal dose for 50% of animals consuming it, is 0.2 mg per kilogram of body weight; it has been used as a rodent poison. The action of fluoroacetate has been traced to aconitase, which is inhibited in vivo by fluorocitrate, which is formed from fluoroacetate in 2 steps: F

FCH2COO– Fluoroacetate

Acetyl-CoA synthetase

O FCH2

C

SCoA

Fluoroacetyl-CoA

Citrate synthase

H

C

COO–

HO

C

COO–

H2C

COO–

(2R, 3S )-Fluorocitrate

Fluoroacetate readily crosses both the cellular and the mitochondrial membranes, and in mitochondria it is converted to fluoroacetyl-CoA by acetyl-CoA synthetase. Fluoroacetyl-CoA is a substrate for citrate synthase, which condenses it with oxaloacetate to form fluorocitrate. Fluoroacetate may thus be viewed as a trojan horse inhibitor. Analogous to the giant Trojan horse of legend—which the soldiers of Troy took into their city, not knowing that Greek soldiers were hidden inside it and waiting to attack—fluoroacetate enters the TCA cycle innocently enough, in the citrate synthase reaction. Citrate synthase converts fluoroacetate to inhibitory fluorocitrate for its TCA cycle partner, aconitase, blocking the cycle.

Isocitrate Dehydrogenase Catalyzes the First Oxidative Decarboxylation in the Cycle In the next step of the TCA cycle, isocitrate is oxidatively decarboxylated to yield !-ketoglutarate, with concomitant reduction of NAD" to NADH in the isocitrate dehydrogenase reaction (Figure 18.10). The reaction has a net ∆G°% of −8.4 kJ/mol, and it is sufficiently exergonic to pull the aconitase reaction forward. This 2-step reaction involves (1) oxidation of the C-2 alcohol of isocitrate to form oxalosuccinate, followed by (2) a "-decarboxylation reaction that expels the central carboxyl group as CO2, leaving the product !-ketoglutarate. Oxalosuccinate, the "-keto acid produced by the initial dehydrogenation reaction, is unstable and thus readily decarboxylated. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18 H2C

COO–

H

C

COO–

H

C

COO–

(a)

The Tricarboxylic Acid Cycle

OH NAD+ Isocitrate dehydrogenase (b)

NADH + H+ H2C

C OO– CO2

O C

H

C

C

H2C

O– H+

COO–

H2C C

C OO–

O

O Oxalosuccinate

FIGURE 18.10 (a) The isocitrate dehydrogenase reaction. (b) The active site of isocitrate dehydrogenase. Isocitrate is shown in blue, NADP+ (as an NAD" analog) in gold, and Ca2" in red (pdb id = 1AI2).

COO–

!-Ketoglutarate

Isocitrate Dehydrogenase Links the TCA Cycle and Electron Transport Isocitrate dehydrogenase provides the first connection between the TCA cycle and the electrontransport pathway and oxidative phosphorylation, via its production of NADH. As a connecting point between 2 metabolic pathways, isocitrate dehydrogenase is a regulated reaction. NADH and ATP are allosteric inhibitors, whereas ADP acts as an allosteric activator, lowering the Km for isocitrate by a factor of 10. The enzyme is virtually inactive in the absence of ADP. Also, the product, !-ketoglutarate, is a crucial !-keto acid for aminotransferase reactions (see Chapters 12 and 23), connecting the TCA cycle (i.e., carbon metabolism) with nitrogen metabolism.

!-Ketoglutarate Dehydrogenase Catalyzes the Second Oxidative Decarboxylation of the TCA Cycle A second oxidative decarboxylation occurs in the !-ketoglutarate dehydrogenase reaction: H2C

COO–

NAD+

CoASH

NADH

H2C C

CO2

H2C

COO–

H2C COO–

O !-Ketoglutarate

!-Ketoglutarate dehydrogenase

C

SCoA

O Succinyl-CoA

Like the pyruvate dehydrogenase complex, !-ketoglutarate dehydrogenase is a multienzyme complex—consisting of !-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase—that employs 5 different coenzymes (Table 18.2). The dihydrolipoyl dehydrogenase in this reaction is identical to that in the pyruvate dehydrogenase reaction. The mechanism is analogous to that of pyruvate dehydrogenase. As with the pyruvate dehydrogenase reaction, this reaction produces NADH and a thioester NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 3 Metabolism and Its Regulation

TABLE 18.2 Composition of the !-Ketoglutarate Dehydrogenase Complex from Escherichia coli Enzyme

Enzyme Mr

Coenzyme

Number of Subunits

Subunit Mr

Number of Subunits per Complex

!-ketoglutarate dehydrogenase

Thiamine pyrophosphate

192 000

2

96 000

24

Dihydrolipoyl transsuccinylase

Lipoic acid, CoASH

1 700 000

24

70 000

24

Dihydrolipoyl dehydrogenase

NAD+

112 000

2

56 000

12

FAD,

product—in this case, succinyl-CoA. Succinyl-CoA and NADH products are energy-rich species that are important sources of metabolic energy in subsequent cellular processes.

18.4 Condensation: A reaction between 2 or more molecules that results in formation of a larger molecule, with elimination of some simpler molecule, such as water (as in dehydration synthesis). Synthase: A condensation reaction that does not require a nucleoside triphosphate as an energy source. Synthetase: A condensation reaction that requires a nucleoside triphosphate (often ATP) as an energy source.

How Is Oxaloacetate Regenerated to Complete the TCA Cycle?

Succinyl-CoA Synthetase Catalyzes Substrate Level Phosphorylation The NADH produced in the foregoing steps can be routed through the electron transport pathway to make high-energy phosphates via oxidative phosphorylation. However, succinyl-CoA is itself a high-energy intermediate and is utilized in the next step of the TCA cycle to drive the phosphorylation of GDP to GTP (in mammals) or ADP to ATP (in plants and bacteria): H2C

COO–

GDP

+

Pi

GTP

+ CoASH

H2C C

SCoA

O Succinyl-CoA

Succinyl-CoA synthetase

H2C

COO–

H2C

COO–

Succinate

The reaction is catalyzed by succinyl-CoA synthetase, sometimes called succinate thiokinase. The free energies of hydrolysis of succinyl-CoA and GTP or ATP are similar, and the net reaction has a ∆G°% of −3.3 kJ/mol. Succinyl-CoA synthetase provides another example of a substrate level phosphorylation (see Chapter 17), in which a substrate, rather than an electron transport chain or proton gradient, provides the energy for phosphorylation. It is the only such reaction in the TCA cycle. The GTP produced by mammals in this reaction can exchange its terminal phosphoryl group with ADP via the nucleoside diphosphate kinase reaction:

The Mechanism of Succinyl-CoA Synthetase Involves a Phosphohistidine The mechanism of succinyl-CoA synthetase is postulated to involve displacement of CoA by phosphate, forming succinyl phosphate at the active site, followed by transfer of the phosphoryl group to an active site histidine (making a phosphohistidine intermediate) and release of succinate. The phosphoryl moiety is then transferred to GDP to form GTP (Figure 18.11). This sequence of steps “preserves” the energy of the thioester bond of succinyl-CoA in a series of high-energy intermediates that lead to a molecule of ATP: Thioester h [succinyl-P] h [phosphohistidine] h GTP h ATP NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

The First 5 Steps of the TCA Cycle Produce NADH, CO2, GTP (ATP), and Succinate

E

+ Succinyl

This is a good point to pause in our trip through the TCA cycle and see what has happened. A 2-carbon acetyl group has been introduced as acetyl-CoA and linked to oxaloacetate, and 2 CO2 molecules have been liberated. The cycle has produced 2 molecules of NADH and 1 of GTP or ATP and has left 1 molecule of succinate. The TCA cycle can now be completed by converting succinate to oxaloacetate. This latter process represents a net oxidation. The TCA cycle breaks it down into (consecutively) an oxidation step, a hydration reaction, and a second oxidation step. The oxidation steps are accompanied by the reduction of an [FAD] and an NAD". The reduced coenzymes, [FADH2] and NADH, subsequently provide reducing power in the electron transport chain. (It will be seen in Chapter 22 that virtually the same chemical strategy is used in "-oxidation of fatty acids.)

CH2

C

–O

Cys

S

Fe

S S

OH

O–

CoASH

H2C

COO –

H2C

O

C

P

O

O

O–

NH N

O– H2C

COO –

COO – H2C Succinate

H O

As will be seen in Chapter 19, succinate dehydrogenase is identical to the succinate– coenzyme Q reductase of the electron transport chain. In contrast to all of the other enzymes of the TCA cycle, which are soluble proteins found in the mitochondrial matrix, succinate dehydrogenase is an integral membrane protein tightly associated with the inner mitochondrial membrane. Succinate oxidation involves removal of H atoms across a C—C bond, rather than a C—O or C—N bond, and produces the trans-unsaturated fumarate. This reaction (the oxidation of an alkane to an alkene) is not sufficiently exergonic to reduce NAD", but it does yield enough energy to reduce [FAD]. (By contrast, oxidations of alcohols to ketones or aldehydes are more energetically favourable and provide sufficient energy to reduce NAD".) Succinate dehydrogenase is a dimeric protein, with subunits of molecular masses 70 and 27 kD. FAD is covalently bound to the larger subunit; the bond involves a methylene group of C-8a of FAD and N-3 of a histidine on the protein (Figure 18.12). Succinate dehydrogenase also contains 3 different iron–sulfur clusters: a 4Fe-4S cluster, a 3Fe-4S cluster, and a 2Fe-2S cluster, shown below: S

P

Fumarate

FAD FADH2

Cys

N

O

C –OOC

COO– Succinate

SCoA

C

Succinate dehydrogenase

CH2

NH

O

COO–

H

CoA

H2C

The oxidation of succinate to fumarate is carried out by succinate dehydrogenase, a membrane-bound enzyme that is actually part of the electron transport chain: COO–

605

COO –

H2C

Succinate Dehydrogenase Is FAD Dependent

FAD FADH2

The Tricarboxylic Acid Cycle

Fe

S S

–O

P

+ NH

N

O– GDP GTP

NH

N

FIGURE 18.11 The mechanism of the succinyl-CoA synthetase reaction.

Cys

Cys

Viewed from either end of the succinate molecule, the reaction involves dehydrogenation of !, " to a carbonyl (actually, a carboxyl) group. The dehydrogenation is stereospecific, with the pro-S hydrogen removed from one carbon atom and the pro-R hydrogen removed from the other. The electrons captured by [FAD] in this reaction are passed directly into the iron–sulfur clusters of the enzyme and on to coenzyme Q (UQ). The covalently bound FAD is first reduced to [FADH2] and then reoxidized to form [FAD] and the reduced form of coenzyme Q, UQH2. Electrons captured by UQH2 then flow through the rest of the electron transport chain in a series of events that will be discussed in detail in Chapter 19. Note that flavin coenzymes can carry out either 1-electron or 2-electron transfers. The succinate dehydrogenase reaction represents a net 2-electron reduction of FAD.

C - 8a HN

N

E

Histidine

R

CH2

N

H3C

N C-6

N

O NH

O

FAD

FIGURE 18.12 The covalent bond between FAD and succinate dehydrogenase involves the C-8a methylene group of FAD and the N-3 of a histidine residue on the enzyme.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

606

Part 3 Metabolism and Its Regulation Carbonium ion mechanism COO– C

H

C

H

HO–

COO–

H

H

C

COO– HO

H

COO–

Carbonium ion

E

C CH2

H

COO–

B

Fumarate

COO– + C H

L-Malate

Carbanion mechanism HO– H

COO– C

COO–

H

C COO–

HO H

H

H

C–

HO H

COO–

B

Fumarate

C

COO– H

CH2 B

COO– E

Carbanion

E

C

L-Malate

FIGURE 18.13 Two possible mechanisms for the fumarase reaction.

Fumarase Catalyzes the Trans-Hydration of Fumarate to Form L-Malate Fumarate is hydrated in a stereospecific reaction by fumarase to give L-malate: COO–

H C –OOC

OH

H2O

C

COO–

H2C

COO–

H Fumarase

C H

Fumarate

L -Malate

The reaction involves trans-addition of the elements of water across the double bond. Recall that aconitase carries out a similar reaction and that trans-addition of —H and —OH occurs across the double bond of cis-aconitate. Although the exact mechanism is uncertain, it may involve protonation of the double bond to form an intermediate carbonium ion (Figure 18.13 above) or possibly attack by water or OH− anion to produce a carbanion, followed by protonation.

Malate Dehydrogenase Completes the Cycle by Oxidizing Malate to Oxaloacetate In the last step of the TCA cycle, L-malate is oxidized to oxaloacetate by malate dehydrogenase. This reaction is very endergonic, with a ∆G°% of +30 kJ/mol: NAD+

NADH + H+

OH H

O

C

COO–

H2C

COO–

L-Malate

Malate dehydrogenase

NAD+ NADH + H+

C

COO–

H2C

COO–

Oxaloacetate NEL

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Chapter 18

The Tricarboxylic Acid Cycle

607

Consequently, the concentration of oxaloacetate in the mitochondrial matrix is usually quite low. The reaction, however, is pulled forward by the favourable citrate synthase reaction. Oxidation of malate is coupled to reduction of yet another molecule of NAD+, the third one of the cycle. Counting the [FAD] reduced by succinate dehydrogenase, this makes the fourth coenzyme reduced through oxidation of a single acetate unit. Malate dehydrogenase is structurally and functionally similar to other dehydrogenases, notably lactate dehydrogenase (Figure 18.14). Both consist of alternating "-sheet and !-helical segments. Binding of NAD" causes a conformational change in the 20-residue segment that connects the D and E strands of the "-sheet. The change is triggered by an interaction between the adenosine phosphate moiety of NAD" and an arginine residue in this loop region. Such a conformational change is consistent with an ordered single-displacement mechanism for NAD"-dependent dehydrogenases (see Chapter 12). FIGURE 18.14 The active site of malate dehydrogenase. Malate is shown in yellow; NAD+ is pink (pdb id = 1EMD).

18.5

What Are the Energetic Consequences of the TCA Cycle?

The net reaction accomplished by the TCA cycle, as follows, shows 2 molecules of CO2, 1 ATP, and 4 reduced coenzymes produced per acetate group oxidized. The cycle is exergonic, with a net ∆G°% for 1 pass around the cycle of approximately −40 kJ/mol. Table 18.1 compares the ∆G°% values for the individual reactions with the overall ∆G°% for the net reaction: Acetyl-CoA + 3 NAD + + [FAD] + ADP + Pi + 2 H2O h 2 CO2 + 3 NADH + 3 H + + [FADH2] + ATP + CoASH &G'% = -40 kJ/mol Glucose metabolized via glycolysis produces 2 molecules of pyruvate and thus 2 molecules of acetyl-CoA, which can enter the TCA cycle. Combining glycolysis and the TCA cycle gives the net reaction shown: Glucose + 2 H2O + 10 NAD + + 2[FAD] + 4 ADP + 4 Pi h 6 CO2 + 10 NADH + 10 H + + 2 [FADH2] + 4 ATP All 6 carbons of glucose are liberated as CO2, and a total of 4 molecules of ATP are formed thus far in substrate level phosphorylations. The 12 reduced coenzymes produced up to this point can eventually produce as many as 34 molecules of ATP in the electron transport and oxidative phosphorylation pathways. A stoichiometric relationship for these subsequent processes would be NADH + H + + 12 O2 + 3 ADP + 3 Pi h NAD + + 3 ATP + 4 H2O [FADH2] + 12 O2 + 2 ADP + 3 Pi h [FAD] + 2 ATP + 3 H2O Thus, a total of 3 ATP per NADH and 2 ATP per FADH2 may be produced through the processes of electron transport and oxidative phosphorylation.

The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle It is instructive to consider how the carbon atoms of a given acetate group are routed through several turns of the TCA cycle. As shown in Figure 18.15 on page 609, neither of the carbon atoms of a labelled acetate unit is lost as CO2 in the first turn of the cycle. The CO2 evolved in any turn of the cycle derives from the carboxyl groups of the oxaloacetate acceptor (from the previous turn), not from incoming acetyl-CoA. On the other hand, succinate labelled on one end from the original labelled acetate forms 2 different labelled oxaloacetates. The carbonyl carbon of acetyl-CoA is evenly distributed between the 2 carboxyl NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 3 Metabolism and Its Regulation

A DEEPER LOOK

Steric Preferences in NAD+-Dependent Dehydrogenases The enzymes that require nicotinamide coenzymes are stereospecific and transfer hydride to either the pro-R or the pro-S position selectively. What accounts for this stereospecificity? It arises from the fact that the enzymes (and especially the active sites of the enzymes) are inherently asymmetric structures. The nicotinamide coenzyme (and the substrate) fit the active site in only one way. Malate H

OH H

C

COO–

H2C

COO–

O C

+

HR HS

O

NH2 Malate dehydrogenase

N+

L-Malate

dehydrogenase, the citric acid cycle enzyme, transfers hydride to the HR position of NADH, but glyceraldehyde-3-P dehydrogenase in the glycolytic pathway transfers hydride to the HS position, as shown in the accompanying figure. Dehydrogenases are stereospecific with respect to the substrates as well. Note that alcohol dehydrogenase removes hydrogen from the pro-R position of ethanol and transfers it to the pro-R position of NADH.

C

COO–

H2C

COO–

H

H

O C C H2C

O C

OH 2– OPO3

+

N+

H

CH3 Ethanol

Pi

Glyceraldehyde3-phosphate dehydrogenase

N+ R NAD+

C H2C

HR H S

O

NH2

OH

C Alcohol dehydrogenase

CH3 Acetaldehyde

NH2

+

H+

NH2

+

H+

N

2– OPO3

H

H+

O C

+ R NADH

O C

+

H

2– OPO3

+

R NADH

1,3-Bisphosphoglycerate

NAD+

HS OH

C

NH2

R

Glyceraldehyde3-phosphate

HR C

+

O

NH2

N

NAD+ H

C

+

Oxaloacetate

R

O

HR HS

O C

+ N R NADH

carbons of oxaloacetate, and the labelled methyl carbon of incoming acetyl-CoA ends up evenly distributed between the methylene and carbonyl carbons of oxaloacetate. When these labelled oxaloacetates enter a second turn of the cycle, both of the carboxyl carbons are lost as CO2, but the methylene and carbonyl carbons survive through the second turn. Thus, the methyl carbon of a labelled acetyl-CoA survives 2 full turns of the cycle. In the third turn of the cycle, one-half of the carbon from the original methyl group of acetyl-CoA has become one of the carboxyl carbons of oxaloacetate and is thus lost as CO2. In the fourth turn of the cycle, further “scrambling” results in loss of half of the remaining labelled carbon (one-fourth of the original methyl carbon label of acetyl-CoA), and so on. It can be seen that the carbonyl and methyl carbons of labelled acetyl-CoA have very different fates in the TCA cycle. The carbonyl carbon survives the first turn intact but is completely lost in the second turn. The methyl carbon survives 2 full turns, then undergoes a 50% loss through each succeeding turn of the cycle. It is worth noting that the carbon–carbon bond cleaved in the TCA pathway entered as an acetate unit in the previous turn of the cycle. Thus, the oxidative decarboxylations that cleave this bond are just a cleverly disguised acetate COC cleavage and oxidation. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

The Tricarboxylic Acid Cycle

(a) Fate of the carboxyl carbon of acetate unit O

O S

CoA

S

O

CoA

O HO

Oxaloacetate

HO

Citrate

HO

1st turn

Malate

Oxaloacetate

Citrate

HO

2nd turn

Malate

HO

HO

Isocitrate

Isocitrate (CO2)

1

/2

(CO2)

Fumarate

Fumarate !-Ketoglutarate

Succinate

!-Ketoglutarate (CO2)

Succinyl-CoA

1

/2

Succinate

All labelled carboxyl carbon removed by these two steps

(CO2)

Succinyl-CoA

(b) Fate of methyl carbon of acetate unit O

O S

CoA

S

O

CoA

O HO

Oxaloacetate

HO

Citrate

HO

1st turn

Malate

Oxaloacetate

Citrate

HO

2nd turn

Malate

HO

HO

Isocitrate

Isocitrate (CO2)

(CO2)

Fumarate

Fumarate !-Ketoglutarate

Succinate

!-Ketoglutarate Succinate

(CO2)

Succinyl-CoA

(CO2)

Succinyl-CoA

O

O S

CoA

S

CoA

O

O HO

Oxaloacetate

HO

3rd turn

Malate

Oxaloacetate

Citrate

HO

Citrate

HO

4th turn

Malate

HO

Isocitrate

HO

Isocitrate

1

/4

(CO2)

Fumarate

1/ 2

!-Ketoglutarate Succinate Succinyl-CoA

1

/4

(CO2)

1

/8

(CO2)

Total methyl C label

Fumarate

1/ 4

!-Ketoglutarate Succinate Succinyl-CoA

1

/8

Total methyl C label

(CO2)

FIGURE 18.15 The fate of the carbon atoms of acetate in successive TCA cycles. Assume at the start that labelled acetate is added to cells containing unlabelled metabolites. (a) The carbonyl carbon of acetyl-CoA is fully retained through 1 turn of the cycle but is lost completely in a second turn of the cycle. (b) The methyl carbon of a labelled acetyl-CoA survives 2 full turns of the cycle but becomes equally distributed among the 4 carbons of oxaloacetate by the end of the second turn. In each subsequent turn of the cycle, one-half of this carbon (the original labelled methyl group) is lost. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

609

610

Part 3 Metabolism and Its Regulation

18.6

Can the TCA Cycle Provide Intermediates for Biosynthesis?

Until now we have viewed the TCA cycle as a catabolic process because it oxidizes acetate units to CO2 and converts the liberated energy to ATP and reduced coenzymes. The TCA cycle is, after all, the end point for breakdown of food materials, at least in terms of carbon turnover. However, as shown in Figure 18.16, 4-, 5-, and 6-carbon species produced in the TCA cycle also fuel a variety of biosynthetic processes. !-ketoglutarate, succinyl-CoA, fumarate, and oxaloacetate are all precursors of important cellular species. (In order to participate in eukaryotic biosynthetic processes, however, they must first be transported out of the mitochondria.) A transamination reaction converts !-ketoglutarate directly to glutamate, which can then serve as a versatile precursor for proline, arginine, and glutamine (as described in Chapter 23). Succinyl-CoA provides most of the carbon atoms of the porphyrins. Oxaloacetate can be transaminated to produce aspartate. Aspartic acid itself is a precursor of the pyrimidine nucleotides and, in addition, is a key precursor for the synthesis of asparagine, methionine, lysine, threonine, and isoleucine. Oxaloacetate can also be decarboxylated to yield PEP, which is a key element of several pathways, namely (1) synthesis (in plants and microorganisms) of the aromatic amino acids phenylalanine, tyrosine, and tryptophan; (2) formation of 3-phosphoglycerate and conversion to the amino acids serine, glycine, and cysteine; and (3) gluconeogenesis, which, as we will see in Chapter 21, is the pathway that synthesizes new glucose and many other carbohydrates. Finally, citrate can be exported from the mitochondria and then broken down by ATP– citrate lyase to yield oxaloacetate and acetyl-CoA, a precursor of fatty acids. Oxaloacetate produced in this reaction is rapidly reduced to malate, which can then be processed in either

FIGURE 18.16 The TCA cycle provides intermediates for numerous biosynthetic processes in the cell. Amino acids are highlighted in orange.

Carbohydrates 3-Phosphoglycerate

Erythrose4-phosphate Phenylalanine Tyrosine

Alanine

Phosphoenolpyruvate

Serine

Glycine

Cysteine

Leucine Valine

Tryptophan

Pyruvate CO2

Malonyl-CoA

Fatty acids

Isopentenyl pyrophosphate

Steroids

CO2

CO2

Acetyl-CoA

lo xa

O

Citr ate

CO2

ac

Asparagine

Acetoacetyl-CoA

at et

cit

Iso

e

Aspartate

Malate

inat e

F

oglu

tara

te

yl-C cin

Aspartyl semialdehyde

Glutamate

Proline

Ornithine

oA

Methionine

Glutamine

CO2

Glycine

Threonine Diaminopimelate

e

rat

a um

Purine nucleotides CO2

!-Ke t

Suc

Aspartyl phosphate

Citric acid cycle

Succ

Pyrimidine nucleotides

e rat

2-Amino3-ketoadipate

Citrulline Arginine

Isoleucine Lysine

#-Aminolevulinate

Porphyrins NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

The Tricarboxylic Acid Cycle

611

HUMAN BIOCHEMISTRY

Mitochondrial Diseases Are Rare Diseases arising from defects in mitochondrial enzymes are quite rare, because major defects in the TCA cycle (and the respiratory chain) are incompatible with life, and affected embryos rarely survive to birth. Even so, about 150 different hereditary mitochondrial diseases have been reported. Even though mitochondria carry their own DNA, many of the reported diseases map to the nuclear genome, because most of the mitochondrial proteins are imported from the cytosol. An interesting disease linked to mitochondrial DNA mutations is Leber’s hereditary optic neuropathy (LHON), in which the genetic defects are located primarily in the mitochondrial DNA coding for the subunits of NADH–CoQ reductase, also known as Complex I of the electron transport chain (see Chapter 19). Leber’s disease is the most common form of blindness in otherwise healthy young men, occurring less often in women. With respect to maternally inherited traits, a particular mitochondrial mutation in the 12S rRNA gene at nucleotide position 1555 has been reported.* The switching of a G for an A in this position results in hypersensitivity to aminoglycosides (antibiotics directed toward bacteria) in susceptible individuals. The resulting consequences of such a transition mutation lead to permanent ototoxicity and hearing loss upon administration of those specific types of antibiotics.*,† Other

studies have also suggested or implicated mitochondrial dysfunction (disruptions in electron transport) and oxidative damage (via reactive oxygen species) in several neurologically based illnesses, such as Alzheimer’s disease, Parkinson’s disease, and Huntington disease.‡,§,||,¶ *Hamasaki, K., and Rando. R., 1997. Specific binding of aminoglycosides to a human rRNA construct based on a DNA polymorphism which causes aminoglycoside-induced deafness. Biochemistry 36:12323–12328. †Hutchin, T., and Cortopassi, G., 1994. Proposed molecular and cellular mechanism for aminoglycoside ototoxicity. Antimicrobial Agents and Chemotherapy 38:2517–2520. ‡Filosto, M., Scarpelli, M., et al., 2011. The role of mitochondria in neurodegenerative diseases. Journal of Neurology 258:1763–1764. §Mungarro-Menchaca, X., Ferrera, P., et al., 2002. Beta-amyloid peptide induces ultrastructural changes in synaptosomes and potentiates mitochondrial dysfunction in the presence of ryanodine. Journal of Neuroscience Research 68:89–96. ||Swerdlow, R. H., 2009. The neurodegenerative mitochondriopathies. Journal of Alzheimer’s Disease 17:737–751. ¶Kwong, J. Q., Beal, M. F., et al., 2006. The role of mitochondria in inherited neurodegenerative diseases. Journal of Neurochemistry 97:1659–1675.

of 2 ways: It may be transported into mitochondria, where it is reoxidized to oxaloacetate, or it may be oxidatively decarboxylated to pyruvate by malic enzyme, with subsequent mitochondrial uptake of pyruvate. This cycle permits citrate to provide acetyl-CoA for biosynthetic processes, with return of the malate and pyruvate by-products to the mitochondria.

18.7

What Are the Anaplerotic, or “Filling-Up,” Reactions?

In a sort of reciprocal arrangement, the cell also feeds many intermediates back into the TCA cycle from other reactions. Because such reactions replenish the TCA cycle intermediates, Hans Kornberg proposed that they be called anaplerotic reactions (literally, the “filling-up” reactions). Thus, PEP carboxylase and pyruvate carboxylase synthesize oxaloacetate from pyruvate (Figure 18.17).

ATP

ADP + Pi O

O COO–

C

CO2

CH3

Pyruvate carboxylase

Pyruvate H2O +

O3PO

CO2

COO–

Phosphoenolpyruvate (PEP) CO2 H H+

C

COO–

H2C

COO–

OH

COO–

Pyruvate

COO–

Oxaloacetate

O

CH3

H2C

O

Pi

PEP carboxylase

CH2

C

COO–

Oxaloacetate

2–

C

C

FIGURE 18.17 Pyruvate carboxylase, phosphoenolpyruvate (PEP) carboxylase, and malic enzyme catalyze anaplerotic reactions, replenishing TCA cycle intermediates.

+

Malic NADPH enzyme

NADP+

C

COO–

H2C

COO–

L-Malate

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Part 3 Metabolism and Its Regulation

Pyruvate carboxylase is the most important of the anaplerotic reactions. It exists in the mitochondria of animal cells but not in plants, and it provides a direct link between glycolysis and the TCA cycle. The enzyme is tetrameric and contains covalently bound biotin and an Mg2+ site on each subunit. (It is examined in greater detail in our discussion of gluconeogenesis in Chapter 21.) Pyruvate carboxylase has an absolute allosteric requirement for acetyl-CoA. Thus, when acetyl-CoA levels exceed the oxaloacetate supply, allosteric activation of pyruvate carboxylase by acetyl-CoA raises oxaloacetate levels, so the excess acetyl-CoA can enter the TCA cycle. PEP carboxylase occurs in yeast, bacteria, and higher plants, but not in animals. The enzyme is specifically inhibited by aspartate, which is produced by transamination of oxaloacetate. Thus, organisms utilizing this enzyme control aspartate production by regulation of PEP carboxylase. Malic enzyme is found in the cytosol or mitochondria of many animal and plant cells and is an NADPH-dependent enzyme. It is worth noting that the reaction catalyzed by PEP carboxykinase could also function as an anaplerotic reaction, were it not for the particular properties of the enzyme: 2–

O3PO

CO2

+

GTP

GDP COO–

C CH2 PEP

O C

COO–

H2C

COO–

Oxaloacetate

A DEEPER LOOK

Fool’s Gold and the Reductive Citric Acid Cycle—The First Metabolic Pathway? How did life arise on Earth? It was once supposed that a reducing atmosphere, together with random synthesis of organic compounds, gave rise to a prebiotic “soup,” in which the first living things appeared. However, certain key compounds, such as arginine, lysine, and histidine; the straight chain fatty acids; porphyrins; and essential coenzymes, have not been convincingly synthesized under simulated prebiotic conditions. This and other problems have led researchers to consider other models for the evolution of life. One of these alternative models, postulated by Günter Wächtershäuser, involves an archaic version of the TCA cycle running in the reverse (reductive) direction. Reversal of the TCA cycle results in assimilation of CO2 and fixation of carbon as shown. For each turn of the reversed cycle, 2 carbons are fixed in the formation of isocitrate and 2 more are fixed in the reductive transformation of acetyl-CoA to oxaloacetate. Thus, for every succinate that enters the reversed cycle, 2 succinates are returned, making the cycle highly autocatalytic. Because TCA cycle intermediates are involved in many biosynthetic pathways (see Section 18.6), a reversed TCA cycle would be a bountiful and broad source of metabolic substrates. A reversed, reductive TCA cycle would require energy input to drive it. What might have been the thermodynamic driving force for such a cycle? Wächtershäuser hypothesizes that the anaerobic reaction of FeS and H2S to form insoluble FeS2 (pyrite, also known as fool’s gold) in the prebiotic milieu could have been the driving reaction:

Wächtershäuser has also suggested that early metabolic processes first occurred on the surface of pyrite and other related mineral materials. The iron–sulfur chemistry that prevailed on these mineral surfaces may have influenced the evolution of the iron– sulfur proteins that control and catalyze many reactions in modern pathways (including the succinate dehydrogenase and aconitase reactions of the TCA cycle). This reductive citric acid cycle has been shown to occur in certain extant archaea and bacteria, where it serves all their carbon needs. CO2

CO2

Acetyl-CoA Oxaloacetate

Oxaloacetate XH2 X Malate

XH2 Malate

Citrate

X Isocitrate X

Fumarate

XH2 X Succinate

CO2

XH2

Fumarate

FeS + H2S h FeS2(pyrite)T + H2 This reaction is highly exergonic, with a standard state freeenergy change (∆G° %) of −38 kJ/mol. Under the conditions that might have existed in a prebiotic world, this reaction would have been sufficiently exergonic to drive the reductive steps of a reversed TCA cycle.

Pyruvate

PEP

XH2 X

X Succinate

!-Ketoglutarate

XH2 SuccinylCoA

CO2

" A reductive, reversed TCA cycle.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

The Tricarboxylic Acid Cycle

CO2 binds weakly to PEP carboxykinase, whereas oxaloacetate binds very tightly (KD = 2 × 10−6 M), and, as a result, the enzyme favours formation of PEP from oxaloacetate. The catabolism of amino acids provides pyruvate, acetyl-CoA, oxaloacetate, fumarate, !-ketoglutarate, and succinate, all of which may be oxidized by the TCA cycle. In this way, proteins may serve as excellent sources of nutrient energy, as seen in Chapter 23.

18.8

How Is the TCA Cycle Regulated?

Situated as it is between glycolysis and the electron transport chain, the TCA cycle must be carefully controlled. If the cycle were permitted to run unchecked, large amounts of metabolic energy could be wasted in overproduction of reduced coenzymes and ATP; conversely, if it ran too slowly, ATP would not be produced rapidly enough to satisfy the needs of the cell. Also, as just seen, the TCA cycle is an important source of precursors for biosynthetic processes and must be able to provide them as needed. What are the sites of regulation in the TCA cycle? Based on our experience with glycolysis (see Figure 17.22), we might anticipate that some of the reactions of the TCA cycle would operate near equilibrium under cellular conditions (with ∆G < 0), whereas others— the sites of regulation—would be characterized by large negative ∆G values. Estimates for the values of ∆G in mitochondria, based on mitochondrial concentrations of metabolites, are summarized in Table 18.1. Three reactions of the cycle—citrate synthase, isocitrate dehydrogenase, and !-ketoglutarate dehydrogenase—operate with large negative ∆G values under mitochondrial conditions and are thus the primary sites of regulation in the cycle. The regulatory actions that control the TCA cycle are shown in Figure 18.18. As one might expect, the principal regulatory “signals” are the concentrations of acetyl-CoA, ATP, NAD", and NADH, with additional effects provided by several other metabolites. The main sites of regulation are pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and !-ketoglutarate dehydrogenase. All of these enzymes are inhibited by NADH, so when the cell has produced all the NADH that can conveniently be turned into ATP, the cycle shuts down. For similar reasons, ATP is an inhibitor of pyruvate dehydrogenase and isocitrate dehydrogenase. The TCA cycle is turned on, however, when either the ADP/ATP or the NAD"/NADH ratio is high, an indication that the cell has run low on ATP or NADH. Regulation of the TCA cycle by NADH, NAD", ATP, and ADP thus reflects the energy status of the cell. On the other hand, succinyl-CoA is an intracycle regulator, inhibiting citrate synthase and !-ketoglutarate dehydrogenase. Acetyl-CoA acts as a signal to the TCA cycle that glycolysis or fatty acid breakdown is producing 2-carbon units. Acetyl-CoA activates pyruvate carboxylase, the anaplerotic reaction that provides oxaloacetate, the acceptor for increased flux of acetyl-CoA into the TCA cycle.

Pyruvate Dehydrogenase Is Regulated by Phosphorylation/ Dephosphorylation As we shall see in Chapter 21, most organisms can synthesize sugars such as glucose from pyruvate. However, animals cannot synthesize glucose from acetyl-CoA. For this reason, the pyruvate dehydrogenase complex, which converts pyruvate to acetyl-CoA, plays a pivotal role in metabolism. Conversion to acetyl-CoA commits nutrient carbon atoms either to oxidation in the TCA cycle or to fatty acid synthesis (see Chapter 9). Because this choice is so crucial to the organism, pyruvate dehydrogenase is a carefully regulated enzyme. It is subject to product inhibition and is further regulated by nucleotides. Finally, activity of pyruvate dehydrogenase is regulated by phosphorylation and dephosphorylation of the enzyme complex itself. High levels of either product, acetyl-CoA or NADH, allosterically inhibit the pyruvate dehydrogenase complex. Acetyl-CoA specifically blocks dihydrolipoyl transacetylase, and NADH acts on dihydrolipoyl dehydrogenase. The mammalian pyruvate dehydrogenase is also regulated by covalent modifications. As shown in Figure 18.19, a Mg2+dependent pyruvate dehydrogenase kinase is associated with the enzyme in mammals. This kinase is allosterically activated by NADH and acetyl-CoA, and when levels of these NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 3 Metabolism and Its Regulation Pyruvate CO2

Acetyl-CoA Pyruvate dehydrogenase

ATP

+ Acetyl-CoA ADP

Pyruvate carboxylase

+

NADH

+

NAD+, CoA

CO2

ATP

Acetyl-CoA Pi

ATP

Citrate synthase

NADH

Oxaloacetate

Succinyl-CoA

H2O Malate

Citrate

H2O

TCA Cycle

Fumarate

ATP

Isocitrate

+

Isocitrate dehydrogenase

NADH ADP

CO2 Succinate

!-Ketoglutarate Pi

GTP

GDP

Succinyl-CoA

!-Ketoglutarate dehydrogenase

+

CO2

AMP NADH Succinyl-CoA

FIGURE 18.18 Regulation of the TCA cycle. High NADH/NAD+ ratio High AcCoA/CoASH ratio ATP

ADP

Pyruvate dehydrogenase kinase Inactive pyruvate dehydrogenase

Active pyruvate dehydrogenase

Pyruvate dehydrogenase phosphatase

Pi

Ca2+

H2O

Low NADH/NAD+ ratio Low AcCoA/CoASH ratio FIGURE 18.19 Regulation of the pyruvate dehydrogenase reaction. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

The Tricarboxylic Acid Cycle

metabolites rise in the mitochondrion, they stimulate phosphorylation of a serine residue on the pyruvate dehydrogenase subunit, blocking the first step of the pyruvate dehydrogenase reaction, the decarboxylation of pyruvate. Inhibition of the dehydrogenase in this manner eventually lowers the levels of NADH and acetyl-CoA in the matrix of the mitochondrion. Reactivation of the enzyme is carried out by pyruvate dehydrogenase phosphatase, a Ca2"-activated enzyme that binds to the dehydrogenase complex and hydrolyzes the phosphoserine moiety on the dehydrogenase subunit. At low ratios of NADH to NAD" and low acetyl-CoA levels, the phosphatase maintains the dehydrogenase in an activated state, but a high level of acetyl-CoA or NADH once again activates the kinase and leads to the inhibition of the dehydrogenase. Insulin and Ca2+ ions activate dephosphorylation, and pyruvate inhibits the phosphorylation reaction. Pyruvate dehydrogenase is also sensitive to the energy status of the cell. AMP activates pyruvate dehydrogenase, whereas GTP inhibits it. High levels of AMP are a sign that the cell may become energy poor. Activation of pyruvate dehydrogenase under such conditions commits pyruvate to energy production.

Isocitrate Dehydrogenase Is Strongly Regulated The mechanism of regulation of isocitrate dehydrogenase is in some respects the reverse of that of pyruvate dehydrogenase. The mammalian isocitrate dehydrogenase is subject only to allosteric activation by ADP and NAD+ and to inhibition by ATP and NADH. Thus, high NAD+/ NADH and ADP/ATP ratios stimulate isocitrate dehydrogenase and TCA cycle activity. It may seem surprising that isocitrate dehydrogenase is strongly regulated, because it is not an apparent branch point within the TCA cycle. However, the citrate/isocitrate ratio controls the rate of production of cytosolic acetyl-CoA, because acetyl-CoA in the cytosol is derived from citrate exported from the mitochondrion. (Breakdown of cytosolic citrate produces oxaloacetate and acetyl-CoA, which can be used in a variety of biosynthetic processes.) Thus, isocitrate dehydrogenase activity in the mitochondrion favours catabolic TCA cycle activity over anabolic utilization of acetyl-CoA in the cytosol. Interestingly, the Escherichia coli isocitrate dehydrogenase is regulated by covalent modification. Serine residues on each subunit of the dimeric enzyme are phosphorylated by a protein kinase, causing inhibition of the isocitrate dehydrogenase activity. Activity is restored by the action of a specific phosphatase. When TCA cycle and glycolytic intermediates—such as isocitrate, 3-phosphoglycerate, pyruvate, PEP, and oxaloacetate—are high, the kinase is inhibited, the phosphatase is activated, and the TCA cycle operates normally. When levels of these intermediates fall, the kinase is activated, isocitrate dehydrogenase is inhibited, and isocitrate is diverted to the glyoxylate pathway, as explained in the next section.

18.9

Can Any Organisms Use Acetate as Their Sole Carbon Source?

Plants (particularly seedlings, which cannot yet accomplish efficient photosynthesis), as well as some bacteria and algae, can use acetate as the only source of carbon for all the carbon compounds they produce. Although we saw that the TCA cycle can supply intermediates for some biosynthetic processes, the cycle gives off 2 CO2 for every 2-carbon acetate group that enters and cannot effect the net synthesis of TCA cycle intermediates. Thus, it would not be possible for the cycle to produce the massive amounts of biosynthetic intermediates needed for acetate-based growth unless alternative reactions were available. In essence, the TCA cycle is geared primarily to energy production, and it “wastes” carbon units by giving off CO2. Modification of the cycle to support acetate-based growth would require eliminating the CO2-producing reactions and enhancing the net production of 4-carbon units (i.e., oxaloacetate). Plants and bacteria employ a modification of the TCA cycle called the glyoxylate cycle to produce 4-carbon dicarboxylic acids (and eventually even sugars) from 2-carbon acetate units. The glyoxylate cycle bypasses the 2 oxidative decarboxylations of the TCA cycle and instead routes isocitrate through the isocitrate lyase and malate synthase reactions (Figure 18.20). Glyoxylate produced by isocitrate lyase reacts with a second molecule of acetyl-CoA to form L-malate. The net effect is to conserve NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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FIGURE 18.20 The glyoxylate cycle. The first 2 steps are identical to TCA cycle reactions. The third step bypasses the CO2-evolving steps of the TCA cycle to produce succinate and glyoxylate. The malate synthase reaction forms malate from glyoxylate and another acetyl-CoA. The result is that 1 turn of the cycle consumes 1 oxaloacetate and 2 acetyl-CoA molecules but produces 2 molecules of oxaloacetate. (Succinate produced in the isocitrate lyase reaction is converted to oxaloacetate by TCA cycle reactions.) The net for this cycle is 1 oxaloacetate from 2 acetyl-CoA molecules.

O C

H3C

SCoA

Acetyl-CoA CoASH

O C

COO–

H2C

COO–

H2O

COO–

H2C C

COO–

H2C

COO–

HO

Oxaloacetate

Citrate HO C

COO–

H2C

COO–

H

GLYOXYLATE CYCLE

Malate CoASH

C

H3C

HC SCoA

Acetyl-CoA

COO–

HC

COO–

HC

COO–

OH

Malate synthase O

H2C

COO–

Isocitrate lyase

Isocitrate

O Glyoxylate

H2C

COO–

H2C

COO–

Succinate

carbon units, using 2 acetyl-CoA molecules per cycle to generate oxaloacetate. Some of this is converted to PEP and then to glucose by pathways discussed in Chapter 21.

The Glyoxylate Cycle Operates in Specialized Organelles The enzymes of the glyoxylate cycle in plants are contained in glyoxysomes, organelles devoted to this cycle. Yeast and algae carry out the glyoxylate cycle in the cytoplasm. The enzymes common to both the TCA and glyoxylate pathways exist as isozymes, with spatially and functionally distinct enzymes operating independently in the 2 cycles.

Isocitrate Lyase Short-Circuits the TCA Cycle by Producing Glyoxylate and Succinate The isocitrate lyase reaction (Figure 18.21) produces succinate, a 4-carbon product of the cycle, as well as glyoxylate, which can then combine with a second molecule of acetyl-CoA.

H H

H2C

COO–

C

COO–

C

COO–

O

H

+H B

HC

COO–

O B

E

Glyoxylate

+

H2C

COO–

B

H2C

COO–

+ H B

Succinate

E

2R, 3S-Isocitrate FIGURE 18.21 The isocitrate lyase reaction. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 18

The Tricarboxylic Acid Cycle

Isocitrate lyase catalyzes an aldol cleavage and is similar to the reaction mediated by aldolase in glycolysis. The malate synthase reaction (see Figure 18.20), a Claisen condensation of acetyl-CoA with the aldehyde of glyoxylate to yield malate, is quite similar to the citrate synthase reaction. Compared with the TCA cycle, the glyoxylate cycle (1) contains only 5 steps (as opposed to 8), (2) lacks the CO2-liberating reactions, (3) consumes 2 molecules of acetylCoA per cycle, and (4) produces 4-carbon units (oxaloacetate) as opposed to 1-carbon units.

The Glyoxylate Cycle Helps Plants Grow in the Dark The existence of the glyoxylate cycle explains how certain seeds grow underground (or in the dark), where photosynthesis is impossible. Many seeds (e.g., peanuts, soybeans, and castor beans) are rich in lipids, and as we will see in Chapter 22, most organisms degrade the fatty acids of lipids to acetyl-CoA. Glyoxysomes form in seeds as germination begins, and the glyoxylate cycle uses the acetyl-CoA produced in fatty acid oxidation to provide large amounts of oxaloacetate and other intermediates for carbohydrate synthesis. Once the growing plant begins photosynthesis and can fix CO2 to produce carbohydrates (see Chapter 20), the glyoxysomes disappear.

Glyoxysomes Must Borrow 3 Reactions from Mitochondria Glyoxysomes do not contain all the enzymes needed to run the glyoxylate cycle: Succinate dehydrogenase, fumarase, and malate dehydrogenase are absent. Consequently, glyoxysomes must cooperate with mitochondria to run their cycle (Figure 18.22). Succinate

Glyoxysome

Mitochondrion

Aspartate

Fatty acids

AcetylCoA Oxaloacetate

!-Ketoglutarate Glutamate Citrate

Glyoxylate Isocitrate Malate cycle

AcetylCoA

Malate

Glyoxylate

Succinate

Cytosol

!-Keto- Aspartate glutarate Glutamate Oxaloacetate Malate TCA Fumarate Succinate

Oxaloacetate

Phosphoenolpyruvate Carbohydrate FIGURE 18.22 Glyoxysomes lack 3 of the enzymes needed to run the glyoxylate cycle. Succinate dehydrogenase, fumarase, and malate dehydrogenase are all “borrowed” from the mitochondria in a shuttle in which succinate and glutamate are passed to the mitochondria and !-ketoglutarate and aspartate are passed to the glyoxysome. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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travels from the glyoxysomes to the mitochondria, where it is converted to oxaloacetate. Transamination to aspartate follows because oxaloacetate cannot be transported out of the mitochondria. Aspartate formed in this way then moves from the mitochondria back to the glyoxysomes, where a reverse transamination with !-ketoglutarate forms oxaloacetate, completing the shuttle. Finally, to balance the transaminations, glutamate shuttles from glyoxysomes to mitochondria.

SUMMARY The glycolytic pathway converts glucose to pyruvate and produces 2 molecules of ATP per glucose—only a small fraction of the potential energy available from glucose. In the presence of oxygen, pyruvate is oxidized to CO2, releasing the rest of the energy available from glucose via the TCA cycle. 18.1 What Is the Chemical Logic of the TCA Cycle? The entry of new carbon units into the cycle is through acetyl-CoA. Transfer of the 2-carbon acetyl group from acetyl-CoA to the 4-carbon oxaloacetate to yield 6-carbon citrate is catalyzed by citrate synthase. A dehydration–rehydration rearrangement of citrate yields isocitrate. Two successive decarboxylations produce !-ketoglutarate and then succinyl-CoA, a CoA conjugate of a 4-carbon unit. Several steps later, oxaloacetate is regenerated and can combine with another 2-carbon unit of acetyl-CoA. 18.2 How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA? The pyruvate dehydrogenase complex (PDC) is a non-covalent assembly of three different enzymes operating in concert to catalyze successive steps in the conversion of pyruvate to acetyl-CoA. 18.3 How Are 2 CO2 Molecules Produced from Acetyl-CoA? Citrate synthase combines acetyl-CoA with oxaloacetate in a Perkin condensation (a carbon–carbon condensation between a ketone or an aldehyde and an ester). A general base on the enzyme accepts a proton from the methyl group of acetyl-CoA, producing a stabilized !-carbanion of acetyl-CoA. This strong nucleophile attacks the !-carbonyl of oxaloacetate, yielding citryl-CoA, which is hydrolyzed to citrate and CoASH. Citrate is isomerized to isocitrate by aconitase in a 2-step process involving aconitate as an intermediate. The elements of water are first abstracted from citrate to yield aconitate, which is then rehydrated with H– and HO– adding back in opposite positions to produce isocitrate. The net effect is the conversion of a tertiary alcohol (citrate) to a secondary alcohol (isocitrate). The 2-step isocitrate dehydrogenase reaction involves (1) oxidation of the C-2 alcohol of isocitrate to form oxalosuccinate, followed by (2) a "-decarboxylation reaction that expels the central carboxyl group as CO2, leaving the product !-ketoglutarate. Oxalosuccinate, the "-keto acid produced by the initial dehydrogenation reaction, is unstable and thus is readily decarboxylated. !-ketoglutarate dehydrogenase is a multienzyme complex— consisting of !-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase—that employs 5 different coenzymes. The dihydrolipoyl dehydrogenase in this reaction is identical to that in the pyruvate dehydrogenase reaction. The mechanism is an oxidative decarboxylation analogous to that of pyruvate dehydrogenase. Succinyl-CoA is the product. 18.4 How Is Oxaloacetate Regenerated to Complete the TCA Cycle? Succinyl-CoA synthetase catalyzes a substrate-level phosphorylation: Succinyl-CoA is a high-energy intermediate and is used to drive the phosphorylation of GDP to GTP (in mammals) or ADP to ATP (in plants and bacteria). Succinate dehydrogenase (succinate–coenzyme Q reductase of the electron transport chain) catalyzes removal of H atoms across a C—C bond and produces the trans-unsaturated fumarate.

Fumarate is hydrated in a stereospecific reaction by fumarase to give L-malate. The reaction involves trans-addition of the elements of water across the double bond. Malate dehydrogenase completes the TCA cycle. This reaction is very endergonic, with a ∆G°%of +30 kJ/mol. Consequently, the concentration of oxaloacetate in the mitochondrial matrix is usually quite low. Oxidation of malate to oxaloacetate is coupled to reduction of yet another molecule of NAD+, the third one of the cycle. 18.5 What Are the Energetic Consequences of the TCA Cycle? The cycle is exergonic, with a net ∆G°% for one pass around the cycle of approximately −40 kJ/mol. Three NADH, 1 [FADH2], and 1 ATP equivalent are produced in each turn of the cycle. 18.6 Can the TCA Cycle Provide Intermediates for Biosynthesis? !-ketoglutarate, succinyl-CoA, fumarate, and oxaloacetate are all precursors of important cellular species. A transamination reaction converts !-ketoglutarate directly to glutamate, which can then serve as a precursor for proline, arginine, and glutamine. Succinyl-CoA provides most of the carbon atoms of the porphyrins. Oxaloacetate can be transaminated to produce aspartate. Aspartic acid itself is a precursor of the pyrimidine nucleotides and, in addition, is a key precursor for the synthesis of asparagine, methionine, lysine, threonine, and isoleucine. Oxaloacetate can also be decarboxylated to yield PEP, which is a key element of several pathways. 18.7 What Are the Anaplerotic, or “Filling-Up,” Reactions? Anaplerotic reactions replenish the TCA cycle intermediates. Examples include PEP carboxylase and pyruvate carboxylase, both of which synthesize oxaloacetate from pyruvate. 18.8 How Is the TCA Cycle Regulated? The main sites of regulation are pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and !-ketoglutarate dehydrogenase. All of these enzymes are inhibited by NADH. ATP is an inhibitor of pyruvate dehydrogenase and isocitrate dehydrogenase. The TCA cycle is turned on, however, when either the ADP/ATP or the NAD"/NADH ratio is high. Regulation of the TCA cycle by NADH, NAD", ATP, and ADP thus reflects the energy status of the cell. Succinyl-CoA is an intracycle regulator, inhibiting citrate synthase and !-ketoglutarate dehydrogenase. Acetyl-CoA activates pyruvate carboxylase, the anaplerotic reaction that provides oxaloacetate, the acceptor for acetyl-CoA entry into the TCA cycle. 18.9 Can Any Organisms Use Acetate as Their Sole Carbon Source? Plants and bacteria employ a modification of the TCA cycle called the glyoxylate cycle to produce 4-carbon dicarboxylic acids (and eventually even sugars) from 2-carbon acetate units. The glyoxylate cycle bypasses the 2 oxidative decarboxylations of the TCA cycle and instead routes isocitrate through the isocitrate lyase and malate synthase reactions. Glyoxylate produced by isocitrate lyase reacts with a second molecule of acetyl-CoA to form L-malate. The net effect is to conserve carbon units, using 2 acetyl-CoA molecules per cycle to generate oxaloacetate.

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Chapter 18

The Tricarboxylic Acid Cycle

619

PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. Describe the labelling pattern that would result from the introduction into the TCA cycle of glutamate labelled at C( with 14C. 2. Considering glycolysis, the PDH complex, and the Kreb’s cycle, calculate how many NADH and ATP are produced in muscle from 1 molecule of a. fructose-6-phosphate. b. glyceraldehyde-3-phosphate. c. phosphoenolpyruvate. 3. Using the same molecules from problem 2, calculate the number of CO2 produced when considering glycolysis, the PDH complex, and the Kreb’s cycle. 4. Describe the effect on the TCA cycle of (a) increasing the concentration of NAD+, (b) reducing the concentration of ATP, and (c) increasing the concentration of isocitrate. 5. (Integrates with Chapter 14) The serine residue of isocitrate dehydrogenase that is phosphorylated by protein kinase lies within the active site of the enzyme. This situation contrasts with most other examples of covalent modification by protein phosphorylation, where the phosphorylation occurs at a site remote from the active site. What direct effect do you think such active site phosphorylation might have on the catalytic activity of isocitrate dehydrogenase? (See Barford, D., 1991. Molecular mechanisms for the control of enzymic activity by protein phosphorylation. Biochimica et Biophysica Acta 1133:55–62.) 6. The first step of the !-ketoglutarate dehydrogenase reaction involves decarboxylation of the substrate and leaves a covalent TPP intermediate. Write a reasonable mechanism for this reaction. 7. In a tissue where the TCA cycle has been inhibited by fluoroacetate, what difference in the concentration of each TCA cycle metabolite would you expect, compared with a normal, uninhibited tissue? 8. On the basis of the descriptions of the physical properties of FAD and FADH2, suggest a method for the measurement of the enzyme activity of succinate dehydrogenase. 9. Starting with citrate, isocitrate, !-ketoglutarate, and succinate, state which of the individual carbons of the molecule undergo oxidation in the next step of the TCA cycle. Which molecules undergo a net oxidation? 10. In addition to fluoroacetate, consider whether other analogs of TCA cycle metabolites or intermediates might be introduced to inhibit other, specific reactions of the cycle. Explain your reasoning. 11. Based on the action of thiamine pyrophosphate in catalysis of the pyruvate dehydrogenase reaction, suggest a suitable chemical mechanism for the pyruvate decarboxylase reaction in yeast: Pyruvate h acetaldehyde + CO2 12. (Integrates with Chapter 3) Aconitase catalyzes the citric acid cycle reaction: Citrate m isocitrate The standard free-energy change, ∆G°%, for this reaction is "6.7 kJ/ mol. However, the observed free-energy change (∆G) for this reaction in pig heart mitochondria is "0.8 kJ/mol. What is the ratio of [isocitrate]/[citrate] in these mitochondria? If [isocitrate] = 0.03 mM, what is [citrate]? 13. Describe the labelling pattern that would result if 14CO2 were incorporated into the TCA cycle via the pyruvate carboxylase reaction.

14. Describe the labelling pattern that would result if the reductive, reversed TCA cycle (see A Deeper Look on page 612) operated with 14CO2. 15. Describe the labelling pattern that would result in the glyoxylate cycle if a plant were fed acetyl-CoA labelled at the OCH3 carbon. 16. The malate synthase reaction, which produces malate from acetylCoA and glyoxylate in the glyoxylate pathway, involves chemistry similar to that of the citrate synthase reaction. Write a mechanism for the malate synthase reaction and explain the role of CoA in this reaction. 17. In most cells, fatty acids are synthesized from acetate units in the cytosol. However, the primary source of acetate units is the TCA cycle in mitochondria, and acetate cannot be transported directly from the mitochondria to the cytosol. Cells solve this problem by exporting citrate from the mitochondria and then converting citrate to acetate and oxaloacetate. Then, because cells cannot transport oxaloacetate into mitochondria directly, they must convert it to malate or pyruvate, both of which can be taken up by mitochondria. Draw a complete pathway for citrate export, conversion of citrate to malate and pyruvate, and import of malate and pyruvate by mitochondria. a. Which of the reactions in this cycle might require energy input? b. What would be the most likely source of this energy? c. Do you recognize any of the enzyme reactions in this cycle? d. What coenzymes might be required to run this cycle? 18. A typical intramitochondrial concentration of malate is 0.22 mM. If the ratio of NAD" to NADH in mitochondria is 20, and if the malate dehydrogenase reaction is at equilibrium, calculate the concentration of oxaloacetate in the mitochondrion at 25°C. A typical mitochondrion can be thought of as a cylinder 1 μm in diameter and 2 μm in length. Calculate the number of molecules of oxaloacetate in a mitochondrion. In analogy with pH (the negative logarithm of [H"]), what is pOAA? 19. Glycolysis, the pyruvate dehydrogenase reaction, and the TCA cycle result in complete oxidation of a molecule of glucose to CO2. Review the calculation of oxidation numbers for individual atoms in any molecule, and then calculate the oxidation numbers of the carbons of glucose, pyruvate, the acetyl carbons of acetyl-CoA, and the metabolites of the TCA cycle to convince yourself that complete oxidation of glucose involves removal of 24 electrons and that each acetyl-CoA through the TCA cycle gives up 8 electrons. 20. Recalling all reactions of the TCA cycle can be a challenging proposition. One way to remember them is to begin with the simplest molecule—succinate—which is a symmetric 4-carbon molecule. Begin with succinate, and draw the 8 reactions of the TCA cycle. Remember that succinate S oxaloacetate is accomplished by a special trio of reactions: oxidation of a single bond to a double bond, hydration across the double bond, and oxidation of an alcohol to a ketone. From there, a molecule of acetyl-CoA is added. If you remember the special function of acetyl-CoA (see A Deeper Look, page 599), this is an easy reaction to draw. From there, you need only isomerize, carry out the 2 oxidative decarboxylations, and remove the CoA molecule to return to succinate. 21. Aconitase is rapidly inactivated by 2R,3R-fluorocitrate, which is produced from fluoroacetate in the citrate synthase reaction. Interestingly, inactivation by fluorocitrate is accompanied by stoichiometric release of fluoride ion (i.e., 1 F-ion is lost per aconitase active site). This observation is consistent with “mechanismbased inactivation” of aconitase by fluorocitrate. Suggest a mechanism for this inactivation, based on formation of 4-hydroxytrans-aconitate, which remains tightly bound at the active site. To assess your answer, consult this reference: Lauble, H., Kennedy, M., et al., 1996. The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex. Proceedings of the National Academy of Sciences 93:13699–13703.

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Part 3 Metabolism and Its Regulation

FURTHER READING General Bodner, G. M., 1986. The tricarboxylic acid (TCA), citric acid or Krebs cycle. Journal of Chemical Education 63:673–677. Dalsgaard, M. K., 2006. Fuelling cerebral activity in exercising man. Journal of Cerebral Blood Flow and Metabolism 26:731–750. Fisher, C. R., and Girguis, P., 2007. A proteomic snapshot of life at a vent. Science 315:198–199. Gibala, M. J., Young, M. E., et al., 2000. Anaplerosis of the citric acid cycle: Role in energy metabolism of heart and skeletal muscle. Acta Physiologica Scandinavica 168:657–665. Holmes, F. L., 1993. Hans Krebs: Architect of Intermediary Metabolism, 1933–1937, Vol. 2. Oxford: Oxford University Press.

Pithukpakorn, M., 2005. Disorders of pyruvate metabolism and the tricarboxylic acid cycle. Molecular Genetics and Metabolism 85:243–246. Regulation of the TCA Cycle Atkinson, D. E., 1977. Cellular Energy Metabolism and Its Regulation. New York: Academic Press. Bott, M., 2007. Offering surprises: TCA cycle regulation in Corynebacterium glutamicum. Trends in Microbiology 15:417–425. Gibson, D., and Harris, R., 2001. Metabolic Regulation in Mammals. New York: Taylor and Francis. Pyruvate Dehydrogenase

Hu, Y., and Holden, J. F., 2006. Citric acid cycle in the hyperthermophilic archaeon Pyrobaculum islandicum grown autotrophically, heterotrophically, and mixotrophically with acetate. Journal of Bacteriology 188:4350–4355.

Brautigam, C. A., Wynn, R. M., et al., 2006. Structural insight into interactions between dihydrolipoamide dehydrogenase (E3) and E3-binding protein of human pyruvate dehydrogenase complex. Structure 14:611–621.

Krebs, H. A., 1981. Reminiscences and Reflections. Oxford: Oxford University Press.

Harris, R. A., Bowker-Kinley, M. M., et al., 2002. Regulation of the activity of the pyruvate dehydrogenase complex. Advances in Enzyme Regulation 42:249–259.

Newsholme, E. A., and Leech, A. R., 1983. Biochemistry for the Medical Sciences. New York: John Wiley and Sons. Schurr, A., 2006. Lactate: The ultimate cerebral oxidative energy substrate? Journal of Cerebral Blood Flow and Metabolism 26:142–152.

Milne, J. L. S., Shi, D., et al., 2002. Molecular architecture and mechanism of an icosahedral pyruvate dehydrogenase complex: A multifunctional catalytic machine. EMBO Journal 21:5587–5598.

Smith, E., and Morowitz, H. J., 2004. Universality in intermediary metabolism. Proceedings of the National Academy of Sciences U.S.A. 101:13168–13173.

Sugden, M. C., and Holdness, M. J., 2006. Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases. Archives of Physiology and Biochemistry 112:139–149.

Enzymes of the TCA Cycle

Zhou, Z. H., McCarthy, D. B., et al., 2001. The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes. Proceedings of the National Academy of Sciences U.S.A. 98:14802–14807.

Fraser, M. E., James, M. N. G., et al., 1999. A detailed structural description of Escherichia coli succinyl-CoA synthetase. Journal of Molecular Biology 285:1633–1653. Perham, R. N., 2000. Swinging arms and swinging domains in multifunctional enzymes: Catalytic machines for multistep reactions. Annual Review of Biochemistry 69:961–1004. St. Maurice, M., Reinhardt, L., et al., 2007. Domain architecture of pyruvate carboxylase, a biotin-dependent multifunctional enzyme. Science 317:1076–1079.

Glyoxylate Cycle Eastmond, P. J., and Graham, I. A., 2001. Re-examining the role of the glyoxylate cycle in oilseeds. Trends in Plant Science 6:72–77.

Diseases of the TCA Cycle Briere, J.-J., Favier, J., et al., 2006. Tricarboxylic acid cycle dysfunction as a cause of human diseases and tumor formation. American Journal of Physiology and Cellular Physiology 291:C1114–C1120.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

19

Electron Transport and Oxidative Phosphorylation

! Wall Piece #IV (1985), a kinetic sculpture

by George Rhoads. This complex mechanical art form can be viewed as a metaphor for the molecular apparatus underlying electron transport and ATP synthesis by oxidative phosphorylation.

In all things of nature there is something of the marvellous.

George Rhoads/ Rock Stream Studios

Aristotle (384–322 BCE)

ESSENTIAL QUESTION

KEY QUESTIONS 19.1

Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur?

19.2

What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions?

19.3

How Is the Electron-Transport Chain Organized?

19.4

What Are the Thermodynamic Implications of Chemiosmotic Coupling?

19.5

How Does a Proton Gradient Drive the Synthesis of ATP?

Living cells save up metabolic energy predominantly in the form of fats and carbohydrates, and they “spend” this energy for biosynthesis, membrane transport, and movement. In both directions, energy is exchanged and transferred in the form of ATP. In Chapters 17 and 18 we saw that glycolysis and the TCA cycle convert some of the energy available from stored and dietary sugars directly to ATP. However, most of the metabolic energy that is obtainable from substrates entering glycolysis and the TCA cycle is funnelled via oxidation– reduction reactions into NADH and reduced flavoproteins, the latter symbolized by [FADH2].

19.6

What Is the P/O Ratio for Mitochondrial Oxidative Phosphorylation?

19.7

How do cells oxidize NADH and [FADH2] and convert their reducing potential into the chemical energy of ATP?

How Are the Electrons of Cytosolic NADH Fed into Electron Transport?

19.8

How Do Mitochondria Mediate Apoptosis?

WHY IT MATTERS Before we begin our discussion of the electron-transport chain and oxidative phosphorylation, we pose the question, “Why does electron transport matter?” As we will see in the upcoming chapter, Peter Mitchell (1920–1992, see photo on the next page) was instrumental in putting forth the “chemiosmotic hypothesis” in 1961 using the fundamental principles of electron transport. From his early days in college, he studied several subjects, including mathematics, physics, chemistry, physiology, and biochemistry, all of which helped in the postulation of his theory. He went on to establish the Chemical Biology Unit in the Department of Zoology, at The University of Edinburgh and received numerous awards for his work, including the Fellowship of the Royal Society (1974), an Honorary Doctoral Degree from the University of Chicago, and, of course, the Nobel Prize in Chemistry in 1978 for his “chemiosmotic hypothesis.” It will become evident in the latter part of the chapter that his theory defined the basic principle of ATP synthesis, which was intrinsically coupled to a proton motive redox system, a system whose dynamics were intertwined with the inner mitochondrial membrane. In later years, this hypothesis would come to be accepted as the method by which cells generate the highly energetic compound ATP for their biological needs.

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

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Part 3 Metabolism and Its Regulation

ASSOCIATED PRESS

" Peter Mitchell in the 1980s

Whereas ATP made in glycolysis and the TCA cycle is the result of substrate-level phosphorylation, NADH-dependent ATP synthesis is the result of oxidative phosphorylation. Electrons stored in the form of the reduced coenzymes, NADH or [FADH2], are passed through an elaborate and highly organized chain of proteins and coenzymes, the so-called electron-transport chain, finally reaching O2 (molecular oxygen), the terminal electron acceptor. Each component of the chain can exist in (at least) 2 oxidation states, and each component is successively reduced and reoxidized as electrons move through the chain from NADH (or [FADH2]) to O2. In the course of electron transport, a proton gradient is established across the inner mitochondrial membrane. It is the energy of this proton gradient that drives ATP synthesis.

19.1

Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur?

The processes of electron transport and oxidative phosphorylation are membrane associated. Prokaryotes are the simplest life form, and prokaryotic cells typically consist of a single cellular compartment surrounded by a plasma membrane and a more rigid cell wall. In such a system, the conversion of energy from NADH and [FADH2] to the energy of ATP via electron transport and oxidative phosphorylation is carried out at (and across) the plasma membrane. In eukaryotic cells, electron transport and oxidative phosphorylation are localized in mitochondria, which are also the sites of TCA cycle activity and (as we shall see in Chapter 22) fatty acid oxidation. Mammalian cells contain 800–2500 mitochondria; other types of cells may have as few as 1 or 2 or as many as half a million mitochondria. Human erythrocytes, whose purpose is simply to transport oxygen to tissues, contain no mitochondria at all. The typical mitochondrion is about 0.5 ; 0.3 micron in diameter and from 0.5 micron to several microns long; its overall shape is sensitive to metabolic conditions in the cell.

Mitochondrial Functions Are Localized in Specific Compartments Mitochondria are surrounded by a simple outer membrane and a more complex inner membrane (Figure 19.1). The space between the inner and outer membranes is referred to as the intermembrane space. Several enzymes that utilize ATP (such as creatine kinase and adenylate kinase) are found in the intermembrane space. The smooth outer membrane is about 30%–40% lipid and 60%–70% protein and has a relatively high concentration of NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 19

Electron Transport and Oxidative Phosphorylation

Outer membrane Inner membrane Intermembrane space

Matrix

Cristae (a)

(b)

FIGURE 19.1 (a) A drawing of a mitochondrion with components labelled. (b) Tomography of a rat liver mitochondrion. The tubular structures in red, yellow, green, purple, and aqua represent individual cristae formed from the inner mitochondrial membrane. (b, Frey, T. G., and Mannella, C. A., 2000. The internal structure of mitochondria. Trends in Biochemical Sciences 25:319–324.)

phosphatidylinositol. The outer membrane contains significant amounts of porin, a transmembrane protein, rich in b-sheets, that forms large channels across the membrane, permitting free diffusion of molecules with molecular weights of about 10 000 or less. The outer membrane plays a prominent role in maintaining the shape of the mitochondrion. The inner membrane is richly packed with proteins, which account for nearly 80% of its weight; thus, its density is higher than that of the outer membrane. The fatty acids of inner membrane lipids are highly unsaturated. Cardiolipin and diphosphatidylglycerol (see Chapter 8) are abundant. The inner membrane lacks cholesterol and is impermeable to molecules and ions. Species that must cross the mitochondrial inner membrane—ions, substrates, fatty acids for oxidation, and so on—are carried by specific transport proteins in the membrane. Notably, the inner membrane is extensively folded (Figure 19.1). The folds, known as cristae, provide the inner membrane with a large surface area in a small volume. During periods of active respiration, the inner membrane appears to shrink significantly, leaving a comparatively large intermembrane space.

The Mitochondrial Matrix Contains the Enzymes of the TCA Cycle The space inside the inner mitochondrial membrane is called the matrix, and it contains most of the enzymes of the TCA cycle and fatty acid oxidation. (An important exception, succinate dehydrogenase of the TCA cycle, is located in the inner membrane itself.) In addition, mitochondria contain circular DNA molecules, along with ribosomes and the enzymes required to synthesize proteins coded within the mitochondrial genome. Although some of the mitochondrial proteins are made this way, most are encoded by nuclear DNA and synthesized by cytosolic ribosomes.

19.2

What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions?

On numerous occasions in earlier chapters, we have stressed that NADH and reduced flavoproteins ([FADH2]) are forms of metabolic energy. These reduced coenzymes have a strong tendency to be oxidized—that is, to transfer electrons to other species. Oxidative NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 3 Metabolism and Its Regulation

phosphorylation converts the energy of electron transfer into the energy of phosphoryl transfer stored in the phosphoric anhydride bonds of ATP. Just as the group transfer potential was used in Chapter 3 to quantitate the energy of phosphoryl transfer, the standard reduction potential, denoted by eo !, quantitates the tendency of chemical species to be reduced or oxidized. The standard reduction potential difference describing electron transfer between 2 species,

Reduced donor Oxidized donor

ne"

Oxidized acceptor Reduced acceptor

(19.1)

is related to the free energy change for the process by #G $ = -nf#eo !

(19.2)

where n represents the number of electrons transferred; f is Faraday’s constant, 96 485 J/V·mol; and #e0! is the difference in reduction potentials between the donor and acceptor. This relationship is straightforward, but it depends on a standard of reference by which reduction potentials are defined.

Standard Reduction Potentials Are Measured in Reaction Half-Cells (a)

Ethanol

acetaldehyde –0.197 V Potentiometer Electron flow

Electron flow Agar bridge

2 H+

Ethanol

H2

acetaldehyde

Sample: acetaldehyde/ ethanol (b)

Fumarate

Reference H+ /1 atm H2

succinate +0.031 V

Electron flow

Electron flow Agar bridge

Succinate

H2

Fumarate

Sample: fumarate/ succinate

2 H+

Reference H+ /1 atm H2

FIGURE 19.2 Experimental apparatus used to measure the standard reduction potential of the indicated redox couples: (a) the acetaldehyde/ethanol couple, (b) the fumarate/succinate couple.

Standard reduction potentials are determined by measuring the voltages generated in reaction half-cells (Figure 19.2). A half-cell consists of a solution containing 1 M concentrations of both the oxidized and the reduced forms of the substance whose reduction potential is being measured and a simple electrode. (Together, the oxidized and reduced forms of the substance are referred to as a redox couple.) Such a sample half-cell is connected to a reference half-cell and electrode via a conductive bridge (usually a salt-containing agar gel). A sensitive potentiometer (voltmeter) connects the 2 electrodes so that the electrical potential (voltage) between them can be measured. The reference half-cell normally contains 1 M H+ in equilibrium with H2 gas at a pressure of 1 atm. The H+/H2 reference half-cell is arbitrarily assigned a standard reduction potential of 0.0 V. The standard reduction potentials of all other redox couples are defined relative to the H+/H2 reference half-cell on the basis of the sign and magnitude of the voltage (electromotive force, emf) registered on the potentiometer (Figure 19.2). If electron flow between the electrodes is toward the sample half-cell, reduction occurs spontaneously in the sample half-cell and the reduction potential is said to be positive. If electron flow between the electrodes is away from the sample half-cell and toward the reference cell, the reduction potential is said to be negative because electron loss (oxidation) is occurring in the sample half-cell. Strictly speaking, the standard reduction potential, eo!, is the electromotive force generated at 25°C and pH 7.0 by a sample half-cell (containing 1 M concentrations of the oxidized and reduced species) with respect to a reference halfcell. (Note that the reduction potential of the hydrogen half-cell is pH dependent. The standard reduction potential, 0.0 V, assumes 1 M H+. The hydrogen half-cell measured at pH 7.0 has an eo! of -0.421 V.) Two Examples Figure 19.2a shows a sample/reference half-cell pair for measurement of the standard reduction potential of the acetaldehyde/ethanol couple. Because electrons flow toward the reference half-cell and away from the sample half-cell, the standard reduction potential is negative, specifically -0.197 V. In contrast, the fumarate/succinate couple (Figure 19.2b) causes electrons to flow from the reference half-cell to the sample half-cell; that is, reduction occurs, and the reduction potential is thus positive. For each half-cell, a half-cell reaction describes the reaction taking place. For the fumarate/succinate half-cell coupled to a H+/H2 reference half-cell, the reaction occurring is indeed a reduction of fumarate: Fumarate + 2 H+ + 2 e- h succinate

eo ! = +0.031 V

(19.3)

However, the reaction occurring in the acetaldehyde/ethanol half-cell is the oxidation of ethanol: Ethanol h acetaldehyde + 2 H+ + 2 e-

eo! = -0.197 V

(19.4) NEL

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Chapter 19

Electron Transport and Oxidative Phosphorylation

eo! Values Can Be Used to Predict the Direction of Redox Reactions Some typical half-cell reactions and their respective standard reduction potentials are listed in Table 19.1. Whenever reactions of this type are tabulated, they are uniformly written as reduction reactions, regardless of what occurs in the given half-cell. The sign of the standard reduction potential indicates which reaction really occurs when the given halfcell is combined with the reference hydrogen half-cell. Redox couples that have large

TABLE 19.1 Standard Reduction Potentials for Several Biological Reduction Half-Reactions Reduction Half-Reaction 1 2

eo! (V) 0.816

O2 + 2 H+ + 2 e- h H2O

Fe3+ + e- h Fe2+

0.771

Photosystem P700

0.430

NO-3 + 2 H+ + 2 e- h NO-2 + H2O Cytochrome f

(Fe3+)

Cytochrome a

+

(Fe3+)

Cytochrome a3

e-

h cytochrome f

e-

+

(Fe3+)

e-

+

0.421 0.365

(Fe2+)

h cytochrome a3 h cytochrome a

0.350

(Fe2+)

0.290

(Fe2+)

Rieske Fe-S (Fe3+) + e- h Rieske Fe-S (Fe2+)

0.280

Cytochrome c (Fe3+) + e- h cytochrome c (Fe2+)

0.254

(Fe3+)

Cytochrome c1 UQH #

+

UQ + 2

H+

H+

e-

+

h cytochrome c1

0.220

(Fe2+)

+

e-

h UQH2 (UQ = coenzyme Q)

0.190

+ 2

e-

h UQH2

0.060 0.050

Cytochrome bH (Fe3+) + e- h cytochrome bH (Fe2+) Fumarate + 2 UQ +

H+

H+

e-

+

+ 2

0.031

h succinate

0.030

h UQH·

(Fe3+)

Cytochrome b5

e-

e-

+

h cytochrome b5

0.020

(Fe2+)

[FAD] + 2 H+ + 2 e- h [FADH2] Cytochrome bL

(Fe3+)

+

H+

Oxaloacetate + 2

e-

+ 2

0.003–0.091*

h cytochrome bL

e-

-0.100

(Fe2+)

-0.166

h malate

Pyruvate + 2 H+ + 2 e- h lactate Acetaldehyde + 2 FMN + 2

H+

FAD + 2

H+

H+

+ 2

e-

-0.185 -0.197

h ethanol

+ 2

e-

h FMNH2

-0.219

+ 2

e-

h FADH2

-0.219

Glutathione (oxidized) + 2 H+ + 2 e- h 2 glutathione (reduced) Lipoic acid + 2

H+

+ 2

e-

1,3-Bisphosphoglycerate + 2

h dihydrolipoic acid

-0.290

H+

-0.290

+ 2

e-

h glyceraldehyde-3-phosphate + Pi

-0.320

NAD+ + 2 H+ + 2 e- h NADH + H+ NADP+

+ 2

H+

+ 2

e-

h NADPH +

Lipoyl dehydrogenase [FAD] + 2 [FADH2]

H+

+ 2

-0.320

H+

e-

h lipoyl dehydrogenase

2

+ 2

e-

-0.421

h H2

Ferredoxin (spinach)

(Fe3+)

-0.340 -0.380

a-Ketoglutarate + CO2 + 2 H+ + 2 e- h isocitrate H+

-0.230

+

e-

h ferredoxin (spinach)

(Fe2+)

Succinate + CO2 + 2 H+ + 2 e- h a-ketoglutarate + H2O

-0.430 -0.670

*Typical values for reduction of bound FAD in flavoproteins such as succinate dehydrogenase. (See Bonomi, F., Pagani, S., Cerletti, P., and Giori, C., 1983. Modification of the thermodynamic properties of the electron-transferring groups in mitochondrial succinate dehydrogenase upon binding of succinate. European Journal of Biochemistry 134:439–445.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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positive reduction potentials have a strong tendency to accept electrons, and the oxidized form of such a couple (e.g., O2) is a strong oxidizing agent. Redox couples with large negative reduction potentials have a strong tendency to undergo oxidation (i.e., donate electrons), and the reduced form of such a couple (e.g., NADPH) is a strong reducing agent.

eo! Values Can Be Used to Analyze Energy Changes in Redox Reactions The half-reactions and reduction potentials in Table 19.1 can be used to analyze energy changes in redox reactions. The oxidation of NADH to NAD+ can be coupled with the reduction of a-ketoglutarate to isocitrate: NAD+ + isocitrate h NADH + H+ + a-ketoglutarate + CO2

(19.5)

This is the isocitrate dehydrogenase reaction of the TCA cycle. Writing the 2 half-cell reactions, we have NAD+ + 2 H+ + 2 e- h NADH + H+ eo! = -0.32 V

(19.6)

a-Ketoglutarate + CO2 + 2 H+ + 2 e- h isocitrate eo! = -0.38 V

(19.7)

In a spontaneous reaction, electrons are donated by (flow away from) the half-reaction with the more negative reduction potential and are accepted by (flow toward) the halfreaction with the more positive reduction potential. Thus, in the present case, isocitrate donates electrons and NAD+ accepts electrons. The convention defines #eo! as #eo! = e%!(acceptor) - e% ! (donor)

(19.8)

In the present case, isocitrate is the donor and NAD+ the acceptor, so we write #eo ! = -0.32 V - (-0.38 V) = +0.06 V

(19.9)

From Equation 19.2, we can now calculate #G°! as #G $! = -(2) (96.485 kJ/V # mol) (0.06 V)

(19.10)

#G $! = -11.58 kJ/mol Note that a reaction with a net positive #eo! yields a negative #G°!, indicating a spontaneous reaction.

The Reduction Potential Depends on Concentration We have already noted that the standard free energy change for a reaction, #G°!, does not reflect the actual conditions in a cell, where reactants and products are not at standardstate concentrations (1 M). Equation 3.13 was introduced to permit calculations of actual free energy changes under non–standard-state conditions. Similarly, standard reduction potentials for redox couples must be modified to account for the actual concentrations of the oxidized and reduced species. For any redox couple, ox + ne- ∆ red

(19.11)

the actual reduction potential is given by e = eo ! + (RT>nf) ln

[ox] [red]

(19.12)

Reduction potentials can also be sensitive to molecular environment. The influence of environment is especially important for flavins, such as FAD/FADH2 and FMN/FMNH2. These species are normally bound to their respective flavoproteins; the reduction potential of bound FAD, for example, can be very different from the value shown in Table 19.1 for the free FAD/FADH2 couple of -0.219 V. Problem 7 at the end of the chapter addresses this case. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 19

19.3

Electron Transport and Oxidative Phosphorylation

627

How Is the Electron-Transport Chain Organized?

As we have seen, the metabolic energy from oxidation of food materials—sugars, fats, and amino acids—is funnelled into formation of reduced coenzymes (NADH) and reduced flavoproteins ([FADH2]). The electron-transport chain reoxidizes the coenzymes and channels the free energy obtained from these reactions into the creation of a proton gradient. This reoxidation process involves the removal of both protons and electrons from the coenzymes. Electrons move from NADH and [FADH2] to molecular oxygen, O2, which is the terminal acceptor of electrons in the chain. The reoxidation of NADH, NADH (reductant) + H+ + O2 (oxidant) h NAD+ + H2O

(19.13)

involves the following half-reactions: NAD+ + 2 H+ + 2 e- h NADH + H+ 1 2

O2 + 2

H+

+ 2

e-

h H2O

eo ! = -0.32 V

(19.14)

eo ! = +0.816 V

(19.15)

Here, half-reaction 19.15 is the electron acceptor, and half-reaction 19.14 is the electron donor. Then #eo ! = 0.816 - (-0.32) = 1.136 V

(19.16)

and, according to Equation 19.2, the standard-state free energy change, #G°!, is -219 kJ/mol. Molecules along the electron-transport chain have reduction potentials between the values for the NAD+/NADH couple and the oxygen/H2O couple, so electrons move down the energy scale toward progressively more positive reduction potentials (Figure 19.3). Although electrons move from more negative to more positive reduction potentials in the electron-transport chain, it should be emphasized that the electron carriers do not operate in a simple linear sequence. This will become evident when the individual components of the electron-transport chain are discussed in the following paragraphs.

FIGURE 19.3 eo! and e values for the components of the mitochondrial electron-transport chain. Values indicated are consensus values for animal mitochondria. Black bars represent eo!; red bars e.

(Fe/S)N3

(Fe/S)N1 (Fe/S)N4

FMN

–400

NAD+/NADH

Complex I

–200

bL

a c

Cu A

c1

Rieske Fe/S

bH UQ10

(Fe/S)S1

Complex IV

a3

+400

(Fe/S)S3

+200

(Fe/S)N2

FAD

0 Fum/Succ

(mV)

Complex II

Complex III

+600 NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 3 Metabolism and Its Regulation

The Electron-Transport Chain Can Be Isolated in Four Complexes The electron-transport chain involves several different molecular species, including 1. Flavoproteins, which contain tightly bound FMN or FAD as prosthetic groups and which may participate in 1- or 2-electron transfer events. 2. Coenzyme Q, also called ubiquinone (and abbreviated CoQ or UQ) (see Figure 19.5 on page 630), which can function in either 1- or 2-electron transfer reactions. 3. Several cytochromes [proteins containing heme prosthetic groups (see Chapter 5), which function by carrying or transferring electrons], including cytochromes b, c, c1, a, and a3. Cytochromes are 1-electron transfer agents in which the heme iron is converted from Fe2+ to Fe3+ and back. 4. A number of iron–sulfur proteins, which participate in 1-electron transfers involving the Fe2+ and Fe3+ states. 5. Protein-bound copper, a 1-electron transfer site that converts between Cu+ and Cu2+. All these intermediates except for cytochrome c are membrane associated (either in the mitochondrial inner membrane of eukaryotes or in the plasma membrane of prokaryotes). Three types of proteins involved in this chain (flavoproteins, cytochromes, and iron–sulfur proteins) possess electron-transferring prosthetic groups. The components of the electron-transport chain can be purified from the mitochondrial inner membrane. Solubilization of the membranes containing the electron-transport chain results in the isolation of 4 distinct protein complexes, and the complete chain can thus be considered to be composed of 4 parts: (I) NADH–coenzyme Q reductase, (II) succinate–coenzyme Q reductase, (III) coenzyme Q–cytochrome c reductase, and (IV) cytochrome c oxidase (Figure 19.4). Complex I accepts electrons from NADH, serving as a link between glycolysis, the TCA cycle, fatty acid oxidation, and the electron-transport chain. Complex II includes succinate dehydrogenase and thus forms a direct link between the TCA cycle and electron transport. Complexes I and II produce a common product, reduced coenzyme Q (UQH2), which is the substrate for coenzyme Q–cytochrome c reductase (Complex III). As shown in Figure 19.4, there are 2 other ways to feed electrons to UQ: the electron-transferring flavoprotein, which transfers electrons from the flavoprotein-linked step of fatty acyl-CoA dehydrogenase; and sn-glycerophosphate dehydrogenase. Complex III oxidizes UQH2 while reducing cytochrome c, which in turn is the substrate for Complex IV, cytochrome c oxidase. Complex IV is responsible for reducing molecular oxygen. Each Fatty acyl-CoA dehydrogenase Complex I Flavoprotein 1

Flavoprotein 3

NADH dehydrogenase, FMN, Fe-S centres

Electron-transferring flavoprotein, FAD, Fe-S centres

NADH–coenzyme Q oxidoreductase

Complex III

UQ /UQH2 pool

Cytochrome bc1 complex, 2 b -type hemes, Rieske Fe-S centre, C-type heme (cyt c1) Coenzyme Q–cytochrome c oxidoreductase

Complex II Flavoprotein 2

Flavoprotein 4

Succinate dehydrogenase, FAD (covalent), Fe-S centres, b-type heme

Sn-glycerophosphate dehydrogenase FAD, Fe-S centres

Succinate–coenzyme Q oxidoreductase

1 2 O2

H2O

Complex IV

Cytochrome c

Cytochrome aa 3 complex, 2 a-type hemes, Cu ions Cytochrome c oxidase

FIGURE 19.4 An overview of the complexes and pathways in the mitochondrial electrontransport chain. (Adapted from Nicholls, D. G., and Ferguson, S. J., 2002. Bioenergetics 3. London: Academic Press.) NEL

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Chapter 19

Electron Transport and Oxidative Phosphorylation

of the complexes shown in Figure 19.4 is a large multisubunit complex embedded within the inner mitochondrial membrane.

Complex I Oxidizes NADH and Reduces Coenzyme Q As its name implies, this complex transfers a pair of electrons from NADH to coenzyme Q, a small, hydrophobic, yellow compound. Another common name for this enzyme complex is NADH dehydrogenase. The complex (with an estimated mass of 980 kD) involves at least 45 polypeptide chains, 1 molecule of flavin mononucleotide (FMN), and 8 or 9 Fe-S clusters, together containing a total of 20–26 iron atoms (Table 19.2). By virtue of its dependence on FMN, NADH–UQ reductase is a flavoprotein. Although the precise mechanism of the NADH–UQ reductase is unknown, the first step involves binding of NADH to the enzyme on the matrix side of the inner mitochondrial membrane and transfer of electrons from NADH to tightly bound FMN: NADH + [FMN] + H+ h [FMNH2] + NAD+

(19.17)

The second step involves the transfer of electrons from the reduced [FMNH2] to a series of Fe-S proteins, including both 2Fe-2S and 4Fe-4S clusters (see page 632). The versatile redox properties of the flavin group of FMN are important here. NADH is a 2-electron donor, whereas the Fe-S proteins are 1-electron transfer agents. The flavin of FMN has 3 redox states: the oxidized, semiquinone, and reduced states. It can act as either a 1-electron or a 2-electron transfer agent and may serve as a critical link between NADH and the Fe-S proteins. The final step of the reaction involves the transfer of 2 electrons from iron–sulfur clusters to coenzyme Q. Coenzyme Q is a mobile electron carrier. Its isoprenoid tail makes it highly hydrophobic, and it diffuses freely in the hydrophobic core of the inner mitochondrial membrane. As a result, it shuttles electrons from Complexes I and II to Complex III. The redox cycle of UQ is shown in Figure 19.5. The structural and functional organization of Complex I is shown in Figure 19.6. Complex I Transports Protons from the Matrix to the Cytosol The oxidation of 1 NADH and the reduction of 1 UQ by NADH–UQ reductase results in a net transport of protons from the matrix side to the cytosolic side of the inner membrane. The cytosolic side, where H+ accumulates, is referred to as the P (for positive) face; similarly, the matrix side is the N (for negative) face. Some of the energy liberated by the flow of electrons through this complex is used in a coupled process to drive the transport of protons across

TABLE 19.2 Protein Complexes of the Mitochondrial Electron-Transport Chain Complex NADH–UQ reductase Succinate–UQ reductase UQ–cyt c reductase

Mass (kD)

Subunits

980

Ú45

140 250

4 9–10

Prosthetic Group

Binding Site for

FMN

NADH (matrix side)

Fe-S

UQ (lipid core)

FAD

Succinate (matrix side)

Fe-S

UQ (lipid core)

Heme bL

Cyt c (intermembrane space side)

Heme bH Heme c1 Fe-S Cytochrome c

13

1

Heme c

Cyt c1 Cyt a

Cytochrome c oxidase

162

13

Heme a Heme a3

Cyt c (intermembrane space side)

CuA CuB NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

629

630

Part 3 Metabolism and Its Regulation (a)

O

H3CO

CH3

H3CO

(CH2

e–

CH3 CH

C

CH2)10

+

O•

H+

H

e–

CH3O

CH3

CH3O

R

+

OH

O Coenzyme Q, oxidized form (Q, ubiquinone)

H3CO

CH3

H3CO

R OH

Semiquinone intermediate (QH •)

(b)

OH

H+

Coenzyme Q, reduced form (QH2, ubiquinol)

FIGURE 19.5 (a) The 3 oxidation states of coenzyme Q. (b) A space-filling model of coenzyme Q. 2 H+

(a)

2 H+

(b) 8/10

2 e– UQH2

UQ

2 Fe-S centres

7/11/14

13

12

2 e–

2 H+ 2 H+

2 Fe-S centres 2 H+ FMNH2

FMN

NADH + H+

NAD+ (c)

N2 N6b N6a N5 N1b N4

N3 N1a

FMN

N7

FIGURE 19.6 (a) Structural organization of mammalian Complex I, based on electron microscopy, showing functional relationships within the L-shaped complex. Electron flow from NADH to UQH2 in the membrane pool is indicated. (b) Structure of the hydrophilic domain of Complex I from Thermus thermophilus is shown on a model of the membrane-associated complex (pdb id = 2FUG). The locations of individual subunits are indicated. (c) Arrangement of the redox centres in Complex I. The various iron–sulfur centres of Complex I are designated by capital N. (Part a adapted from Janssen, R. J., Nijtmans, L. G., van den Heuvel, L. P., and Smeitink, J. A., 2006. Mitochondrial complex I: Structure, function, and pathology. Journal of Inherited Metabolic Diseases 29:499–515; and parts b and c adapted from Figure 1 of Sazanov, L., and Hinchliffe, P., 2006. Structure of the hydrophilic domain of respiratory Complex I from Thermus thermophilus. Science 311:1430–1436.) NEL

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Chapter 19

Electron Transport and Oxidative Phosphorylation

631

HUMAN BIOCHEMISTRY

Solving a Medical Mystery Revolutionized Our Treatment of Parkinson’s Disease A tragedy among illegal drug users was the impetus for a revolutionary treatment of Parkinson’s disease. In 1982, several mysterious cases of paralysis came to light in southern California. The victims, some of them teenagers, were frozen like living statues, unable to talk or move. The case was baffling at first, but it was soon traced to a batch of synthetic heroin that contained MPTP (1-methyl-4phenyl-1,2,3,6-tetrahydropyridine) as a contaminant. MPTP is rapidly converted in the brain to MPP+(1-methyl-4-phenyl pyridine) by CH3

H CH3 H H

N+

H H H H

N+ Monoamine oxidase B

MPP+

MPTP

Cell death in substantia nigra

the enzyme monoamine oxidase B. MPP+is a potent inhibitor of mitochondrial Complex I (NADH–UQ reductase), and it acts preferentially in the substantia nigra, an area of the brain that is essential to movement and also the region of the brain that deteriorates slowly in Parkinson’s disease. Parkinson’s disease results from the inability of the brain to produce sufficient quantities of dopamine, a neurotransmitter. Neurologist J. William Langston, asked to consult on the treatment of some of these patients, recognized that the symptoms of this druginduced disorder were in fact similar to those of Parkinsonism. He began treatment of the patients with L-dopa, which is decarboxylated in the brain to produce dopamine. The treated patients immediately regained movement. Langston then took a bold step. He implanted fetal brain tissue into the brains of several of the affected patients, prompting substantial recovery from the Parkinson-like symptoms. Langston’s innovation sparked a revolution in the use of tissue implantation for the treatment of neurodegenerative diseases. Other toxins may cause similar effects in neural tissue. Timothy Greenamyre at Emory University showed that rats exposed to the pesticide rotenone (see Figure 19.27 on page 646) over a period of weeks experience a gradual loss of function in dopaminergic neurons and then develop symptoms of Parkinsonism, including limb tremors and rigidity. This finding supports earlier research that links long-term pesticide exposure to Parkinson’s disease.

the membrane. (This is an example of active transport, a phenomenon examined in detail in Chapter 10.) Available experimental evidence suggests a stoichiometry of 4 H+ transported per 2 electrons passed from NADH to UQ.

Complex II Oxidizes Succinate and Reduces Coenzyme Q Complex II is perhaps better known by its other name, succinate dehydrogenase, the only TCA cycle enzyme that is an integral membrane protein in the inner mitochondrial membrane. This complex (Figure 19.7) has a mass of 124 kD and is composed of 2 hydrophilic subunits, a flavoprotein (Fp, 68 kD) and an iron–sulfur protein (Ip, 29 kD), and 2 hydrophobic, membrane-anchored subunits (15 kD and 11 kD), which contain 1 heme b and provide the binding site for UQ. Fp contains an FAD covalently bound to a His residue (see Figure 18.12), and Ip contains 3 Fe-S centres: a 4Fe-4S cluster, a 3Fe-4S cluster, and a 2Fe-2S cluster. When succinate is converted to fumarate in the TCA cycle, concomitant reduction of bound FAD to FADH2 occurs in succinate dehydrogenase. This FADH2 transfers its electrons immediately to Fe-S centres, which pass them on to UQ. Electron flow from succinate to UQ, Succinate h fumarate + 2 H+ + 2 eUQ + 2

H+

+ 2

e-

h UQH2

Net rxn: Succinate + UQ h fumarate + UQH2

(19.18) (19.19)

#eo ! = 0.029 V

(19.20)

yields a net reduction potential of 0.029 V. (Note that the first half-reaction is written in the direction of the e- flow. As always, #e0! is calculated according to Equation 19.8.) The small free energy change of this reaction does not contribute to the transport of protons across the inner mitochondrial membrane. This is a crucial point because (as we will see) proton transport is coupled with ATP synthesis. Oxidation of 1 FADH2 in the electron-transport chain results in synthesis of approximately 2 molecules of ATP, compared with the approximately 3 ATPs produced by NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

632

Part 3 Metabolism and Its Regulation

FIGURE 19.7 (a) A scheme for electron flow in Complex II. Oxidation of succinate occurs with reduction of [FAD]. Electrons are then passed to Fe-S centres and then to coenzyme Q (UQ). Proton transport does not occur in this complex. (b) The structure of Complex II from pig heart (pdb id = 1ZOY). (c) The arrangement of redox centres. Electron flow is from bottom to top.

(a)

Intermembrane space

Complex II Complex III UQH2

2Fe3+

UQ

Heme b

2Fe2+

3Fe4S 4Fe4S 2Fe2S Matrix

2 H+ FAD

Succinate (b)

FADH2

Fumarate

(c)

Heme b

Fe-S centres

FAD

the oxidation of 1 NADH. Other flavoproteins can also supply electrons to UQ, including mitochondrial sn-glycerophosphate dehydrogenase, an inner membrane-bound shuttle enzyme, and the fatty acyl-CoA dehydrogenases: 3 soluble matrix enzymes involved in fatty acid oxidation (Figure 19.8; also see Chapter 22). The path of electrons from succinate to UQ is shown in Figure 19.7.

FIGURE 19.8 The fatty acyl-CoA dehydrogenase reaction, emphasizing that the reaction involves reduction of enzyme-bound FAD (indicated by brackets).

O C

H3C

SCoA [FAD] UQ

H3C

[FADH2]

O C SCoA

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Chapter 19

633

Electron Transport and Oxidative Phosphorylation

Complex III Mediates Electron Transport from Coenzyme Q to Cytochrome c In the third complex of the electron-transport chain, reduced coenzyme Q (UQH2) passes its electrons to cytochrome c via a unique redox pathway known as the Q cycle. UQ– cytochrome c reductase (UQ–cyt c reductase), as this complex is known, involves 3 different cytochromes and an Fe-S protein. In the cytochromes of these and similar complexes, the iron atom at the centre of the porphyrin ring cycles between the reduced Fe2+ (ferrous) and oxidized Fe3+ (ferric) states. Cytochromes were first named and classified on the basis of their absorption spectra (Figure 19.9), which depend upon the structure and environment of their heme groups. The b cytochromes contain iron protoporphyrin IX (Figure 19.10), the same heme found in hemoglobin and myoglobin. The c cytochromes contain heme c, derived from iron protoporphyrin IX by the covalent attachment to cysteine residues from the associated protein. [One other heme variation, heme a, contains a 15-carbon isoprenoid chain on a modified vinyl group and a formyl group in place of 1 of the methyls (see Figure 19.10). Cytochrome a is found in 2 forms in Complex IV of the electron-transport chain, as we shall see.] UQ–cyt c  reductase (Figure 19.11) contains a b-type cytochrome, of 30–40 kD, with 2 different heme sites and 1 c-type cytochrome. The 2 hemes on the b cytochrome polypeptide in UQ– cyt c reductase are distinguished by their reduction potentials and the wavelength (lmax) of the so-called a-band. One of these hemes, known as bL or b566, has a standard reduction potential, eo !, of -0.100 V and a wavelength of maximal absorbance (lmax) of 566 nm. The other, known as bH or b562, has a standard reduction potential of +0.050 V and a lmax of 562 nm. (H and L here refer to high and low reduction potential.) The structure of the UQ–cyt c reductase, also known as the cytochrome bc1 complex, has been determined by Johann Deisenhofer and his colleagues. (Deisenhofer was a co-recipient of the Nobel Prize in Chemistry for his work on the structure of a photosynthetic reaction centre; see Chapter 20.) The complex is a dimer, with each monomer consisting of 11 protein subunits and 2165 amino acid residues (monomer mass, 248 kD). The dimeric structure is pear shaped and consists of a large domain that extends 75 Å into the mitochondrial matrix, a transmembrane domain consisting of 13 transmembrane a-helices in each monomer and a small domain that extends 38 Å into the intermembrane space (Figure 19.11). Most of the Rieske protein (an Fe-S protein named for its discoverer) is mobile in the crystal (only 62 of its 196 residues are shown in the structure in Figure 19.11), and Deisenhofer postulated that mobility of this subunit could be required for electron transfer in the function of this complex.

H3 C CH2

CH

CH

CH2 CH3

N N

Fe

N

N

H3C

CH2CH2COO

_

_ CH2CH2COO

H3C

Iron protoporphyrin IX (found in cytochrome b, myoglobin, and hemoglobin)

S H3C

S

CHCH3

CH3CH

CH3

N N

Fe

N

N

H3C

CH2CH2COO

H3 C

CH2CH2COO

_

_

Heme c (found in cytochrome c)

Complex III Drives Proton Transport As with Complex I, passage of electrons through the Q cycle of Complex III is accompanied by proton transport across the inner mitochondrial membrane. The postulated pathway for electrons in this system is shown in Figure 19.12. A large pool of UQ and UQH2 exists in the inner mitochondrial membrane. The Q cycle is

α β OH

H3 C

CH

CH2CH

(a) (a) Cytochrome c: reduced spectrum Absorbance

(b) Cytochrome c: oxidized spectrum O

500 550 600 Wavelength (nm)

650

FIGURE 19.9 Visible absorption spectra of cytochrome c.

Fe

N

N

H3C

450

CH3

N N

(b)

CH2

CH

CH2CH2COO CH2CH2COO

Heme a (found in cytochrome a)

FIGURE 19.10 The structures of iron protoporphyrin IX, heme c, and heme a.

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_

_

634

Part 3 Metabolism and Its Regulation

FIGURE 19.11 The structure of UQ–cyt c reductase, also known as the cytochrome bc1 complex. The a-helical bundle near the top of the structure defines the transmembrane domain of the protein (pdb id = 1BE3).

FIGURE 19.12 The Q cycle in mitochondria. (a) The electron-transfer pathway following oxidation of the first UQH2 at the Qp site near the cytosolic face of the membrane. (b) The pathway following oxidation of a second UQH2.

(a) First half of Q cycle

Intermembrane space (P-phase)

UQH2 UQ–

UQH2

UQH2

2 H+

Qp site

UQ

UQ

UQ

Cyt c

First UQH2 from pool

e–

2 e – oxidation 1 e– at Qp site

e–

UQ UQ–

Matrix (N-phase)

FeS

2 H+ out

Cyt bL

UQ

Pool

Cyt c1

e–

Synopsis

Cyt c

UQ to pool

Cyt bH

1 e– UQ at Qn site

Qn site

(b) Second half of Q cycle

Qp site

Intermembrane space (P-phase) UQH2 UQH2

UQ

UQ Pool

e–

UQ

e–

+

2 H+in

FeS

2 H+ out

e– Cyt bH

UQH2 to pool

Qn site

+ 2 Cyt cox

2 H+ – 2e 4 H+ out

Cyt c

Second UQH2 from pool UQ to pool

UQ– UQH2

Net UQH2

Cyt c1

UQH2 UQ–

Synopsis

Cyt c

Cyt bL

UQH2

Matrix (N-phase)

2 H+

+ 2 Cyt cred + UQ

2 e – oxidation 1 e– at Qp site

UQ.– at Qn site

1 e–

2 H+ NEL

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Chapter 19

Electron Transport and Oxidative Phosphorylation

635

initiated when a molecule of UQH2 from this pool diffuses to a site (called Qp) on Complex III near the cytosolic face of the membrane. Oxidation of this UQH2 occurs in 2 steps: First, an electron from UQH2 is transferred to the Rieske protein and then to cytochrome c1. This releases 2 H+ to the cytosol and leaves UQ # -, a semiquinone anion form of UQ, at the Qp site. Next, the second electron is transferred to the bL heme, converting UQ # - to UQ. The Rieske protein and cytochrome c1 are similar in structure; each has a globular domain and is anchored to the inner mitochondrial membrane by a hydrophobic segment. However, the hydrophobic segment is N-terminal in the Rieske protein and C-terminal in cytochrome c1. The electron on the bL heme facing the cytosolic side of the membrane is now passed to the bH heme on the matrix side of the membrane. This electron transfer occurs against a membrane potential of 0.15 V and is driven by the loss of redox potential as the electron moves from bL (eo= = -0.100 V)) to bH (eo= = +0.050 V)). The electron is then passed from bH to a molecule of UQ at a second quinone-binding site, Qn, converting this UQ to UQ # -. The resulting UQ # remains firmly bound to the Qn site. This completes the first half of the Q cycle (Figure 19.12a). The second half of the cycle (Figure 19.12b) is similar to the first half, with a second molecule of UQH2 oxidized at the Qp site, 1 electron being passed to cytochrome c1 and the other transferred to heme bL and then to heme bH. In this latter half of the Q cycle, however, the bH electron is transferred to the semiquinone anion, UQ # -, at the Qn site. With the addition of 2 H+ from the mitochondrial matrix, this produces a molecule of UQH2, which is released from the Qn site and returns to the coenzyme Q pool, completing the Q cycle. The Q Cycle Is an Unbalanced Proton Pump Why has nature chosen this convoluted path for electrons in Complex III? First, Complex III takes up 2 protons on the matrix side of the inner membrane and releases 4 protons on the cytoplasmic side for each pair of electrons that passes through the Q cycle. The other significant feature of this mechanism is that it offers a convenient way for a 2-electron carrier, UQH2, to interact with the bL and bH hemes, the Rieske protein Fe-S cluster, and cytochrome c1, all of which are 1-electron carriers. Cytochrome c Is a Mobile Electron Carrier Electrons traversing Complex III are passed through cytochrome c1 to cytochrome c. Cytochrome c is the only one of the mitochondrial cytochromes that is water soluble. Its structure (Figure 19.13) is globular; the planar heme group lies near the centre of the protein, surrounded predominantly by hydrophobic amino acid residues. The iron in the porphyrin ring is coordinated to both a histidine nitrogen and the sulfur atom of a methionine residue. Coordination with ligands in this manner on both sides of the porphyrin plane precludes the binding of oxygen and other ligands, a feature that distinguishes cytochrome c from hemoglobin (see Chapter 14). Cytochrome c, like UQ, is a mobile electron carrier. It associates loosely with the inner mitochondrial membrane (in the intermembrane space on the cytosolic side of the inner membrane) to acquire electrons from the Fe-S–cyt c1 aggregate of Complex III, and then it migrates along the membrane surface in the reduced state, carrying electrons to cytochrome c oxidase, the fourth complex of the electron-transport chain.

FIGURE 19.13 The structure of mitochondrial cytochrome c. The heme is shown at the centre of the structure. It is covalently linked to the protein via 2 sulfur atoms. A third sulfur from a methionine residue coordinates the iron (pdb id = 2B4Z).

Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side Complex IV is called cytochrome c oxidase because it accepts electrons from cytochrome c and directs them to the 4-electron reduction of O2 to form H2O: 4 cyt c (Fe2+) + 4 H+ + O2 h 4 cyt c (Fe3+) + 2 H2O

(19.21)

Thus, cytochrome c oxidase and O2 are the final destination for the electrons derived from the oxidation of food materials. In concert with this process, cytochrome c oxidase also drives transport of protons across the inner mitochondrial membrane. The combined processes of oxygen reduction and proton transport involve a total of 8 H+ in each catalytic cycle: 4 H+ for O2 reduction and 4 H+ transported from the matrix to the intermembrane space. The total number of subunits in cytochrome c oxidase varies from 2–4 (in bacteria) to 13 (in mammals). Three subunits (I, II, and III) are common to most organisms (Figure 19.14).

FIGURE 19.14 Bovine cytochrome c oxidase consists of 13 subunits. The 3 largest subunits—I (purple), II (yellow), and III (blue)—contain the proton channels and the redox centres (pdb id = 2EIJ).

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 3 Metabolism and Its Regulation

FIGURE 19.15 The complete structure of bovine cytochrome c oxidase (pdb id = 2EIJ).

This minimal complex, which contains 2 hemes (termed a and a3) and 3 copper ions (2 in the CuA centre and 1 in the CuB site), is sufficient to carry out both oxygen reduction and proton transport. The total mass of the protein in mammalian Complex IV (Figure 19.15 above) is 204 kD. In mammals, subunits I through III, the largest ones, are encoded by mitochondrial DNA, synthesized in the mitochondrion, and inserted into the inner membrane from the matrix side. The 10 smaller subunits are coded by nuclear DNA, are synthesized in the cytosol, and are presumed to play regulatory roles in the complex. In the bovine structure, subunit I is cylindrical in shape and consists of 12 transmembrane helices, without any significant extramembrane parts. Hemes a and a3, which lie perpendicular to the membrane plane, and CuB are cradled by the helices of subunit I (Figure 19.16). Subunits II and III lie on opposite sides of subunit I and do not contact each other (see Figure 19.14). Subunit II has an extramembrane domain on the outer face FIGURE 19.16 The electron-transfer pathway for cytochrome oxidase. Cytochrome c binds on the cytosolic side, transferring electrons through the copper and heme centres to reduce O2 on the matrix side of the membrane (pdb id = 2EIJ).

2 H+

2 & Cyt c 2 & e–

CuA 2 & e–

Cyt a

CuB 2&

e– Cyt a3

1 – 2 O2

2 H+

+ 2 H+

H2O

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 19

Electron Transport and Oxidative Phosphorylation

of the inner mitochondrial membrane. This domain consists of a 10-strand b-barrel that holds the 2 copper ions of the CuA site 7 Å from the nearest surface atom of the subunit. Subunit III consists of 7 transmembrane helices with no significant extramembrane domains. Electron Transfer in Complex IV Involves Two Hemes and Two Copper Sites Electron transfer through Complex IV begins with binding of cytochrome c to the b-barrel of subunit II. Four electrons are transferred sequentially (1 each from 4 molecules of cytochrome c) first to the CuA centre, next to heme a, and finally to the CuB/heme a3 active site, where O2 is reduced to H2O (Figure 19.16): Cyt c h CuA h heme a h CuB >heme a3 h O2

(19.22)

A tryptophan residue, which lies 5 Å above the CuA site (Figure 19.17a), is the entry point for electrons from cytochrome c. It lies in a hydrophobic patch on subunit II, surrounded by a ring of negatively charged Asp and Glu residues. Electrons flow rapidly from CuA to heme a, which is coordinated by a pair of His residues (Figure 19.17b), and then to the CuB/heme a3 complex. The Fe atom in heme a3 is 5-coordinate (Figure 19.17c), with 4 ligands from the heme plane and 1 from His376. This leaves a sixth position free, and this is the catalytic site where O2 binds and is reduced. CuB is about 5 Å from the Fe atom of heme a3 and is coordinated by 3 histidine ligands, including His240, His290, and His291 (Figure 19.17c). An unusual cross-link between His240 and Tyr244 lowers the pKa of the Tyr hydroxyl so that it can participate in proton transport across the membrane.

Proton Transport across Cytochrome c Oxidase Is Coupled to Oxygen Reduction Proton transport in Rhodobacter sphaeroides cytochrome c oxidase takes place via 2 channels denoted the D- and K-pathways (Figure 19.18a). Both channels contain water molecules, and they are lined with polar residues that can either protonate and deprotonate or form hydrogen bonds. The D-pathway is named for Asp132 at the channel opening, and the K-pathway is named for Lys362, a critical residue located midway in the channel. These 2 channels converge at the binuclear CuB/heme a3 site midway across the complex and the membrane. Here, Glu286 serves as a branch point, shuttling protons either to the catalytic site for O2 reduction (to form H2O) or to the exit channel (residues 320–340) that leads protons to the intermembrane space (Figure 19.18a). In each catalytic cycle, 2 H+ pass through the K-pathway and 6 H+ traverse the D-pathway. The K-pathway protons and 2 of the D-pathway protons participate in the reduction of 1 O2 to 2 H2O, and the remaining 4 D-pathway protons are passed across the membrane and released to the intermembrane space.

(a)

(b) C196

(c)

H204

H61

H161 E198

C200

H290

H240

M207

H378

H291

H376

FIGURE 19.17 Structures of the redox centres of bovine cytochrome c oxidase. (a) The CuA site, (b) the heme a site, and (c) the binuclear CuB/heme a3 site (pdb id = 2EIJ). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 3 Metabolism and Its Regulation (a)

(b)

CuA D!229

K'227

R482

H+

T337

R481 W172 Heme a

H

O

H333

H

H

O

H

H334 CuB/ heme a3

E286 E286 S201 N121

C

Y288 K362

C

T359 S365

N139

E!101

H+

D132 D-pathway

K-pathway FIGURE 19.18 (a) The proton channels of cytochrome c oxidase from R. sphaeroides. Functional residues in the D- and K-pathways are indicated. The D- and K-pathways converge at the CuB/heme a3 centre. The proton exit channel is lined by residues 320–340 of subunit I (pdb id = 1M56). (b) Protons are presumed to “hop” along arrays of water molecules in the proton transport channels of cytochrome c oxidase. Such a chain of protonation and deprotonation events means that the proton eventually released from the exit channel is far removed from the proton that entered the D-pathway and initiated the cascade.

How are protons driven across cytochrome c oxidase? The mechanism involves 3 key features: • The pKa values of protein side chains in the proton channels are shifted (by the local environment) to make them effective proton donors or acceptors during transport. For example, the pKa of Glu286 is unusually high, at 9.4. (This is similar to the behaviour of Asp85 and Asp96 in bacteriorhodopsin; see page 343, Chapter 10.) • Electron transfer events induce conformation changes that control proton transport. For example, redox events at the CuB/heme a3 site are sensed by Glu286 and an adjacent proton-gating loop (residues 169–175), controlling H+ binding and release by Glu286 and proton movement through the exit channel. • Protons are “transported” via chains of hydrogen-bonded water molecules in the proton channels (Figure 19.18b). Sequential hopping of protons along these “proton wires” essentially transfers a “positive charge” between distant residues in the channel. (Note that the H+ that arrives at an accepting residue is not the same proton that left the donating residue.)

The Four Electron-Transport Complexes Are Independent It should be emphasized here that the 4 major complexes of the electron-transport chain operate independently in the inner mitochondrial membrane. Each is a multi-protein aggregate maintained by numerous strong associations between peptides of the complex, but there is no evidence that the complexes associate with one another in the membrane. Measurements of the lateral diffusion rates of the 4 complexes, of coenzyme Q, and of cytochrome c in the inner mitochondrial membrane show that the rates differ considerably, indicating that these complexes do not move together in the membrane. Kinetic studies with reconstituted systems show that electron transport does not operate by means of connected sets of the 4 complexes. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 19

Electron Transport and Oxidative Phosphorylation

The Model of Electron Transport Is a Dynamic One The model that emerges for electron transport is shown in Figure 19.19. The 4 complexes are independently mobile in the membrane. Coenzyme Q collects electrons from NADH–UQ reductase and succinate–UQ reductase and delivers them (by diffusion through the membrane core) to UQ–cyt c reductase. Cytochrome c is water soluble and moves freely in the intermembrane space, carrying electrons from UQ–cyt c reductase to cytochrome c oxidase. In the process of these electron transfers, protons are driven across the inner membrane (from the matrix side to the intermembrane space). The proton gradient generated by electron transport represents an enormous source of potential energy. As seen in the next section, this potential energy is used to synthesize ATP as protons flow back into the matrix.

Electron Transfer Energy Stored in a Proton Gradient: The Mitchell Hypothesis As we briefly alluded to at the beginning of this chapter, in 1961, Peter Mitchell proposed that the energy stored in a proton gradient across the inner mitochondrial membrane by electron transport drives the synthesis of ATP in cells. The proposal became known as Mitchell’s chemiosmotic hypothesis. In this hypothesis, protons are driven across the membrane from the matrix to the intermembrane space and cytosol by the events of electron transport. This mechanism stores the energy of electron transport in an electrochemical potential. As protons are driven out of the matrix, the pH rises and the matrix becomes negatively charged with respect to the cytosol (Figure 19.20). Electron transport-driven proton pumping thus creates a pH gradient and an electrical gradient across the inner membrane, both of which tend to attract protons back into the matrix from the cytoplasm. Flow of protons down this electrochemical gradient, an energetically favourable process, drives the synthesis of ATP.

4 H+

4 H+

2 H+ Cyt cox

Cyt cred Cyt cox

Intermembrane space

Cyt cred

III I

UQH2

UQH2

UQ

UQ

II

IV

Matrix Succinate

Fumarate

1 – 2 O2

+ 2 H+ H2O

NADH

+

H+

NAD+ 4 H+

2 H+

2 H+

FIGURE 19.19 A model for the electron-transport pathway in the mitochondrial inner membrane. UQ/UQH2 and cytochrome c are mobile electron carriers and function by transferring electrons between the complexes. The proton transport driven by Complexes I, III, and IV is indicated. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 3 Metabolism and Its Regulation

Cytosol

H+ H+ H+ H+ H+

H+

H+

H+

H+

H+ H+

Intermembrane space (high [H+], low pH)

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

III

II

H+

H+

H+ H+

I

H+

IV

F0

H+

Matrix (low [H+], high pH)

F1

FIGURE 19.20 The proton and electrochemical gradients existing across the inner mitochondrial membrane. The electrochemical gradient is generated by the transport of protons across the membrane by Complexes I, III, and IV in the inner mitochondrial membrane.

The ratio of protons transported per pair of electrons passed through the chain (the so-called H" >2e# ratio) has been an object of great interest for many years. Nevertheless, the ratio has remained extremely difficult to determine. The consensus estimate for the electron-transport pathway from succinate to O2 is 6 H+ >2 e-. The ratio for Complex I by itself remains uncertain, but recent best estimates place it as high as 4 H+ >2 e-. On the basis of this value, the stoichiometry of transport for the pathway from NADH to O2 is 10 H+ >2 e-. Although this is the value assumed in Figure 19.19, it is important to realize that this represents a consensus drawn from many experiments.

19.4

What Are the Thermodynamic Implications of Chemiosmotic Coupling?

Mitchell’s chemiosmotic hypothesis revolutionized our thinking about the energy coupling that drives ATP synthesis by means of an electrochemical gradient. How much energy is stored in this electrochemical gradient? For the transmembrane flow of protons across the inner membrane [from inside (matrix) to outside], we could write H+in h H+out

(19.23)

The free energy difference for protons across the inner mitochondrial membrane includes a term for the concentration difference and a term for the electrical potential. This is expressed as #G = RT ln

[c2] [c1]

+ Zf#c

(19.24) NEL

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Chapter 19

Electron Transport and Oxidative Phosphorylation

641

where c1 and c2 are the proton concentrations on the 2 sides of the membrane, Z is the charge on a proton, F is Faraday’s constant, and #c is the potential difference across the membrane. For the case at hand, this equation becomes #G = RT ln

[H+out] [H+in]

+ Zf#c

(19.25)

In terms of the matrix and cytoplasm pH values, the free energy difference is #G = -2.303 RT(pHout - pHin) + f#c

(19.26)

Reported values for #c and #pH vary, but the membrane potential is always found to be positive outside and negative inside, and the pH is always more acidic outside and more basic inside. Taking typical values of #c = 0.18 V and #pH = 1 unit, the free energy change associated with the movement of 1 mole of protons from inside to outside is #G = 2.3 RT + f(0.18 V)

(19.27)

With f = 96.485 kJ/V # mol, the value of #G at 37°C is #G = 5.9 kJ + 17.4 kJ = 23.3 kJ

(19.28)

which is the free energy change for movement of a mole of protons across an inner membrane. Note that the free energy terms for both the pH difference and the potential difference are unfavourable for the outward transport of protons, with the latter term making the greater contribution. On the other hand, the #G for inward flow of protons is -23.3 kJ/mol. It is this energy that drives the synthesis of ATP, in accord with Mitchell’s model. As mentioned in the introductory paragraph, Peter Mitchell was awarded the Nobel Prize in Chemistry in 1978 for this work.

19.5

How Does a Proton Gradient Drive the Synthesis of ATP?

The great French chemist Antoine Lavoisier showed in 1777 that foods undergo combustion in the body. Since then, chemists and biochemists have wondered how energy from food oxidation is captured by living things. Mitchell paved the way by suggesting that a proton gradient across the inner mitochondrial membrane could drive the synthesis of ATP. But how could the proton gradient be coupled to ATP production? The answer lies in a mitochondrial complex called ATP synthase, or sometimes F1F0–ATPase (for the reverse reaction it catalyzes). The F1 portion of the ATP synthase was first identified in early electron micrographs of mitochondrial preparations as spherical, 8.5 nm projections or particles on the inner membrane. The purified particles catalyze ATP hydrolysis, the reverse reaction of the ATP synthase. Stripped of these particles, the membranes can still carry out electron transfer but cannot synthesize ATP. In one of the first reconstitution experiments with membrane proteins, Efraim Racker showed that adding the particles back to stripped membranes restored electron transfer-dependent ATP synthesis.

Rotor

Rotor shaft Stator

ATP Synthase Is Composed of F1 and F0 ATP synthase is a remarkable molecular machine. It is an enzyme, a proton pump, and a rotating molecular motor. Nearly all the ATP that fuels our cellular processes is made by this multifaceted molecular superstar. The spheres observed in electron micrographs make up the F1 unit, which catalyzes ATP synthesis (Figure 19.21). These F1 spheres are attached to an integral membrane protein aggregate called the F0 unit. F1 consists of 5 polypeptide chains named a, b, g, d, and e, with a subunit stoichiometry a3b3gde (Table 19.3 and Figure 19.22). F0 includes 3 hydrophobic subunits denoted by a, b, and c, with an apparent

FIGURE 19.21 The ATP synthase, a rotating molecular motor. The c-, g-, and e-subunits constitute the rotating portion (the rotor) of the motor. Flow of protons from the a-subunit through the c-subunit turns the rotor and drives the cycle of conformational changes in a and b that synthesize ATP (pdb id = 1C17; 1E79; 2A7U; 2CLY; and 2BO5).

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Part 3 Metabolism and Its Regulation

TABLE 19.3 Yeast F1F0–ATP Synthase Subunit Organization Complex F1

F0

Protein Subunit Function

Mass (kD)

a

55.4

3

stator

b

51.3

3

stator

g

30.6

1

rotor

d

14.6

1

rotor**

Stoichiometry

e

6.6

1

rotor

a

27.9

1

stator

b

23.3

1

stator

c

7.8

10–15*

rotor

d

19.7

1

stator

h

10.4

1

stator

OSCP

20.9

1

stator

*The number of c subunits varies among organisms: yeast mitochondria, 10; Ilyobacter tartaricus, 11; Escherichia coli, 12; spinach chloroplasts, 14; Spirulina platensis, 15. **The subunit nomenclature can be confusing. E. coli ATP synthase lacks a d-subunit in its rotor; its d-subunit is analogous structurally and functionally to the mitochondrial OSCP.

stoichiometry of a1b2c10–15. F0 forms the transmembrane pore or channel through which protons move to drive ATP synthesis. The a- and b-subunits of F0 form part of the stator—a stationary component anchored in the membrane—and a ring of 10–15 c-subunits (see Table 19.3) constitutes a major component of the rotor of the motor. Protons flowing through the a–c complex cause the c-ring to rotate in the membrane. Each c-subunit is a folded pair of a-helices joined by a short loop, whereas the a-subunit is presumed to be a cluster of a-helices. The b-subunit, together with the d- and h-subunits and the oligomycin sensitivity-conferring protein (OSCP), form a long, slender stalk that connects F0 in the membrane with F1, which extends out into the matrix. The b-, d-, and h-subunits form long a-helical segments that comprise the stalk, and OSCP adds a helical bundle cap that sits at the bottom of an a-subunit of F1 (Figure 19.21). The stalk is a stable link between F0 and F1, essentially joining the two, both structurally and functionally. (a)

(b)

FIGURE 19.22 (a) An axial view of the F1 unit of the F1F0-ATP synthase, showing alternating a- and b-subunits in a hexameric array, with the g-subunit (purple) visible in the centre of the structure. (b) A side view of the F1 unit, with 1 a-subunit and 1 b-subunit removed to show how the g-subunit (red) extends through the centre of the a3b3-hexamer. Also shown are the d-subunit (aqua) and the e-subunit (pink), which link the g-subunit to the F0 unit (pdb id = 1E79). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 19

Electron Transport and Oxidative Phosphorylation

The Catalytic Sites of ATP Synthase Adopt Three Different Conformations The F1 structure appears at first to be a symmetric hexamer of a- and b-subunits. However, it is asymmetric in several ways. The a- and b-subunits, arranged in an alternating pattern in the hexamer, are similar but not identical. The hexamer contains 6 ATPbinding sites, each arranged at the interface of adjacent subunits. Three of these, each located mostly on a b-subunit but with some residues contributed by an a-subunit, are catalytic sites for ATP synthesis. The other 3, each located mostly on an a-subunit but with residues contributed by a b-subunit, are non-catalytic and inactive. The non-catalytic a-sites have similar structures, but the 3 catalytic b-sites have 3 different conformations. In the crystal structure first characterized by John Walker, 1 of the b-subunit ATP sites contains AMP-PNP (a non-hydrolyzable analog of ATP), another contains ADP, and the third site is empty. NH2 # O

N

N N

N

CH2O O H

H

P

! O O

O–

P

" O H N

O–

O–

P

(19.29)

O–

H

OH OH

Nonhydrolyzable !–" bond

H

!,"-Imidoadenosine 5!-triphosphate (AMP-PNP)

Walker’s work provided structural verification for a novel hypothesis first advanced by Paul Boyer, the binding change mechanism for ATP synthesis. Walker and Boyer, whose efforts provided complementary insights into the workings of this molecular motor, shared in the Nobel Prize for Chemistry in 1997.

Boyer’s 18O Exchange Experiment Identified the Energy-Requiring Step The elegant studies by Boyer of 18O exchange in ATP synthase provided important insights into the mechanism of the enzyme. Boyer and his colleagues studied the ability of the synthase to incorporate labelled oxygen from H218O into Pi. This reaction (Figure 19.23) occurs via synthesis of ATP from ADP and Pi, followed by hydrolysis of ATP with incorporation of oxygen atoms from the solvent. Although net production of ATP requires coupling with a proton gradient, Boyer observed that this exchange reaction occurs readily, even in the absence of a proton gradient. The exchange reaction was so facile that, eventually, all 4 oxygens of phosphate were labelled with 18O. This important observation indicated that the formation of enzyme-bound ATP does not require energy. The experiments that followed, by Boyer, Harvey Penefsky, and others, showed clearly that the energyrequiring step in the ATP synthase was actually the release of newly synthesized ATP from In the absence of a proton gradient: H+ ADP

+

Pi

H218O

H2O [ ATP ] Enzyme bound

H+ ADP

+

–

18O 18O

18OH

P 18O

–

FIGURE 19.23 ATP–ADP exchange in the absence of a proton gradient. Exchange leads to incorporation of 18O in phosphate as shown. Boyer’s experiments showed that 18O could be incorporated into all 4 positions of phosphate, demonstrating that the free energy change for ATP formation from enzyme-bound ADP + Pi is close to zero. (Mitochondrial Structure: Two Types of Subunits on Negatively Stained Mitochondrial Membranes Donald F. Parsons, Science 31 May 1963: 140 (3570), 985–987. [DOI:10.1126/science.140.3570.985]) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

643

Part 3 Metabolism and Its Regulation

the enzyme (Figure 19.24). Eventually, it would be shown that flow of protons through F0 drives the enzyme conformational changes that result in the binding of substrates on ATP synthase, ATP synthesis, and the release of products.

Boyer’s Binding Change Mechanism Describes the Events of Rotational Catalysis Boyer proposed that these conformational changes occurred in a rotating fashion. His rotational catalysis model, the binding change mechanism (Figure 19.24), suggested that at any instant the 3 b-subunits of F1 existed in 3 different conformations, that these different states represented the 3 steps of ATP synthesis, and that each site stepped through the 3 states to make ATP. A site beginning with ADP and phosphate bound (the first state) would synthesize ATP (producing the second state) and then release ATP, leaving an empty site (the third state). In the binding change mechanism, the 3 catalytic sites thus cycle concertedly through the 3 intermediate states of ATP synthesis.

L T

AT P

O

ADP

ADP

+

Pi

+ Pi

AT P

644

Energy

ADP

+

Pi

P AT

ATP H2O ATP T L

O

ADP

Cycle repeats

+

Pi

Proton Flow through F0 Drives Rotation of the Motor and Synthesis of ATP How might the cycling proposed by Boyer’s binding change mechanism occur? Important clues have emerged from several experiments that show that the g-subunit rotates with respect to the ab-complex. How such rotation might be linked to transmembrane proton flow and ATP synthesis is shown in Figure 19.25. The ring of c-subunits is a rotor that turns with respect to the a-subunit, a stator component consisting of 5 transmembrane a-helices with proton access channels on either side of the membrane. The g-subunit is the link between the functions of F1 and F0. In 1 complete rotation, the g-subunit drives conformational changes in each b-subunit that lead to ATP synthesis. Thus, 3 ATPs are synthesized per turn. But how does the F0 complex couple the events of proton transport and ATP synthesis? The a-subunit contains 2 half-channels, a proton inlet channel that opens to the intermembrane space, and a proton outlet channel that opens to the matrix. The c-subunits are proton carriers that transfer protons from the inlet channel to the outlet channel only by rotation of the c-ring. Each c-subunit contains a protonatable residue, Asp61. Protons flowing from the intermembrane space through the inlet half-channel protonate the Asp61 of a passing c-subunit and ride the rotor around the ring until they reach the outlet channel and flow out into the matrix. The molecular details of this process are shown in Figure 19.25. Each c-subunit in the c-ring has an inner helix and an outer helix. Asp61 is located midway along the outer a-helix. When protonated, the Asp carboxyl faces into the adjacent subunit. Rotation of the entire outer a-helix exposes Asp61 to the outside when it is deprotonated. Arg210, located midway on a transmembrane helix of the a-subunit, forms hydrogen bonds with Asp61 residues on 2 adjacent c-subunits. The half-channels of the a-subunit extend up and down from Arg210. The inlet channel terminates in Asn214, whereas the outlet channel terminates at Ser206. The structure of the c-subunit complex is exquisitely suited for proton transport. When a proton enters the a-subunit inlet channel and is transferred from a-subunit Asn214 to c-subunit Asp61, the a-helix of that c-subunit rotates clockwise to bury the Asp carboxyl group (Figure 19.25c). Each Asp61 remains protonated once it leaves the a-subunit interface, because the hydrophobic environment of the membrane interior makes deprotonation

FIGURE 19.24 The binding change mechanism for ATP synthesis by ATP synthase. This model assumes that F1 has 3 interacting and conformationally distinct active sites: an open (O) conformation with almost no affinity for ligands, a loose (L) conformation with low affinity for ligands, and a tight (T) conformation with high affinity for ligands. Synthesis of ATP is initiated (step 1) by binding of ADP and Pi to an L site. In the second step, an energy-driven conformational change converts the L site to a T conformation and converts T to O and O to L. In the third step, ATP is synthesized at the T site and released from the O site. Two additional passes through this cycle produce 2 more ATPs and return the enzyme to its original state. NEL

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Chapter 19 (a)

ATP

(b)

OSCP

#

Electron Transport and Oxidative Phosphorylation

c2!L

a Ser206 ! ADP

+

Pi

#

#

!

c2L

#3

!

c1L

645

c1!L

#2 #4 #5

F1

c1R

c2R

c2!R

c1!R

a Arg210

Stalk

H+ "

%$Cytoplasm

b2

a Asn214 a Asp61

+ Periplasm

C10"15

F0

a

Asp61

H+

Arg210

(c) c-subunits

H

H

D61

D61 D61

D61

D61

# " #

#

H+

R210

S 206 N 214

a-subunit

D61

H

" R 210

D61

"

R 210

N214

S206

H

D61

#

S 206 N 214

H

D61

FIGURE 19.25 (a) Protons entering the inlet half-channel in the a-subunit are transferred to binding sites on c-subunits. Rotation of the c-ring delivers protons to the outlet half-channel in the a-subunit. Flow of protons through the structure turns the rotor and drives the cycle of conformational changes in b that synthesize ATP. (b) Arg210 on the a-subunit lies between the end of the inlet half-channel (Asn214) and the end of the outlet half-channel (Ser206) (pdb id = 1C17). (c) A view looking down into the plane of the membrane. Transported protons flow from the inlet halfchannel to Asp61 residues on the c-ring, around the ring, and then into the outlet half-channel. When Asp61 is protonated, the outer helix of the c-subunit rotates clockwise to bury the protonated carboxyl group for its trip around the c-ring. Counterclockwise ring rotation then brings another protonated Asp61 to the a-subunit, where an exiting proton is transferred to the outlet half-channel.

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(and charge formation) highly unfavourable. However, when a protonated Asp residue approaches the a-subunit outlet channel, the proton is transferred to Ser206 and exits through the outlet channel. The a-subunit Arg210 side chain orients adjacent Asp61 groups and promotes transfers of entering protons from a-subunit Asn214 to Asp61 and transfers of exiting protons from Asp61 to a-subunit Ser206. Arg210, because it is protonated, also prevents direct proton transfer from Asn214 to Ser206, which would circumvent ring rotation and motor function. ATP synthesis occurs in concert with rotation of the c-ring because the g-subunit is anchored to the rotating c-ring and rotates with it. Rotation causes the g-subunit to turn relative to the 3 b-subunit nucleotide sites of F1, changing the conformation of each in sequence, so ADP is first bound, then phosphorylated, then released, according to Boyer’s binding change mechanism.

Racker and Stoeckenius Confirmed the Mitchell Model in a Reconstitution Experiment When Mitchell first described his chemiosmotic hypothesis in 1961, little evidence existed to support it and it was met with considerable skepticism by the scientific community. Eventually, however, considerable evidence accumulated to support this model. It is now clear that the electron-transport chain generates a proton gradient, and careful measurements have shown that ATP is synthesized when a pH gradient is applied to mitochondria that cannot carry out electron transport. Even more relevant is a simple but crucial experiment reported in 1974 by Efraim Racker and Walther Stoeckenius, which provided specific confirmation of the Mitchell hypothesis. In this experiment, the bovine mitochondrial ATP synthase was reconstituted in simple lipid vesicles with bacteriorhodopsin, a light-driven proton pump from Halobacterium halobium. As shown in Figure 19.26, upon illumination, bacteriorhodopsin pumped protons into these vesicles, and the resulting proton gradient was sufficient to drive ATP synthesis by the ATP synthase. Because the only 2 kinds of proteins present were one that produced a proton gradient and one that used such a gradient to make ATP, this experiment essentially verified Mitchell’s chemiosmotic hypothesis.

Light H+

Bacteriorhodopsin

H+

H+

Lipid vesicle

ADP

Mitochondrial F1F0–ATP synthase

+

Pi

Inhibitors of Oxidative Phosphorylation Reveal Insights about the Mechanism Many details of electron transport and oxidative phosphorylation mechanisms have been gained from studying the effects of particular electron transport and oxidative phosphorylation inhibitors (Figure 19.27). The sites of inhibition by these agents are indicated in Figure 19.28.

H+

ATP

FIGURE 19.26 The reconstituted vesicles containing ATP synthase and bacteriorhodopsin used by Stoeckenius and Racker to confirm the Mitchell chemiosmotic hypothesis.

Inhibitors of Complexes I, II, and III Block Electron Transport Rotenone is a common insecticide that strongly inhibits the NADH–UQ reductase. Rotenone is obtained from the roots of several species of plants. Aborigines in certain parts of the world have made a practice of beating the roots of trees along riverbanks to release rotenone into the water, where it paralyzes fish and makes them easy prey. Amytal and other barbiturates H

O

O

H

CH3O OCH3

CH3

O

H N

C2H5 (CH3)2CHCH2CH2

...

O

...

H

CH2 C

O

O

N

NH O

Rotenone

CH3

Amytal (amobarbital)

C6H5

COOC2H5

Demerol (meperidine)

FIGURE 19.27 The structures of several inhibitors of electron transport and oxidative phosphorylation. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 19

Electron Transport and Oxidative Phosphorylation

Proton gradient Cyanide Azide Carbon monoxide

Cyt c Cyt c Cyt c

NADH– coenzyme Q reductase

e– e–

UQ

UQ Coenzyme Q– cytochrome c reductase

ATP synthase

e– 1 – 2 O2

Complex III

Succinate

NADH

Cyt c oxidase

Succinate– coenzyme Q reductase

e–

Oligomycin

Cyt c

Complex II

Complex I

+ 2 H+ H2O

Rotenone Amytal Demerol

Uncouplers: 2,4-Dinitrophenol Dicumarol FCCP FIGURE 19.28 The sites of action of several inhibitors of electron transport and/or oxidative phosphorylation.

and the widely prescribed painkiller Demerol also inhibit Complex I. All these substances appear to inhibit the reduction of coenzyme Q and the oxidation of the Fe-S clusters of NADH–UQ reductase. Cyanide, Azide, and Carbon Monoxide Inhibit Complex IV Complex IV, the cytochrome c oxidase, is specifically inhibited by cyanide, azide, and carbon monoxide (Figure 19.28). Cyanide and azide bind tightly to the ferric form of cytochrome a3, whereas carbon monoxide binds only to the ferrous form. The inhibitory actions of cyanide and azide at this site are very potent, whereas the principal toxicity of carbon monoxide arises from its affinity for the iron of hemoglobin. Herein lies an important distinction between the poisonous effects of cyanide and carbon monoxide. Because animals (including humans) carry many, many hemoglobin molecules, they must inhale a large quantity of carbon monoxide to die from it. These same organisms, however, possess comparatively few molecules of cytochrome a3. Consequently, a limited exposure to cyanide can be lethal. The sudden action of cyanide attests to the organism’s constant and immediate need for the energy supplied by electron transport. Oligomycin Is an ATP Synthase Inhibitor Inhibitors of ATP synthase include oligomycin. Oligomycin is a polyketide antibiotic that acts directly on ATP synthase by binding to the OSCP subunit of F0. Oligomycin also blocks the movement of protons through F0.

Uncouplers Disrupt the Coupling of Electron Transport and ATP Synthase Another important class of reagents affects ATP synthesis, but in a manner that does not involve direct binding to any of the proteins of the electron-transport chain or the F1F0– ATPase. These agents are known as uncouplers because they disrupt the tight coupling NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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between electron transport and the ATP synthase. Uncouplers act by dissipating the proton gradient across the inner mitochondrial membrane created by the electron-transport system. Typical examples include 2,4-dinitrophenol, dicumarol, and carbonyl cyanide-p(trifluoromethoxy) phenylhydrazone (perhaps better known as fluoro carbonyl cyanide phenylhydrazone, or FCCP) (Figure 19.29). These compounds share 2 common features: a hydrophobic character and a dissociable proton. As uncouplers, they function by carrying protons across the inner membrane. Their tendency is to acquire protons on the cytosolic surface of the membrane (where the proton concentration is high) and carry them to the matrix side, thereby destroying the proton gradient that couples electron transport and the ATP synthase. In mitochondria treated with uncouplers, electron transport continues and protons are driven out through the inner membrane. However, they leak back in so rapidly via the uncouplers that ATP synthesis does not occur. Instead, the energy released in electron transport is dissipated as heat.

Dinitrophenol O2N

OH NO2

Dicumarol O

OO

OH

O

OH

Carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone —best known as FCCP; for Fluoro Carbonyl Cyanide Phenylhydrazone F3C

O

N H

N

C

N

C

N

C

FIGURE 19.29 Structures of several uncouplers, molecules that dissipate the proton gradient across the inner mitochondrial membrane and thereby destroy the tight coupling between electron transport and the ATP synthase reaction.

ATP–ADP Translocase Mediates the Movement of ATP and ADP across the Mitochondrial Membrane ATP, the cellular energy currency, must exit the mitochondria to carry energy throughout the cell, and ADP must be brought into the mitochondria for reprocessing. Neither of these processes occurs spontaneously because the highly charged ATP and ADP molecules do not readily cross biological membranes. Instead, these processes are mediated by a single transport system, the ATP–ADP translocase. This protein tightly couples the exit of ATP with the entry of ADP so that the mitochondrial nucleotide levels remain approximately constant. For each ATP transported out, 1 ADP is transported into the matrix. The translocase, which accounts for approximately 14% of the total mitochondrial membrane protein, is a homodimer of 30 kD subunits. The structure of the bovine translocase consists of 6 transmembrane a-helices. The helices are all tilted with respect to the membrane, and the first, third, and fifth helices are bent or kinked at proline residues in the middle of the membrane (Figure 19.30). Transport occurs via a single nucleotide-binding site, which alternately faces the matrix and the intermembrane space. It binds ATP on the matrix side, reorients to face the outside, and exchanges ATP for ADP, with subsequent rearrangement to face the matrix side of the inner membrane. Outward Movement of ATP Is Favoured over Outward ADP Movement The charge on ATP at pH 7.2 or so is about -4, and the charge on ADP at the same pH is about -3. Thus, net exchange of an ATP (out) for an ADP (in) results in the net movement of 1 negative charge from the matrix to the cytosol. (This process is equivalent to the movement of a proton from the cytosol to the matrix.) Recall that the inner membrane is positive outside

(a)

Intermembrane space

N

(b) Matrix

– –

+ +

Cytosol

– ATP 4 ADP3–

+ + +

– – – H+

1

ATP out for 1 ADP in

– – –

= 1 H+

in

(= 1

chargeout)

+ + +

Matrix FIGURE 19.30 (a) The bovine ATP–ADP translocase (pdb id = 2C3E). (b) Outward transport of ATP (via the ATP–ADP translocase) is favoured by the membrane electrochemical potential. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 19

Electron Transport and Oxidative Phosphorylation

649

HUMAN BIOCHEMISTRY

Endogenous Uncouplers Enable Organisms to Generate Heat

(see Figure 19.20), and it becomes clear that outward movement of ATP is favoured over outward ADP transport, ensuring that ATP will be transported out (Figure 19.30). Inward movement of ADP is favoured over inward movement of ATP for the same reason. Thus, the membrane electrochemical potential itself controls the specificity of the ATP–ADP translocase. However, the electrochemical potential is diminished by the ATP–ADP translocase cycle and therefore operates with an energy cost to the cell. The cell must compensate by passing yet more electrons down the electron-transport chain. What is the cost of ATP–ADP exchange relative to the energy cost of ATP synthesis itself ? We already noted that moving 1 ATP out and 1 ADP in is the equivalent of 1 proton moving from the cytosol to the matrix. Synthesis of an ATP results from the movement of approximately 3 protons from the cytosol into the matrix through F0. Altogether, this means that approximately 4 protons are transported into the matrix per ATP synthesized. Thus, approximately one-fourth of the energy derived from the respiratory chain (electron transport and oxidative phosphorylation) is expended as the electrochemical energy devoted to mitochondrial ATP–ADP transport.

19.6

What Is the P/O Ratio for Mitochondrial Oxidative Phosphorylation?

The P/O ratio is the number of molecules of ATP formed in oxidative phosphorylation per 2 electrons flowing through a defined segment of the electron-transport chain. In spite of intense study of this ratio, its actual value remains a matter of contention. The P/O ratio depends on the ratio of H+ transported out of the matrix per 2 e- passed from NADH to O2 in the electron-transport chain, and on the number of H+ that pass NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Philodendron © W. Wayne Lockwood, MD/CORBIS

Chipmunk © Joe McDonald/CORBIS

UCP2, and UCP3 as metabolic regulators and as factors in obesity. Under fasting conditions, expression of UCP1 mRNA is decreased, but expression of UCP2 and UCP3 is increased. There is no indication, however, that UCP2 and UCP3 actually function as uncouplers. There has also been interest in the possible roles of UCP2 and UCP3 in the development of obesity, especially because the genes for these proteins lie on chromosome 7 of the mouse, close to other genes linked to obesity. Certain plants use the heat of uncoupled proton transport for a special purpose. Skunk cabbage and related plants contain floral spikes that are maintained as much as 20° above ambient temperature in this way. The warmth of the spikes serves to vaporize odoriferous molecules, which attract insects that fertilize the flowers. Red tomatoes have very small mitochondrial membrane proton gradients compared with green tomatoes, evidence that uncouplers are more active in red tomatoes.

Thinkstock

Alaskan Brown Bear © Charles Mauzy/CORBIS

Certain cold-adapted animals, hibernating animals, and newborn animals generate large amounts of heat by uncoupling oxidative phosphorylation. These organisms have a type of fat known as brown adipose tissue, so called for the colour imparted by the many mitochondria this adipose tissue contains. The inner membrane of brown adipose tissue mitochondria contains large amounts of an endogenous protein called thermogenin (literally, “heat maker”) or uncoupling protein 1 (UCP1). UCP1 creates a passive proton channel through which protons flow from the cytosol to the matrix. Mice that lack UCP1 cannot maintain their body temperature in cold conditions, whereas normal animals produce larger amounts of UCP1 when they are cold adapted. Two other mitochondrial proteins, designated UCP2 and UCP3, have sequences similar to UCP1. Because the function of UCP1 is so closely linked to energy utilization, there has been great interest in the possible roles of UCP1,

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Part 3 Metabolism and Its Regulation

through the ATP synthase to synthesize an ATP. The latter number depends on the number of c-subunits in the F0 ring of the synthase. As noted in Table 19.3, the number of c-subunits in the ATP synthase ranges from 10 to 15, depending on the organism. This would correspond to ratios of H+ consumed per ATP from about 3 to 5, respectively, since each rotation of the ATP synthase rotor drives the formation of 3 ATP. Adding 1 H+ for the action of the ATP–ADP translocase raises these values to about 4 and 6 respectively. a

10 10 H+ P 1 ATP b = ba = + 4H 4 O 2 e [NADH h 1⁄2O2]

If we accept the value of 10 H+ transported out of the matrix per 2 e- passed from NADH to O2 through the electron-transport chain, and agree that 4 H+ are transported into the matrix per ATP synthesized (and translocated), then the mitochondrial P/O ratio is 10/4, or 2.5, for the case of electrons entering the electron-transport chain as NADH. This is somewhat lower than earlier estimates, which placed the P/O ratio at 3 for mitochondrial oxidation of NADH. For the portion of the chain from succinate to O2, the H+/2 e- ratio is 6 (as noted previously), and the P/O ratio in this case would be 6/4, or 1.5; earlier estimates placed this number at 2. The consensus of more recent experimental measurements of P/O ratios for these 2 cases has been closer to the values of 2.5 and 1.5. Many chemists and biochemists, accustomed to the integral stoichiometries of chemical and metabolic reactions, were once reluctant to accept the notion of non-integral P/O ratios. At some point, as we learn more about these complex coupled processes, it may be necessary to reassess the numbers.

19.7

How Are the Electrons of Cytosolic NADH Fed into Electron Transport?

Most of the NADH used in electron transport is produced in the mitochondrial matrix, an appropriate site because NADH is oxidized by Complex I on the matrix side of the inner membrane. Furthermore, the inner mitochondrial membrane is impermeable to NADH. Recall, however, that NADH is produced in glycolysis by glyceraldehyde-3-P dehydrogenase in the cytosol. If this NADH were not oxidized to regenerate NAD+, the glycolytic pathway would cease to function due to NAD+ limitation. Eukaryotic cells have a number of shuttle systems that harvest the electrons of cytosolic NADH for delivery to mitochondria without actually transporting NADH across the inner membrane (Figures 19.31 and 19.32).

Glycerol3-phosphate

Dihydroxyacetone phosphate

CH2OH HO

C

NAD+

NADH

+

CH2OH

H+

H

C

CH2OPO–2 3

O

CH2OPO–2 3

Periplasm Inner mitochondrial membrane

FAD

E

FADH2

E

Electrontransport chain

Flavoprotein 4 Mitochondrial matrix FIGURE 19.31 The glycerophosphate shuttle (also known as the glycerol phosphate shuttle) couples the cytosolic oxidation of NADH with mitochondrial reduction of [FAD].

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Chapter 19

Electron Transport and Oxidative Phosphorylation COO–

COO– O

C

Cytosol

O

Matrix

C CH2

CH2 CH2 COO– #-Ketoglutarate

#-Ketoglutarate– malate carrier

COO– CH

HO

HO

CH2

COO– Malate

COO– Malate NAD+

NAD+ Malate dehydrogenase

Malate dehydrogenase

+ H+

COO–

C

C

O

CH2 + H3N

COO–

+ H3N

CH

CH

COO– Glutamate

Aspartate– glutamate carrier

CH2 COO– Aspartate

CH

COO– Glutamate

+

H+

O

CH2 COO– Oxaloacetate Aspartate aminotransferase

CH2

CH2 COO–

COO–

CH2

CH2

Aspartate aminotransferase

+ H3N

NADH

COO–

COO– Oxaloacetate

COO– #-Ketoglutarate

CH

CH2

NADH

CH2

COO–

+ H3N

COO– CH CH2

Mitochondrial membrane

COO– Aspartate

FIGURE 19.32 The malate (oxaloacetate)–aspartate shuttle, which operates across the inner mitochondrial membrane.

The Glycerophosphate Shuttle Ensures Efficient Use of Cytosolic NADH In the glycerophosphate shuttle, 2 different glycerophosphate dehydrogenases, 1 in the cytosol and 1 on the outer face of the mitochondrial inner membrane, work together to carry electrons into the mitochondrial matrix (see Figure 19.31). NADH produced in the cytosol transfers its electrons to dihydroxyacetone phosphate, thus reducing it to glycerol-3-phosphate. This metabolite is reoxidized by the FAD+-dependent mitochondrial membrane enzyme to reform dihydroxyacetone phosphate and enzyme-bound FADH2. The 2 electrons of [FADH2] are passed directly to UQ, forming UQH2. Thus, via this shuttle, cytosolic NADH can be used to produce mitochondrial [FADH2] and, subsequently, UQH2. As a result, cytosolic NADH oxidized via this shuttle route yields only 1.5 molecules of ATP. The cell “pays” with a potential ATP molecule for the convenience of getting cytosolic NADH into the mitochondria. Although this may seem wasteful, there is an important payoff. The glycerophosphate shuttle is essentially irreversible, and even when NADH levels are very low relative to NAD+, the cycle operates effectively.

The Malate–Aspartate Shuttle Is Reversible The second electron shuttle system, called the malate–aspartate shuttle, is shown in Figure 19.32. Oxaloacetate is reduced in the cytosol, acquiring the electrons of NADH (which is NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 3 Metabolism and Its Regulation

oxidized to NAD+). Malate is transported across the inner membrane, where it is reoxidized by malate dehydrogenase, converting NAD+ to NADH in the matrix. This mitochondrial NADH readily enters the electron-transport chain. The oxaloacetate produced in this reaction cannot cross the inner membrane and must be transaminated to form aspartate, which can be transported across the membrane to the cytosolic side. Transamination in the cytosol recycles aspartate back to oxaloacetate. In contrast to the glycerol phosphate shuttle, the malate–aspartate cycle is reversible, and it operates as shown in Figure 19.32 only if the NADH/NAD+ ratio in the cytosol is higher than the ratio in the matrix. Because this shuttle produces NADH in the matrix, the full 2.5 ATPs per NADH are recovered.

The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used The complete route for the conversion of the metabolic energy of glucose to ATP was described in Chapters 17 through 19. Assuming appropriate P/O ratios, the number of ATP molecules produced by the complete oxidation of a molecule of glucose can be estimated. Keeping in mind that P/O ratios must be viewed as approximate, for all the reasons previously cited we will assume the values of 2.5 and 1.5 for the mitochondrial oxidation of NADH and succinate respectively. In eukaryotic cells, the combined pathways of glycolysis, the TCA cycle, electron transport, and oxidative phosphorylation then yield a net of approximately 30–32 molecules of ATP per molecule of glucose oxidized, depending on the shuttle route used (Table 19.4).

TABLE 19.4 Yield of ATP from Glucose Oxidation ATP Yield per Glucose Glycerol Phosphate Shuttle

Malate– Aspartate Shuttle

Glycolysis: Glucose to Pyruvate (Cytosol) Phosphorylation of glucose

-1

-1

Phosphorylation of fructose-6-phosphate

-1

-1

Dephosphorylation of 2 molecules of 1,3-BPG

+2

+2

Dephosphorylation of 2 molecules of PEP

+2

+2

+2

+2

+3

+5

+5

+5

+3

+3

+15

+15

30

32

Pathway

Oxidation of 2 molecules of glyceraldehyde-3-phosphate yields 2 NADH Pyruvate Conversion to Acetyl-CoA (Mitochondria) 2 NADH Citric Acid Cycle (Mitochondria) 2 molecules of GTP from 2 molecules of succinyl-CoA Oxidation of 2 molecules each of isocitrate, a-ketoglutarate, and malate yields 6 NADH Oxidation of 2 molecules of succinate yields 2 [FADH2] Oxidative Phosphorylation (Mitochondria) 2 NADH from glycolysis yield 1.5 ATPs each if NADH is oxidized by glycerol phosphate shuttle; 2.5 ATPs by malate–aspartate shuttle Oxidative decarboxylation of 2 pyruvate to 2 acetyl-CoA: 2 NADH produce 2.5 ATPs each 2 [FADH2] from each citric acid cycle produce 1.5 ATPs each 6 NADH from citric acid cycle produce 2.5 ATPs each Net Yield

Note: These P/O ratios of 2.5 and 1.5 for mitochondrial oxidation of NADH and [FADH2] are “consensus values.” Because they may not reflect actual values and because these ratios may change depending on metabolic conditions, these estimates of ATP yield from glucose oxidation are approximate. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 19

Electron Transport and Oxidative Phosphorylation

653

The net stoichiometric equation for the oxidation of glucose, using the glycerol phosphate shuttle, is Glucose + 6 O2 + ! 30 ADP + ! 30 Pi h 6 CO2 + ! 30 ATP + ! 36 H2O

(19.30)

Because the 2 NADH formed in glycolysis are “transported” by the glycerol phosphate shuttle in this case, they each yield only 1.5 ATP, as already described. On the other hand, if these 2 NADH take part in the malate–aspartate shuttle, each yields 2.5 ATPs, giving a total (in this case) of 32 ATPs formed per glucose oxidized. Most of the ATP—26 of 30 or 28 of 32—is produced by oxidative phosphorylation; only 4 ATP molecules result from direct synthesis during glycolysis and the TCA cycle. The situation in bacteria is somewhat different. Prokaryotic cells need not carry out ATP–ADP exchange. Thus, bacteria have the potential to produce approximately 38 ATPs per glucose.

3.5 Billion Years of Evolution Have Resulted in a Very Efficient System Hypothetically speaking, how much energy does a eukaryotic cell extract from the glucose molecule? Taking a value of 50 kJ/mol for the hydrolysis of ATP under cellular conditions (see Chapter 3), the production of 32 ATPs per glucose oxidized yields 1600 kJ/mol of glucose. The cellular oxidation (combustion) of glucose yields #G = -2937 kJ/mol. We can calculate an efficiency for the pathways of glycolysis, the TCA cycle, electron transport, and oxidative phosphorylation of 1600/2937 * 100% = 54%.

19.8

How Do Mitochondria Mediate Apoptosis?

Mitochondria not only are the home of the TCA cycle and oxidative phosphorylation but also are a crossroads for several cell signalling pathways. Mitochondria take up Ca2+ ions released from the endoplasmic reticulum, thus helping control intracellular Ca2+ signals. They produce reactive oxygen species (ROS) that play signalling roles in cells, although ROS can also cause cellular damage. Mitochondria also participate in the programmed death of cells, a process known as apoptosis (the second “p” is silent in this word). Apoptosis is a mechanism through which certain cells are eliminated from higher organisms. It is central to the development and homeostasis of multicellular organisms, and it is the route by which unwanted or harmful cells are eliminated. Under normal circumstances, apoptosis is suppressed through compartmentation of the involved activators and enzymes. Mitochondria play a major role in this subcellular partitioning of the apoptotic activator molecules. One such activator is cytochrome c, which normally resides in the intermembrane space, bound tightly to a lipid chain of cardiolipin in the membrane (Figure 19.33). A variety of triggering agents, including Ca2+, ROS, certain lipid molecules, and certain protein kinases, can induce the opening of pores in the mitochondrial membrane. For example, using hydrogen peroxide as a substrate, cytochrome c can oxidize its bound cardiolipin chain, releasing itself from the membrane. When the outer membrane is made permeable by other apoptotic signals, cytochrome c can enter the cytosol. Permeabilization events, which occur at points where outer and inner mitochondrial membranes are in contact, involve association of the ATP–ADP translocase in the inner membrane and the voltage-dependent anion channel (VDAC) in the outer membrane. This interaction leads to the opening of protein-permeable pores. Cytochrome c, as well as several other proteins, can pass through these pores. Pore formation is carefully regulated by the BCL-2 family of proteins, which includes both pro-apoptotic members (proteins known as Bax, Bid, and Bad) and anti-apoptotic members (BCL-2 itself, as well as BCL-XL and BCL-W).

ROS: Reactive oxygen species, such as oxygen ions, free radicals, and peroxides.

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654

Part 3 Metabolism and Its Regulation (a) Cyt c Cardiolipin

OOH H2O2

OOH

C

(b)

C

C CL

C CO- POX

C

C

C

C

CO- POX

FIGURE 19.33 (a) Cytochrome c is anchored at the inner mitochondrial membrane by association with cardiolipin (diphosphatidylglycerol). The peroxidase activity of cytochrome c oxidizes a cardiolipin lipid chain, releasing cytochrome c from the membrane. (b) The opening of pores in the outer membrane, induced by a variety of triggering agents, releases cytochrome c to the cytosol, where it initiates the events of apoptosis.

Cytochrome c Triggers Apoptosome Assembly But how is the release of cytochrome c translated into the activation of the death cascade, a point of no return for the cell? The answer lies in the assembly, in the cytosol, of a signalling platform called the apoptosome (Figure 19.34). The function of the apoptosome is to activate a cascade of proteases called caspases. (Here, “c” is for cysteine and “asp” is for aspartic acid. Caspases have Cys at the active site and cleave their peptide substrates after Asp residues.) The apoptosome is a wheel-shaped, heptameric platform that looks like (and in some ways behaves like) an earth-orbiting space station. It is assembled from 7 subunits of the apoptotic proteaseactivating factor 1 (Apaf-1), a multi-domain protein. Apaf-1 contains an ATPase domain (which prefers dATP over ATP in some organisms), a caspase-recruitment domain (CARD), and a WD40 repeat domain. Normally (before the death-signalling cytochrome c is released from mitochondria), these 3 domains are folded against each other (Figure 19.34b), with dATP tightly bound, and Apaf-1 is “locked” in an inactive monomeric state. Binding of cytochrome c to the WD40 domain, followed by dATP hydrolysis, converts Apaf-1 to an extended conformation. Then, exchange of dADP for a new molecule of dATP prompts assembly of the heptameric platform (Figure 19.34), which goes on to activate the death-dealing caspase cascade. Mitochondria-mediated apoptosis is the mode of cell death for many neurons in the brain during strokes and other brain-trauma injuries. When a stroke occurs, the neurons at the site of oxygen deprivation die within minutes by a non-specific process of necrosis, but cells adjacent to the immediate site of injury die more slowly by apoptosis. These latter cells have been saved by a variety of therapeutic interventions that suppress apoptosis in laboratory studies, raising the hope that strokes and other neurodegenerative conditions may someday be treated clinically in similar ways. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 19

Electron Transport and Oxidative Phosphorylation

655

(a) Apaf-1 CARD

NOD

WD40 WD40

(b) Locked form

Semi-open, autoinhibited form

Cytochrome c

Apoptosome

WD40 dATP-dADP exchange

Cytochrome c binding dATP hydrolysis CARD NOD

(c)

FIGURE 19.34 (a) Apaf-1 is a multi-domain protein consisting of an N-terminal CARD, a nucleotide-binding and oligomerization domain (NOD), and several WD40 domains. (b) Binding of cytochrome c to the WD40 domains and ATP hydrolysis unlocks Apaf-1 to form the semi-open conformation. Nucleotide exchange leads to oligomerization and apoptosome formation. (c) A model of the apoptosome, a wheel-like structure with molecules of cytochrome c bound to the WD40 domains, which extend outward like spokes.

SUMMARY 19.1 Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur? The processes of electron transport and oxidative phosphorylation are membrane associated. In prokaryotes, the conversion of energy from NADH and [FADH2] to the energy of ATP via electron transport and oxidative phosphorylation is carried out at (and across) the plasma membrane. In eukaryotic cells, electron transport and oxidative phosphorylation are localized in mitochondria. Mitochondria are surrounded by a simple outer membrane and a more complex inner membrane. The space between the inner and outer membranes is referred to as the intermembrane space. 19.2 What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? Just as the group transfer potential is used to quantitate the energy of phosphoryl transfer, the standard reduction potential, denoted by eo !, quantitates the tendency of chemical species to be reduced or oxidized. Standard reduction potentials are determined by measuring the voltages generated in reaction half-cells. A half-cell consists of a solution containing 1 M concentrations of both the oxidized and the reduced forms of the substance whose reduction potential is being measured and a simple electrode. 19.3 How Is the Electron-Transport Chain Organized? The components of the electron-transport chain can be purified from the mitochondrial

inner membrane as 4 distinct protein complexes: (I) NADH–coenzyme Q reductase, (II) succinate–coenzyme Q reductase, (III) coenzyme Q– cytochrome c reductase, and (IV) cytochrome c oxidase. In Complexes I, II, and IV, electron transfer drives the movement of protons from the mitochondrial matrix to the intermembrane space. Complex I (NADH dehydrogenase) involves more than 45 polypeptide chains, 1 molecule of flavin mononucleotide (FMN), and as many as 9 Fe-S clusters, together containing a total of 20–26 iron atoms. The complex transfers electrons from NADH to FMN, then to a series of FeS proteins, and finally to coenzyme Q. Complex II (succinate dehydrogenase) oxidizes succinate to fumarate, with concomitant reduction of bound FAD to FADH2. This FADH2 transfers its electrons immediately to Fe-S centres, which pass them on to UQ. Electrons flow from succinate to UQ. Complex III drives electron transport from coenzyme Q to cytochrome c via a unique redox pathway known as the Q cycle. UQ–cytochrome c reductase (UQ–cyt c reductase), as this complex is known, involves 3 different cytochromes and an Fe-S protein. In the cytochromes of these and similar complexes, the iron atom at the centre of the porphyrin ring cycles between the reduced Fe2+ (ferrous) and oxidized Fe3+ (ferric) states. Complex IV transfers electrons from cytochrome c to reduce oxygen on the matrix side. Complex IV (cytochrome c oxidase)

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accepts electrons from cytochrome c and directs them to the 4-electron reduction of O2 to form 2 H2O via CuA sites, the heme iron of cytochrome a, CuB, and the heme iron of a3. 19.4 What Are the Thermodynamic Implications of Chemiosmotic Coupling? Peter Mitchell was the first to propose that electron transport leads to formation of a proton gradient that drives ATP synthesis. The free energy difference for protons across the inner mitochondrial membrane includes a term for the concentration difference and a term for the electrical potential. It is this energy that drives the synthesis of ATP, in accord with Mitchell’s model. 19.5 How Does a Proton Gradient Drive the Synthesis of ATP? The mitochondrial complex that carries out ATP synthesis is ATP synthase (F1F0–ATPase). ATP synthase consists of 2 principal complexes, designated F1 and F0. Protons taken up from the cytosol by 1 of the proton access channels in the a-subunit of F0 ride the rotor of c-subunits until they reach the other proton access channel on a, from which they are released into the matrix. Such rotation causes the g-subunit of F1 to turn relative to the 3 b-subunit nucleotide sites of F1, changing the conformation of each in sequence, so ADP is first bound, then phosphorylated, then released, according to Boyer’s binding change mechanism. The inhibitors of oxidative phosphorylation include rotenone, a common insecticide that strongly inhibits the NADH–UQ reductase. Complex IV is specifically inhibited by cyanide (CN-), azide (N-3 ), and carbon monoxide (CO). Cyanide and azide bind tightly to the ferric form of cytochrome a3, whereas carbon monoxide binds only to the ferrous form. Uncouplers disrupt the coupling of electron transport and ATP synthase. Uncouplers share 2 common features: hydrophobic character and a dissociable proton. They function by carrying protons across the inner membrane, acquiring protons on the outer surface of the membrane (where the proton concentration is high) and carrying them to the matrix side. Uncouplers destroy the proton gradient that couples electron transport and the ATP synthase. ATP–ADP translocase mediates the movement of ATP and ADP across the mitochondrial membrane. The ATP–ADP translocase is an inner membrane protein that tightly couples the exit of ATP with the entry of ADP so that the mitochondrial nucleotide

levels remain approximately constant. For each ATP transported out, 1 ADP is transported into the matrix. ATP–ADP translocase binds ATP on the matrix side, reorients to face the intermembrane space, and exchanges ATP for ADP, with subsequent reorientation back to the matrix face of the inner membrane. 19.6 What Is the P/O Ratio for Mitochondrial Oxidative Phosphorylation? The P/O ratio is the number of molecules of ATP formed in oxidative phosphorylation per 2 electrons flowing through a defined segment of the electron-transport chain. The consensus value for the mitochondrial P/O ratio is 10/4, or 2.5, for electrons entering the electron-transport chain as NADH. For succinate to O2, the P/O ratio in this case would be 6/4, or 1.5. 19.7 How Are the Electrons of Cytosolic NADH Fed into Electron Transport? Eukaryotic cells have a number of shuttle systems that collect the electrons of cytosolic NADH for delivery to mitochondria without actually transporting NADH across the inner membrane. In the glycerophosphate shuttle, 2 different glycerophosphate dehydrogenases, 1 in the cytosol and 1 on the outer face of the mitochondrial inner membrane, work together to carry electrons into the mitochondrial matrix. In the malate–aspartate shuttle, oxaloacetate is reduced in the cytosol, acquiring the electrons of NADH (which is oxidized to NAD+). Malate is transported across the inner membrane, where it is reoxidized by malate dehydrogenase, converting NAD+ to NADH in the matrix. 19.8 How Do Mitochondria Mediate Apoptosis? Mitochondria are a crossroads for several cell signalling pathways. Mitochondria take up Ca2+ ions released from the endoplasmic reticulum, helping control intracellular Ca2+ signals. They produce ROS that play signalling roles in cells. They also participate in apoptosis, the programmed death of cells. Triggering agents, including Ca2+, ROS, and certain lipid molecules and protein kinases, can induce the opening of pores in the mitochondrial membrane, releasing cytochrome c, which then binds to the WD40 domain of Apaf-1, activating formation of the heptameric apoptosome platform. Mitochondria-mediated apoptosis is the mode of cell death of many neurons in the brain during strokes and other brain trauma injuries, and interventions that suppress apoptosis may eventually be useful in clinical settings.

PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. For the following reaction, [FAD] + 2 cyt c (Fe2+) + 2 H+ h [FADH2] + 2 cyt c (Fe3+) determine which of the redox couples is the electron acceptor and which is the electron donor under standard-state conditions. Calculate the value of #eo=, and determine the free energy change for the reaction. 2. Calculate the value of #eo= for the glyceraldehyde-3-phosphate dehydrogenase reaction, and calculate the free energy change for the reaction under standard-state conditions. 3. For the following redox reaction, NAD+ + 2 H+ + 2 e- h NADH + H+ suggest an equation (analogous to Equation 19.12) that predicts the pH dependence of this reaction, and calculate the reduction potential for this reaction at pH 8. 4. Sodium nitrite (NaNO2) is used by emergency medical personnel as an antidote for cyanide poisoning (for this purpose, it must

be administered immediately). Based on the discussion of cyanide poisoning in Section 19.5, suggest a mechanism for the life-saving effect of sodium nitrite. 5. A wealthy investor has come to you for advice. She has been approached by a biochemist who seeks financial backing for a company that would market dinitrophenol and dicumarol as weightloss medications. The biochemist has explained to her that these agents are uncouplers and that they would dissipate metabolic energy as heat. The investor wants to know if you think she should invest in the biochemist’s company. How do you respond? 6. Assuming that 3 H+ are transported per ATP synthesized in the mitochondrial matrix, the membrane potential difference is 0.18 V (negative inside), and the pH difference is 1 unit (acid outside, basic inside), calculate the largest ratio of [ATP]/[ADP][Pi] under which synthesis of ATP can occur. 7. Of the dehydrogenase reactions in glycolysis and the TCA cycle, all but 1 use NAD+ as the electron acceptor. The lone exception is the succinate dehydrogenase reaction, which uses covalently bound FAD of a flavoprotein as the electron acceptor. The standard reduction potential for this bound FAD is in the range of 0.003–0.091 V (see Table 19.1). Compared with the other dehydrogenase reactions of glycolysis and the TCA cycle, what is unique about succinate NEL

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Chapter 19

dehydrogenase? Why is bound FAD a more suitable electron acceptor in this case? 8. a. What is the standard free energy change (#G°!) for the reduction of coenzyme Q by NADH as carried out by Complex I (NADH– coenzyme Q reductase) of the electron-transport pathway if #eo=(NAD+ >NADH) = -0.320 V and #eo=(CoQ>CoQH2) = -0.060 V. b. What is the equilibrium constant (Keq) for this reaction? c. Assume (1) that the actual free energy release accompanying the NADH–coenzyme Q reductase reaction is equal to the amount released under standard conditions (as calculated in part a); (2) that this energy can be converted into the synthesis of ATP with an efficiency is 0.75 (i.e., 75% of the energy released upon NADH oxidation is captured in ATP synthesis); and (3) that the oxidation of 1 equivalent of NADH by coenzyme Q leads to the phosphorylation of 1 equivalent of ATP. Under these conditions, what is the maximum ratio of [ATP]/[ADP] attainable by oxidative phosphorylation when [Pi] = 1 mM? (Assume #G°! for ATP synthesis is -30.5 kJ/mol.) 9. Consider the oxidation of succinate by molecular oxygen as carried out via the electron-transport pathway succinate + 12O2 h fumarate + H2O. a. What is the standard free energy change (#G°!) for this reaction if #eo= (Fum>Succ = -0.031 V and #eo= (12O2 >H2O) = +0.816 V? b. What is the equilibrium constant (Keq) for this reaction? c. Assume (1) that the actual free energy release accompanying succinate oxidation by the electron-transport pathway is equal to the amount released under standard conditions (as calculated in part a); (2) that this energy can be converted into the synthesis of ATP with an efficiency of 0.7 (i.e., 70% of the energy released upon succinate oxidation is captured in ATP synthesis); and (3) that the oxidation of 1 succinate leads to the phosphorylation of 2 equivalents of ATP. Under these conditions, what is the maximum ratio of [ATP]/[ADP] attainable by oxidative phosphorylation when [Pi] = 1 mM? (Assume #G°! for ATP synthesis is -30.5 kJ>mol.) 10. Consider the oxidation of NADH by molecular oxygen as carried out via the electron-transport pathway NADH + H+ + 12O2 h NAD+ + H2O. a. What is the standard free energy change (#G°!) for this reaction if eo= (NAD+/NADH) = -0.320 V and eo= (O2/H2O) = +0.816 V? b. What is the equilibrium constant (Keq) for this reaction? c. Assume (1) that the actual free energy release accompanying NADH oxidation by the electron-transport pathway is equal to the amount released under standard conditions (as calculated in part a); (2) that this energy can be converted into the synthesis of ATP with an efficiency of 0.75 (i.e., 75% of the energy released upon NADH oxidation is captured in ATP synthesis); and (3) that the oxidation of 1 NADH leads to the phosphorylation of 3 equivalents of ATP. Under these conditions, what is the maximum ratio of [ATP]/[ADP] attainable by oxidative phosphorylation when [Pi] = 2 mM? (Assume #G°! for ATP synthesis is -30.5 kJ/mol.) 11. Write a balanced equation for the reduction of molecular oxygen by reduced cytochrome c as carried out by Complex IV (cytochrome oxidase) of the electron-transport pathway. a. What is the standard free energy change (#G°!) for this reaction if #eo= cyt c(Fe3+)>cyt c(Fe2+) = +0.254 volts and eo ! (12O2 >H2O) = 0.816 volts

Electron Transport and Oxidative Phosphorylation

657

b. What is the equilibrium constant (Keq) for this reaction? c. Assume (1) that the actual free energy release accompanying cytochrome c oxidation by the electron-transport pathway is equal to the amount released under standard conditions (as calculated in part a); (2) that this energy can be converted into the synthesis of ATP with an efficiency of 0.6 (i.e., 60% of the energy released upon cytochrome c oxidation is captured in ATP synthesis); and (3) that the reduction of 1 molecule of O2 by reduced cytochrome c leads to the phosphorylation of 2 equivalents of ATP. Under these conditions, what is the maximum ratio of [ATP]/[ADP] attainable by oxidative phosphorylation when [Pi] = 3 mM? (Assume #G°! for ATP synthesis is -30.5 kJ/mol.) 12. The standard reduction potential for (NAD+/NADH) is -0.320 V, and the standard reduction potential for (pyruvate/lactate) is -0.185 V. a. What is the standard free energy change (#G°!) for the lactate dehydrogenase reaction NADH + H+ + pyruvate h lactate + NAD+ b. What is the equilibrium constant (Keq) for this reaction? c. If [pyruvate] is 0.05 mM and [lactate] is 2.9 mM, and #G for the lactate dehydrogenase reaction is -15 kJ>mol in erythrocytes, what is the NAD+/[NADH] ratio under these conditions? 13. Assume that the free energy change (#G) associated with the movement of 1 mole of protons from the outside to the inside of a bacterial cell is -23 kJ/mol and that 3 H+ must cross the bacterial plasma membrane per ATP formed by the bacterial F1F0–ATP synthase. ATP synthesis thus takes place by the coupled process 3 H+out + ADP + Pi ∆ 3 H+in + ATP + H2O. a. If the overall free energy change (#Goverall) associated with ATP synthesis in these cells by the coupled process is –21 kJ/mol, what is the equilibrium constant (Keq) for the process? b. What is #Gsynthesis, the free energy change for ATP synthesis, in these bacteria under these conditions? c. The standard free energy change for ATP hydrolysis (#G°!hydrolysis) is –30.5 kJ/mol. If [Pi] = 2 mM in these bacterial cells, what is the [ATP]/[ADP] ratio in these cells? 14. Describe in your own words the path of electrons through the Q cycle of Complex III. 15. Describe in your own words the path of electrons through the copper and iron centres of Complex IV. 16. In the course of events triggering apoptosis, a fatty acid chain of cardiolipin undergoes peroxidation to release the associated cytochrome c. Lipid peroxidation occurs at a double bond. Suggest a mechanism for the reaction of hydrogen peroxide with an unsaturation in a lipid chain, and identify a likely product of the reaction. 17. In Problem 20 at the end of Chapter 18, you might have calculated the number of molecules of oxaloacetate in a typical mitochondrion. What about protons? A typical mitochondrion can be thought of as a cylinder 1 mm in diameter and 2 mm in length. If the pH in the matrix is 7.8, how many protons are contained in the mitochondrial matrix? 18. Considering that all other dehydrogenases of glycolysis and the TCA cycle use NADH as the electron donor, why does succinate dehydrogenase, a component of the TCA cycle and the electron transfer chain, use FAD as the electron acceptor from succinate, rather than NAD+? Note that there are 2 justifications for the choice of FAD here: 1 based on energetics and 1 based on the mechanism of electron transfer for FAD versus NAD+.

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Part 3 Metabolism and Its Regulation

FURTHER READING Apoptosis Cereghetti, G. M., and Scorrano, L., 2006. The many shapes of mitochondrial death. Oncogene 25:4717–4724. Cerveny, K. L., Tamura, Y., et al., 2007. Regulation of mitochondrial fusion and division. Trends in Cell Biology 17:563–569. Chan, D. C., 2006. Mitochondrial fusion and fission in mammals. Annual Review of Cell and Developmental Biology 22:79–99. Orrenius, S., and Zhivotovsky, B., 2005. Cardiolipin oxidation sets cytochrome c free. Nature Chemical Biology 1:188–189. Orrenius, S., 2007. Reactive oxygen species in mitochondria-mediated cell death. Drug Metabolism Reviews 39:443–455. Riedl, S. J., and Salvesen, G. S., 2007. The apoptosome: Signalling platform of cell death. Nature Reviews Molecular Cell Biology 8:405–413. ATP–ADP Translocase Nury, H., Dahout-Gonzalez, C., et al., 2006. Relations between structure and function of the mitochondrial ADP/ATP carrier. Annual Review of Biochemistry 75:713–741. Bioenergetics

Lenaz, G., Fato, R., et al., 2006. Mitochondrial Complex I: Structural and functional aspects. Biochimica et Biophysica Acta 1757:1406–1420. Sazanov, L. A., 2007. Respiratory Complex I: Mechanistic and structural insights provided by the crystal structure of the hydrophilic domain. Biochemistry 46:2275–2288. Seibold, S. A., Mills, D. A., et al., 2005. Water chain formation and possible proton pumping routes in Rhodobacter sphaeroides cytochrome c oxidase: A molecular dynamics comparison of the wild type and R481K mutant. Biochemistry 44:10475–10485. Slater, E. C., 1983. The Q cycle, an ubiquitous mechanism of electron transfer. Trends in Biochemical Sciences 8:239–242. Sun, F., Huo, X., et al., 2005. Crystal structure of mitochondrial respiratory membrane protein Complex II. Cell 121:1043–1057. Trumpower, B. L., 1990. The protonmotive Q cycle: Energy transduction by coupling of proton translocation to electron transfer by the cytochrome bc1 complex. Journal of Biological Chemistry 265:11409–11412. Tsukihara, T., Aoyama, H., et al., 1996. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å. Science 272:1136–1144.

Babcock, G. T., and Wikstrom, M., 1992. Oxygen activation and the conservation of energy in cell respiration. Nature 356:301–309.

Wikstrom, M., and Verkhovsky, M. I., 2007. Mechanism and energetics of proton translocation by the respiratory heme-copper oxidases. Biochimica et Biophysica Acta 1767:1200–1214.

Merz, S., Hammermeister, M., et al., 2007. Molecular machinery of mitochondrial dynamics in yeast. Biological Chemistry 388:917–926.

Xia, D., Yu, C.-A., et al., 1997. The crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 277:60–66.

Mitchell, P., 1961. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191:144–148.

Yoshikawa, S., Muramoto, K., et al., 2006. Reaction mechanism of bovine heart cytochrome c oxidase. Biochimica et Biophysica Acta 1757:395–400.

Mitchell, P., and Moyle, J., 1965. Stoichiometry of proton translocation through the respiratory chain and adenosine triphosphatase systems of rat mitochondria. Nature 208:147–151.

F 1 F 0 –ATPase

Mitchell, P., 1978. The Nobel Prize in Chemistry 1978. Nobelprize.org. 27 Jul 2012 www.nobelprize.org/nobel_prizes/chemistry/laureates/1978/ Mitchell, P., 1979. Keilin’s respiratory chain concept and its chemiosmotic consequences. Science 206:1148–1159. Rich, P.R., 2008. A perspective on Peter Mitchell and the chemiosmotic theory. Journal of Bioenergetics and Biomembranes 40:407–410. Electron Transfer Belevich, I., and Verkhovsky, M. I., 2008. Molecular mechanism of proton translocation by cytochrome c oxidase. Antioxidants and Redox Signaling 10:1–29. Boekema, E. J., and Braun, H-P., 2007. Supramolecular structure of the mitochondrial oxidative phosphorylation system. Journal of Biological Chemistry 282:1–4. Brandt, U., 2006. Energy converting NADH:quinone oxidoreductase (Complex I). Annual Review of Biochemistry 75:69–72. Brzezinski, P., and Adelroth, P., 2006. Design principles of protonpumping haem-copper oxidases. Current Opinion in Structural Biology 16:465–472. Busenlehner, L. S., Branden, G., et al., 2008. Structural elements involved in proton translocation by cytochrome c oxidase as revealed by backbone amide hydrogen–deuterium exchange of the E286H mutant. Biochemistry 47:73–83. Cecchini, G., 2003. Function and structure of Complex II of the respiratory chain. Annual Review of Biochemistry 72:77–109. Hunte, C., Koepke, J., et al., 2000. Structure at 2.3 Å resolution of the cytochrome bc1 complex from the yeast Saccharomyces cerevisiae cocrystallized with an antibody Fv fragment. Structure 8:669–684. Iwata, S., Ostermeier, C., et al., 1995. Structure at 2.8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376:660–669.

Adachi, K., Oiwa, K., et al., 2007. Coupling of rotation and catalysis in F1-ATPase revealed by single-molecule imaging and manipulation. Cell 130:309–321. Aksimentiev, A., Balabin, I. A., et al., 2004. Insights into the molecular mechanism of rotation in the F0 sector of ATP synthase. Biophysical Journal 66:1332–1344. Boyer, P. D., 2002. A research journey with ATP synthase. Journal of Biological Chemistry 277:39045–39061. Dickson, V. K., Silvester, J. A., et al., 2006. On the structure of the stator of the mitochondrial ATP synthase. The EMBO Journal 25:2911–2918. Rastogi, V. K., and Girvin, M. E., 1999. Structural changes linked to proton translocation by subunit c of the ATP synthase. Nature 402:263–268. Senior, A. E., and Weber, J., 2004. Happy motoring with ATP synthase. Nature Structural and Molecular Biology 11:110–112. Senior, A. E., 2007. ATP synthase: Motoring to the finish line. Cell 130:220–221. Stock, D., Leslie, A. G. W., et al., 1999. Molecular architecture of the rotary motor in ATP synthase. Science 286:1700–1705. Weber, J., 2007. ATP synthase: The structure of the stator stalk. Trends in Biochemical Sciences 32:53–55. Wilkins, S., 2005. Rotary molecular motors. Advances in Protein Chemistry 71:345–382. Uncouplers Fogelman, A. M., 2005. When pouring water on the fire makes it burn brighter. Cell Metabolism 2:6–7. Nedergaard, J., Ricquier, D., et al., 2005. Uncoupling proteins: Current status and therapeutic prospects. EMBO Reports 6:917–921.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

20

Photosynthesis

© Richard Hamilton Smith/CORBIS

! Field of goldenrod.

ESSENTIAL QUESTIONS Photosynthesis is the primary source of energy for all life forms except chemolithotrophic prokaryotes. Much of the energy of photosynthesis is used to drive the synthesis of organic molecules from atmospheric CO2. How is solar energy captured and transformed into metabolically useful chemical energy? How is the chemical energy produced by photosynthesis used to create organic molecules from carbon dioxide?

WHY IT MATTERS Before we begin our discussion of photosynthesis, we pose the question: Why does photosynthesis matter? Photosynthesis can be defined as the production of carbon compounds (such as cellulose) and oxygen at the expense of CO2 and water.* It is fundamental for aerobic life, and we can even state that our very existence relies on it. The increased overproduction of CO2 and other greenhouse gases resulting from fossil fuel combustion can be counterproductive to the process of photosynthesis, basically disrupting the delicate balance established in nature.* In today’s environmentally challenged world, news media have brought to the forefront the issue of global warming as a result of elevated CO2 emissions.† In 1997, the Kyoto protocol was set up to bring about international targets for the reduction of these greenhouse gases to fight climate change.‡ Canada alone (although not the biggest contributor to greenhouse emissions†) has spent approximately $1 billion since 1995 in an effort to reduce the amount of these gases; however, despite this, levels keep rising.§ We can start to appreciate how photosynthesis *Hayami,

H., and Nakamura, M., 2007. Greenhouse gas emissions in Canada and Japan: Sector-specific estimates and managerial and economic implications. Journal of Environmental Management 85:371–392. †National Resources Defense Council, 2005. Global warming basics. http://www.nrdc.org/globalwarming/f101.asp. ‡Weaver, A.J., 2011. Toward the Second Commitment Period of the Kyoto Protocol. Science 332:795–796. §Spurgeon, D., 2000. Canada plans reduction in greenhouse-gas emissions. Nature 407:824.

In a sun-flecked lane, Beside a path where cattle trod, Blown by wind and rain, Drawing substance from air and sod; In ruggedness, it stands aloof, The ragged grass and puerile leaves, Lending a hand to fill the woof In the pattern that beauty makes. What mystery this, hath been wrought; Beauty from sunshine, air, and sod! Could we thus gain the ends we sought— Tell us thy secret, Goldenrod. Rosa Staubus Oklahoma pioneer (1886–1966)

KEY QUESTIONS 20.1

What Are the General Properties of Photosynthesis?

20.2

How Is Solar Energy Captured by Chlorophyll?

20.3

What Kinds of Photosystems Are Used to Capture Light Energy?

20.4

What Is the Molecular Architecture of Photosynthetic Reaction Centres?

20.5

What Is the Quantum Yield of Photosynthesis?

20.6

How Does Light Drive the Synthesis of ATP?

20.7

How Is Carbon Dioxide Used to Make Organic Molecules?

20.8

How Does Photorespiration Limit CO2 Fixation?

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

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Part 3 Metabolism and Its Regulation

" Inhabitants of Antarctica. Melting ice due to

Thinkstock

climate change has a negative impact on Adélie penguins.

is necessary to help counteract the increasing global warming problem, the effects of which include the extinction of animal habitats, increased tropical storm/hurricane strengths, pronounced forest fires, and melting glaciers leading to rising sea water levels and coastal flooding.* For example, we can witness the negative effects of thawing in the Antarctic, which directly impacts the Adélie penguin (Pygoscelis adeliae, pictured), causing it to migrate farther from its resting ground in order to obtain nourishment.†

The vast majority of energy consumed by living organisms stems from solar energy captured by the process of photosynthesis. Only chemolithotrophic prokaryotes are independent of this energy source. Of the 1.5 * 1022 kJ of energy reaching Earth each day from the Sun, 1% is absorbed by photosynthetic organisms and transduced into chemical energy.† This energy, in the form of biomolecules, becomes available to other members of the biosphere through food chains. The transduction of solar, or light, energy into chemical energy is often expressed in terms of carbon dioxide fixation, in which hexose is formed from carbon dioxide and oxygen is evolved: Light

6CO2 + 6H2O h C6H12O6 + 6O2

(20.1)

1011

Estimates indicate that tonnes of carbon dioxide are fixed globally per year, of  which one-third is fixed in the oceans, primarily by photosynthetic marine microorganisms. Although photosynthesis is traditionally equated with CO2 fixation, light energy (or rather the chemical energy derived from it) drives all endergonic processes in phototrophic cells. The assimilation of inorganic forms of nitrogen and sulfur into organic molecules (see Chapter 23) represents 2 other metabolic conversions closely coupled to light energy in green plants. Our previous considerations of aerobic metabolism (Chapters 17–19) treated cellular respiration (precisely the reverse of Equation 20.1) as the central energy-releasing process in life. It necessarily follows that the formation of hexose from carbon dioxide and water, the products of cellular respiration, must be endergonic. The necessary energy comes from light. Note that in the carbon dioxide fixation reaction described, light is used to drive a chemical reaction against its thermodynamic potential.

20.1

What Are the General Properties of Photosynthesis?

Photosynthesis Occurs in Membranes Organisms capable of photosynthesis are very diverse, ranging from simple prokaryotic forms to the largest organisms of all, Sequoia gigantea, the giant redwood trees of California. Despite this diversity, we find certain generalities regarding photosynthesis. An *National †Of

Georgraphic News, 2004. Climate change: Pictures of a warming world. http://news.nationalgeographic.com/news/2004/12/photogalleries/global_warming/. the remaining 99%, two-thirds is absorbed by land and oceans, thereby heating the planet; the remaining one-third is lost as light reflected back into space. NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 20

Photosynthesis

661

James Dennis/CNRI/Phototake Inc., NYC

FIGURE 20.1 Electron micrograph of a representative chloroplast.

important one is that photosynthesis occurs in membranes. In photosynthetic prokaryotes, the photosynthetic membranes fill up the cell interior; in photosynthetic eukaryotes, the photosynthetic membranes are localized in large organelles known as chloroplasts (Figures 20.1 above and 20.2). Chloroplasts are one member in a family of related plantspecific organelles known as plastids. Chloroplasts themselves show a range of diversity, from the single spiral chloroplast that gives Spirogyra its name to the multitude of ellipsoidal plastids typical of higher plant cells (Figure 20.3). Characteristic of all chloroplasts, however, is the organization of the inner membrane system, the thylakoid membrane. The thylakoid membrane is organized into paired folds that extend throughout the organelle, as in Figure 20.2. These paired folds, or lamellae, give rise to flattened sacs or discs, thylakoid vesicles (from the Greek thylakos, meaning “sack”), which occur in stacks called grana. A single stack, or granum, may contain dozens of thylakoid vesicles, and different grana are joined by lamellae that run through the soluble portion, or stroma, of the organelle. Chloroplasts thus possess 3 membrane-bound aqueous compartments: the intermembrane space, the stroma, and the interior of the thylakoid

Thylakoid vesicle Outer membrane

FIGURE 20.2 Schematic diagram of an idealized chloroplast.

Inner membrane

Intermembrane space

Granum (stack of thylakoids)

Stroma Thylakoid lumen

Lamella

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Part 3 Metabolism and Its Regulation

(a)

Biophoto Associates/Science Source

© Perennou Nuridsany/Photo Researchers, Inc.

662

(b) FIGURE 20.3 (a) Spirogyra—a freshwater green alga. (b) A higher plant cell.

vesicles, the thylakoid space (also known as the thylakoid lumen). As we shall see, this third compartment serves an important function in the transduction of light energy into ATP formation. The thylakoid membrane has a highly characteristic lipid composition and, like the inner membrane of the mitochondrion, is impermeable to most ions and molecules. Chloroplasts, like their mitochondrial counterparts, possess DNA, RNA, and ribosomes and consequently display a considerable amount of autonomy. However, many critical chloroplast components are encoded by nuclear genes, so autonomy is far from absolute.

Photosynthesis Consists of Both Light Reactions and Dark Reactions If a chloroplast suspension is illuminated in the absence of carbon dioxide, oxygen is evolved. Furthermore, if the illuminated chloroplasts are now placed in the dark and supplied with CO2, net hexose synthesis can be observed (Figure 20.4). Thus, the evolution of oxygen can be temporally separated from CO2 fixation and also has a light dependency that CO2 fixation lacks. The light reactions of photosynthesis, of which O2 evolution is only one part, are associated with the thylakoid membranes. In contrast, the light-independent

O2

Chloroplast suspension

CO2

O2

CO2

Into dark

Light

CO2

O2 Absence of CO2

O2 evolved

O2

CO2

CO2

CO2 fixation into sugars

FIGURE 20.4 The light-dependent and light-independent reactions of photosynthesis. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 20

Photosynthesis

reactions, or dark reactions, notably CO2 fixation, are located in the stroma. A concise summary of the photosynthetic process is that radiant electromagnetic energy (light) is transformed by a specific photochemical system located in the thylakoids to yield chemical energy in the form of reducing potential (NADPH) and high-energy phosphate (ATP). NADPH and ATP can then be used to drive the endergonic process of hexose formation from CO2 by a series of enzymatic reactions found in the stroma (see Equation 20.3, which follows).

Water Is the Ultimate e! Donor for Photosynthetic NADP" Reduction In green plants, water serves as the ultimate electron donor for the photosynthetic generation of reducing equivalents. The reaction sequence nhv

2 H2O + 2 NADP+ + xADP + xPi h O2 + 2 NADPH + 2 H+ + x ATP + x H2O

(20.2)

describes the process, where nhy symbolizes light energy (n is some number of photons of energy hy, where h is Planck’s constant and y is the frequency of the light). Light energy is necessary to make the unfavourable reduction of NADP+ by H2O a #eo= = -1.136V:#G$% = + 219

kJ NADP+ b mol

thermodynamically favourable. Thus, the light energy input, nhy, must exceed 219 kJ/mol NADP+. The stoichiometry of ATP formation depends on the pattern of photophosphorylation operating in the cell at the time and on the ATP yield in terms of the chemiosmotic ratio, ATP/H+, as we will see later. Nevertheless, the stoichiometry of the metabolic pathway of CO2 fixation is certain: 12 NADPH + 12 H+ + 18 ATP + 6 CO2 + 12 H2O S C6H12O6 + 12 NADP+ + 18 ADP + 18 Pi

(20.3)

A More Generalized Equation for Photosynthesis In 1931, comparative study of photosynthesis in bacteria led van Niel to a more general formulation of the overall reaction: Light

CO2 + 2 H2A h (CH2O) + 2A + Hydrogen Hydrogen Reduced Oxidized acceptor donor acceptor donor

H2O

(20.4)

In photosynthetic bacteria, H2A is variously H2S (photosynthetic green and purple sulfur bacteria), isopropanol, or some similar oxidizable substrate [(CH2O) symbolizes a carbohydrate unit]: CO2 + 2 H2S S (CH2O) + H2O + 2S

O ‘ CO2 + 2 CH3 i CHOH i CH3 S (CH2O) + H2O + 2 CH3 i C i CH3 In cyanobacteria and the eukaryotic photosynthetic cells of algae and higher plants, H2A is H2O, as implied earlier, and 2 A is O2. The accumulation of O2 to constitute 21% of Earth’s atmosphere is the direct result of eons of global oxygenic photosynthesis.

20.2

How Is Solar Energy Captured by Chlorophyll?

Photosynthesis depends on the photoreactivity of chlorophyll. Chlorophylls are magnesiumcontaining substituted tetrapyrroles whose basic structure is reminiscent of heme, the iron-containing porphyrin (see Chapters 5 and 19). Chlorophylls differ from heme in a number of properties: Magnesium instead of iron is coordinated in the centre of the planar NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

663

Part 3 Metabolism and Its Regulation

(a)

CH3 CH2 II

R

H

N

N

N H2C

III V

N

I

CH

(b)

O

CH3

H

IV CH3

H

O C

Mg

H

R= Chlorophyll a —CH3 Chlorophyll b —CHO

CH3

OCH3

H

CH2

H2 C

O CH2

C

H

O

HC

C

CH3

H2C CH2 H2C

Chlorophyll a

Chlorophyll b

CH

Solar spectrum

CH3

H2C CH2

Carotenoids

H2C

Bacteriochlorophyll a

Phycoerythrin

CH

CH3

Absorption

H2C CH2 H2C

Phycocyanin

CH

CH3

H3C Hydrophobic phytyl side chain

400

500 Wavelength (nm)

600

700

Fundamentals of Biochemistry, Third edition, Donald Voet, Judith G. Voet and Charlotte W. Pratt. ©2012 John Wiley & Sons, Inc. Reproduced with permission Hohn Wiley & Sons Inc.

Energy

Second excited state

Internal conversion (radiationless) First excited state

Absorption of blue light hv

Absorption of red light hv

Fluorescence Photooxidation (chemical reactions) hv Ground state

FIGURE 20.5 Structures (a) and absorption spectra (b) of chlorophyll a and b as well as that of accessory pigments (carotenoids, phycocyanin, phycoerythrin). The phytyl side chain of ring IV provides a hydrophobic tail to anchor the chlorophyll in membrane–protein complexes. (c) The energy diagram of the electronic states of chlorophyll. Absorbance of blue light raises electrons to the second excited state, while absorption of red light raises electrons to the first excited state. This energy can be emitted as fluorescence or utilzed for photooxidation reactions.

conjugated ring structure; a long chain alcohol, phytol, is esterified to a pyrrole ring substituent; and the methine bridge linking pyrroles III and IV is substituted and cross-linked to ring III, leading to the formation of a fifth 5-membered ring. The structures of chlorophyll a and b are shown in Figure 20.5a. Chlorophylls are excellent light absorbers because of their aromaticity. That is, they possess delocalized p electrons above and below the planar ring structure. The energy differences between electronic states in these p orbitals correspond to the energies of visible light photons. When light energy is absorbed, an electron is promoted to a higher orbital, enhancing the potential for transfer of this electron to a suitable acceptor. Loss of such a photoexcited electron to an acceptor is an oxidation–reduction (redox) reaction. The net result is the transduction of light energy into the chemical energy of a redox reaction.

Chlorophylls and Accessory Light-Harvesting Pigments Absorb Light of Different Wavelengths The absorption spectra of chlorophylls a and b (Figure 20.5) differ somewhat. Plants that possess both chlorophylls can harvest a wider spectrum of incident energy. Other pigments in photosynthetic organisms, accessory light-harvesting pigments (Figure 20.6), increase the possibility for absorption of incident light of wavelengths not absorbed by the chlorophylls. Carotenoids and phycocyanobilins, like chlorophyll, possess many conjugated double bonds and thus absorb visible light. Carotenoids have 2 primary roles in photosynthesis—light harvesting and photoprotection through destruction of reactive oxygen species that arise as by-products of photoexcitation.

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Fundamentals of Biochemistry: Life at the Molecular Level, 4th Edition, Donald Voet, Judith G. Voet and Charlotte W. Pratt. ©2013 John Wiley & Sons, Inc. Reproduced with permission of John Wiley & Sons, Inc.

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Chapter 20

(a) H3C

CH3

CH3

CH3

CH3

CH3

H3C

CH3

!-Carotene (b) O

H N

H N

H N

N

O

H CH3

CH CH3

CH3

CH2

CH2

CH2

CH2

C OH

OC

CH3

CH3

CH2

665

FIGURE 20.6 Structures of representative accessory light-harvesting pigments in photosynthetic cells. (a) b-carotene, an accessory light-harvesting pigment in leaves. (b) Phycocyanobilin, a blue pigment found in cyanobacteria.

H3C

CH3

Photosynthesis

CH3

O

OH Phycocyanobilin

The Light Energy Absorbed by Photosynthetic Pigments Has Several Possible Fates Each photon represents a quantum of light energy. A quantum of light energy absorbed by a photosynthetic pigment has 4 possible fates (Figure 20.7): 1. Loss as heat. The energy can be dissipated as heat through redistribution into atomic vibrations within the pigment molecule. 2. Loss as light. Energy of excitation reappears as fluorescence (light emission); a photon of fluorescence is emitted as the e- returns to a lower orbital. This fate is common only in saturating light intensities. For thermodynamic reasons, the photon of fluorescence has a longer wavelength and hence lower energy than the quantum of excitation. 3. Resonance energy transfer. The excitation energy can be transferred by resonance energy transfer to a neighbouring molecule if the energy level difference between the 2 corresponds to the quantum of excitation energy. In this process, the energy transferred raises an electron in the receptor molecule to a higher-energy state as the photoexcited e- in the original absorbing molecule returns to ground state. This Förster resonance energy transfer is the mechanism whereby quanta of light falling anywhere within an array of pigment molecules can be transferred ultimately to specific photochemically reactive sites. 4. Energy transduction. The energy of excitation, in raising an electron to a higher-energy orbital, dramatically changes the standard reduction potential, Po= , of the pigment such that it becomes a much more effective electron donor. That is, the excited-state species, by virtue of having an electron at a higher-energy level through light absorption, has become a more potent electron donor. Reaction of this excited-state electron donor with an electron acceptor situated in its vicinity leads to the transformation, or transduction, of light energy (photons) to chemical energy (reducing power, the potential for electron transfer reactions). Transduction of light energy into chemical energy, the photochemical event, is the essence of photosynthesis.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

666

Part 3 Metabolism and Its Regulation

FIGURE 20.7 Possible fates of the quantum of light energy absorbed by photosynthetic pigments.

Light energy (hv) e–

+

Pigment molecule (P)

Excited state (P*)

+

e–

Qox Q–red

e–

Thermal dissipation

Fluorescence

Energy transfer

+

+

+

e–

e–

e–

+ e–

Oxidized P (P+)

hn

Heat

Transfer

e–

Photon of fluorescence

+

P* Energy transfer to neighbouring P molecule

The Transduction of Light Energy into Chemical Energy Involves Oxidation–Reduction The diagram presented in Figure 20.8 illustrates the fundamental transduction of light energy into chemical energy (an oxidation–reduction reaction) that is the basis of photosynthesis. Chlorophyll (Chl) resides in a membrane in close association with molecules competent in e- transfer, symbolized here as A and B. Chl absorbs a photon of light,

FIGURE 20.8 Model for light absorption by chlorophyll and transduction of light energy into an oxidation– reduction reaction. I: Photoexcitation of Chl creates Chl*. II: Electron transfer from Chl* to A yields oxidized Chl (Chl") and reduced A (A!) III: An electron transfer pathway from A! to NADP" leads to NADPH formation and restoration of oxidized A (A). IV: Chl" accepts an electron from B, restoring Chl and generating oxidized B (B"). V: B" is reduced back to B by an electron originating in H2O. Water oxidation is the source of O2 formation.

1 2

1 2

NADP+

NADPH

hn Chl*

Chl B

A–

A

A I

B

Chl II

B

A +

Chl III

B

A +

A

Chl IV

1 2

B+

H2O

Chl V

B

1 4

O2 NEL

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Chapter 20

Photosynthesis

667

becoming activated to Chl* in the process. Electron transfer from Chl* to A leads to oxidized Chl (Chl+, a cationic free radical) and reduced A (A- in the diagram). Subsequent oxidation of A- eventually culminates in reduction of NADP+ to NADPH. The electron “hole” in oxidized Chl (Chl+) is filled by transfer of an electron from B to Chl+, restoring Chl and creating B+. B+ is restored to B by an e- donated by water. O2 is the product of water oxidation. Note that the system is restored to its original state once NADPH is formed and H2O is oxidized. Proton translocations accompany these light-driven electron transport reactions. Such H+ translocations establish a chemiosmotic gradient across the photosynthetic membrane that can drive ATP synthesis.

Photosynthetic Units Consist of Many Chlorophyll Molecules but Only a Single Reaction Centre In the early 1930s, Emerson and Arnold (1932) investigated the relationship between the amount of incident light energy, the amount of chlorophyll present, and the amount of oxygen evolved by illuminated algal cells. Emerson and Arnold were seeking to determine the quantum yield of photosynthesis: the number of electrons transferred per photon of light. Their studies gave an unexpected result: When algae were illuminated with very brief light flashes that could excite every chlorophyll molecule at least once, only 1 molecule of O2 was evolved per 2400 chlorophyll molecules. This result implied that not all chlorophyll molecules are photochemically reactive, and it led to the concept that photosynthesis occurs in functionally discrete units. Chlorophyll serves 2 roles in photosynthesis. It is involved in light harvesting and the transfer of light energy to photoreactive sites by exciton transfer, and it participates directly in the photochemical events whereby light energy becomes chemical energy. A photosynthetic unit (Figure 20.9) can be envisioned as an antenna of several hundred light-harvesting chlorophyll molecules (green) plus a special pair of photochemically reactive chlorophyll a molecules called the reaction centre (orange). The purpose of the vast majority of chlorophyll in a photosynthetic unit is to harvest light incident within the unit and funnel it, via resonance energy transfer, to the reaction centre chlorophyll dimers that are photochemically active. Most chlorophyll thus acts as a large light-collecting antenna, and it is at the reaction centres that the photochemical event occurs. Oxidation of chlorophyll leaves a cationic free radical, Chl+, whose properties as an electron acceptor have important consequences for photosynthesis. Note that the Mg2+ ion does not change in valence during these redox reactions.

20.3

h"

Light-harvesting pigment (antenna molecules)

Reaction centre FIGURE 20.9 Schematic diagram of a photosynthetic unit.

What Kinds of Photosystems Are Used to Capture Light Energy?

All photosynthetic cells contain some form of photosystem. Photosynthetic bacteria have only 1 photosystem; furthermore, they lack the ability to use light energy to split H2O and release O2. Cyanobacteria, green algae, and higher plants are oxygenic phototrophs because they can generate O2 from water. Oxygenic phototrophs have 2 distinct photosystems: photosystem I (PSI) and photosystem II (PSII). Type I photosystems use ferredoxins as terminal electron acceptors; type II photosystems use quinones as terminal electron acceptors. PSI is defined by reaction centre chlorophylls with maximal red light absorption at 700 nm; PSII uses reaction centres that exhibit maximal red light absorption at 680 nm. The reaction centre Chl of PSI is referred to as P700 because it absorbs light of 700-nm wavelength; the reaction centre Chl of PSII is called P680 for analogous reasons. Both P700 and P680 are chlorophyll a dimers situated within specialized protein complexes. A distinct property of PSII is its role in light-driven O2 evolution. Interestingly, the photosystems of photosynthetic bacteria are type II photosystems that resemble eukaryotic PSII more than PSI, even though these bacteria lack O2-evolving capacity. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

668

Part 3 Metabolism and Its Regulation

Chlorophyll Exists in Plant Membranes in Association with Proteins Detergent treatment of a suspension of thylakoids dissolves the membranes, releasing complexes containing both chlorophyll and protein. These chlorophyll–protein complexes represent integral components of the thylakoid membrane, and their organization reflects their roles as one of light-harvesting complexes (LHC), PSI complexes, or PSII complexes. All chlorophyll is apparently localized within these 3 macromolecular assemblies.

PSI and PSII Participate in the Overall Process of Photosynthesis What are the roles of the 2 photosystems, and what is their relationship to each other? PSI provides reducing power in the form of NADPH. PSII splits water, producing O2, and feeds the electrons released into an electron transport chain that couples PSII to PSI. Electron transfer between PSII and PSI pumps protons for chemiosmotic ATP synthesis. As summarized by Equation 20.2, photosynthesis involves the reduction of NADP+, using electrons derived from water and activated by light, hy. ATP is generated in the process. The standard reduction potential for the NADP+/NADPH couple is - 0.32 V. Thus, a strong reductant with an !o% more negative than -0.32 V is required to reduce NADP+ under standard conditions. By similar reasoning, a very strong oxidant will be required to oxidize water to oxygen because !o% 1 12O2/H2O 2 is + 0.82 V. Separation of the oxidizing and reducing aspects of Equation 20.2 is accomplished in nature by devoting PSI to NADP+ reduction and PSII to water oxidation. PSI and PSII are linked via an electron transport chain so that the weak reductant generated by PSII can provide an electron to reduce the weak oxidant side of P700 (Figure 20.10). Thus, electrons flow from H2O to NADP+, driven by light energy absorbed at the reaction centres. Oxygen is a by-product of the photolysis, literally “light-splitting,” of water. Accompanying electron flow is production of a proton gradient and ATP synthesis (see Section 20.6). This light-driven phosphorylation is termed photophosphorylation.

The Pathway of Photosynthetic Electron Transfer Is Called the Z Scheme

Ferredoxin (Fd): A generic term for small proteins possessing iron–sulfur clusters that participate in various electron transfer reactions.

Photosystems I and II contain unique complements of electron carriers, and these carriers mediate the stepwise transfer of electrons from water to NADP+. When the individual redox components of PSI and PSII are arranged as an e- transport chain according to their standard reduction potentials, the zigzag result resembles the letter Z laid sideways (Figure 20.11). The various electron carriers are indicated as follows: “Mn complex” symbolizes the manganese-containing oxygen-evolving complex; D is its e- acceptor and the immediate e- donor to P680+; QA and QB represent special plastoquinone molecules (see Figure 20.13 on page 670) and PQ the plastoquinone pool; Fe-S stands for the Rieske iron–sulfur centre, and cyt f, cytochrome f. PC is the abbreviation for plastocyanin, the immediate e- donor to P700+, and FA, FB, and FX represent the membrane-associated ferredoxins downstream from A0 (a specialized Chl a) and A1 (a specialized PSI quinone). Fd is the soluble ferredoxin pool that serves as the e- donor to the flavoprotein (Fp), called ferredoxin!NADP " reductase, which catalyzes reduction of NADP+ to NADPH. Cyt(b6) N,(b6)P symbolizes the cytochrome b6 moieties of the cytochrome b6 f complex. PQ and the cytochrome b6 f complex also serve to transfer e- from FA/FB back to P700+ during cyclic photophosphorylation (the pathway symbolized by the dashed arrow).

FIGURE 20.10 Roles of the 2 photosystems, PSI and PSII.

PSII “blue” light < 680 nm

PSI “red” light 700 nm

P680

P700 Weak oxidant Strong reductant % ≅ 0.45 V % < –0.6 V

Strong oxidant Weak reductant % > +0.8 V %≅0V °

H2O

°

1 2

O2

ADP

°

+

Pi

ATP

°

NADP+

NADPH NEL

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Chapter 20 (a)

Photosynthesis

Photosystem I

–1.20

P700* A0 A1 FA

–0.80

FB FX

Photosystem II –0.40

P680* Chl a Pheo QA

Fp (FAD)

H+

+

NADP+

PQ

QB

0

Fd

o'

(Cyt b6)N (Cyt b6)P

PQ

Fe-S

+0.40

Cyt f hn

PC P700 +0.80

H2O Mn complex

+1.20

hn

D

1 O 2 2

Protons Protons taken up released from stroma into lumen

P680

Protons released in lumen

+1.60 (b)

2 H+

4 H+

ADP + Pi

Stroma H+

hn

hn

Fd

Pheo

QB

Fe

Pheo

FeSA FeSB

Cyt b6 Cyt b6

PQ

FeSX

Fe-S

H2O

2 H+

+

1 2

O2

NADPH CF1CF0– ATP synthase

Cyt f

Fd

Fp (FAD)

A1 A0

P700

P680

Mn complex

NADP+

Photosystem I

Photosystem II

QA

+

ATP

PC

PC

4 H+

4 H+

Lumen

FIGURE 20.11 The Z scheme of photosynthesis. (a) The Z scheme is a diagrammatic representation of photosynthetic electron flow from H2O to NADP". The energy relationships can be derived from the !o% scale beside the Z diagram. Energy input as light is indicated by 2 broad arrows, 1 photon appearing in P680 and the other in P700. P680* and P700* represent photoexcited states. The 3 supramolecular complexes (PSI, PSII, and the cytochrome b6f complex) are in shaded boxes. Proton translocations that establish the proton-motive force driving ATP synthesis are illustrated as well. (b) The functional relationships among PSII, the cytochrome bf complex, PSI, and the photosynthetic CF1CF0–ATP synthase within the thylakoid membrane. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

NADPH

669

670

Part 3 Metabolism and Its Regulation

Overall photosynthetic electron transfer is accomplished by 3 membrane-spanning supramolecular complexes composed of intrinsic and extrinsic polypeptides (shown as shaded boxes bounded by solid black lines in Figure 21.11). These complexes are the PSII complex, the cytochrome b6f complex, and the PSI complex. The PSII complex is aptly described as a light-driven water:plastoquinone oxidoreductase; it is the enzyme system responsible for photolysis of water, and, as such, it is also referred to as the oxygen-evolving complex, or OEC. PSII possesses a metal cluster containing 4 Mn2+ atoms that coordinate 2 water molecules. As P680 undergoes 4 cycles of light-induced oxidation, 4 protons and 4 electrons are removed from the 2 water molecules and their O atoms are joined to form O2. A tyrosyl side chain of the PSII complex (see following discussion) mediates electron 24 transfer between the Mn2+ cluster and P680. The O -evolving reaction requires Ca2+ and 2 Cl- ions in addition to the (Mn2+)4 cluster.

O2 evolved/flash

(a)

4 (b) h"

S0

H+

8

12 16 Flash number H+

h"

+ e–

+ e–

S1

h"

20

H+

+ h" e–

S2

H+

+ e–

S3

S4

O2

2 H 2O

FIGURE 20.12 Oxygen evolution requires the accumulation of 4 oxidizing equivalents in PSII. (a) O2 evolution after brief light flashes. (b) The cycling of the PSII reaction centre through 5 different oxidation states, S0 to S4. One e! is removed photochemically at each light flash, moving the reaction centre successively through S1, S2, S3, and S4. S4 decays spontaneously to S0 by oxidizing 2 H2O to O2.

O H3C

H (CH2

H3C

CH3 CH

C

CH2)9 H

O Plastoquinone A +2 H+ , 2 e–

–2 H+ , 2 e–

Oxygen Evolution Requires the Accumulation of 4 Oxidizing Equivalents in PSII When isolated chloroplasts that have been held in the dark are illuminated with very brief flashes of light, O2 evolution reaches a peak on the third flash and every fourth flash thereafter (Figure 20.12a). The oscillation in O2 evolution damps over repeated flashes and converges to an average value. These data are interpreted to mean that the P680 reaction centre complex cycles through 5 different oxidation states, numbered S0 to S4. One electron and 1 proton are removed photochemically in each step. When S4 is attained, an O2 molecule is released (Figure 20.12b) as PSII returns to oxidation state S0 and 2 new water molecules bind. (The reason the first pulse of O2 release occurred on the third flash [Figure 20.12a] is that the PSII reaction centres in the isolated chloroplasts were already poised at S1 reduction level.)

Electrons Are Taken from H2O to Replace Electrons Lost from P680 The events intervening between H2O and P680 involve D, the name assigned to a specific protein tyrosine residue that mediates e- transfer from H2O via the Mn complex to P680+ (see Figure 20.11). The oxidized form of D is a tyrosyl free-radical species, D+. To begin the cycle, an exciton of energy excites P680 to P680*, whereupon P680* transfers an electron to a nearby Chl a molecule, which is the direct electron acceptor from P680*. This Chl a then reduces a molecule of pheophytin, symbolized by “Pheo” in Figure 20.11. Pheophytin is like chlorophyll a, except 2 H+ replace the centrally coordinated Mg2+ ion. This special pheophytin is the direct electron acceptor from P680*. Loss of an electron from P680* creates P680+, the electron acceptor for D. Electrons flow from Pheo via specialized molecules of plastoquinone, represented by “Q” in Figure 20.11, to a plastoquinone pool (PQ) within the membrane. Because of its lipid nature, plastoquinone is mobile within the membrane and hence serves to shuttle electrons from the PSII supramolecular complex to the cytochrome b6 f complex. Alternate oxidation–reduction of plastoquinone to its hydroquinone form involves the uptake of protons (Figure 20.13). The asymmetry of the thylakoid membrane is designed to exploit this proton uptake and release so that protons (H+) accumulate within the lumen of thylakoid vesicles, establishing an electrochemical gradient. Note that plastoquinone is an analog of coenzyme Q, the mitochondrial electron carrier (see Chapter 19).

OH H3C

H (CH2

H3C

CH3 CH

C

CH2)9 H

OH Plastohydroquinone A FIGURE 20.13 The structures of plastoquinone A and its reduced form, plastohydroquinone (or plastoquinol). Plastoquinone A has 9 isoprene units and is the most abundant plastoquinone in plants and algae.

Electrons from PSII Are Transferred to PSI via the Cytochrome b6f Complex The cytochrome b6 f or plastoquinol:plastocyanin oxidoreductase is a large (210-kD) multimeric protein possessing 26 transmembrane a-helices. This membrane protein complex is structurally and functionally homologous to the cytochrome bc1 complex (Complex III) of mitochondria (see Chapter 19). It includes the 2 heme-containing electron transfer proteins for which it is named, as well as iron–sulfur clusters, which also participate in electron transport. The purpose of this complex is to mediate the transfer of electrons from PSII to PSI and to pump protons across the thylakoid membrane via a plastoquinone-mediated Q NEL

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Chapter 20

Photosynthesis

671

Structure of the cyanobacterial cytochrome b6f complex. The heme groups of cytochromes b6n, b6p, and f are shown in red; the iron–sulfur clusters are blue (pdb id = 1BF5). The upper bundle of a-helices defines the transmembrane domain.

cycle, analogous to that found in mitochondrial e- transport (see Chapter 19). Cytochrome f ( f from the Latin folium, meaning “foliage”) is a c-type cytochrome, with a reduction potential of + 0.365 V. Cytochrome b6 in 2 forms (low- and high-potential) participates in the oxidation of plastoquinol and the Q cycle of the b6 f complex. The cytochrome b6 f complex can also serve in an alternative cyclic electron transfer pathway. Under certain conditions, electrons derived from P700* are not passed on to NADP+ but instead cycle down an alternative path, whereby reduced ferredoxin contributes its electron to PQ. This electron is then passed to the cytochrome b6 f complex, and then back to P700+. This cyclic flow yields no O2 evolution or NADP+ reduction but can lead to ATP synthesis via socalled cyclic photophosphorylation, discussed in Section 33.1 in Chapter 33.

Plastocyanin Transfers Electrons from the Cytochrome b6f Complex to PSI Plastocyanin (PC in Figure 20.11) is an electron carrier capable of diffusion along the inside of the thylakoid and migration in and out of the membrane, aptly suited to its role in shuttling electrons between the cytochrome b6 f complex and PSI. Plastocyanin is a low-molecularweight (10.4-kD) protein containing a single copper atom. PC functions as a single-electron carrier (!o% = +0.32V) as its copper atom undergoes alternate oxidation–reduction between the cuprous (Cu+) and cupric (Cu2+) states. PSI is a light-driven plastocyanin:ferredoxin oxidoreductase. When P700, the specialized chlorophyll a dimer of PSI, is excited by light and oxidized by transferring its e- to an adjacent chlorophyll a molecule that serves as its immediate e- acceptor, P700+ is formed. (The standard reduction potential for the P700+/P700 couple is about +0.45 V.) P700+ readily gains an electron from plastocyanin. The immediate electron acceptor for P700* is a special molecule of chlorophyll. This unique Chl a (A0) rapidly passes the electron to a specialized quinone (A1), which in turn passes the e- to the first in a series of membrane-bound ferredoxins. This Fd series ends with a soluble form of ferredoxin, Fds, which serves as the immediate electron donor to the flavoprotein (Fp) that catalyzes NADP+ reduction, namely, ferredoxin:NADP+ reductase.

20.4

What Is the Molecular Architecture of Photosynthetic Reaction Centres?

What molecular architecture couples the absorption of light energy to rapid electron transfer events, in turn coupling these e- transfers to proton translocations so that ATP synthesis is possible? Part of the answer to this question lies in the membrane-associated NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

672

Part 3 Metabolism and Its Regulation

(a)

(b)

Cytochrome with 4 heme groups

(c)

hν

M

L P870 50–55

NMD

1067

FIGURE 31.13 The 2 positional rules for NMD. (a) The exon junction complex (EJC), which originally binds to premRNA and plays a role in splicing, remains bound to the processed mRNA 20–24 nucleotides upstream of an exon–exon junction. (b) A premature stop codon occurring in an aberrant transcript must be located further than 50–55 nucleotides away from an exon–exon junction in order to be recognized. If it is within 50 nucleotides, NMD will not be triggered. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

1068

Part 5 Information Transfer

(a) Normal stop codon

Premature stop codon

CBC (b)

AAAAA

PP2A S5 S7

eRF

2 3b

CBC

EJC core

AAAAA

eRF

CBC

P

(d)

1

2 3b

CBC

eRF

S5 P S1 S7 1 2 3b

AAAAA CBC

Phosphorylation S1 1 2 3b

PP2A

(h)

EJC core

S1 1 2 3b

Premature stop codon

S1

(c)

P

(g)

1

EJC core

CBC (e)

eRF

CBC 60S 40S

(f)

P

eRF

S1 1 2 3b

P

Dissociation

CBC EJC core

S1 1 2 3b

PP2A

AAAAA

EJC core

(j)

Normal stop codon

EJC core

AAAAA

AAAAA

Dephosphorylation S5 P PP2A S7 1 2 3b

Dcp

(i)

AAAAA

EJC core

Xrm

S5 S7

EJC core

AAAAA

EJC core

AAAAA

P

Dissociation 1 2 3b

AAAAA

CBC FIGURE 31.14 Mechanism of action of NMD. If an active ribosome encounters a PTC along an mRNA, it will interact with the downstream EJC through direct interaction with UPF1 and adapter proteins UPF2 and UPF3 (A–C). This interaction, along with phosphorylation of UPF1, serves to promote activation of NMD (D–F). Once NMD is triggered, ribosome dissociation occurs, and dephosphorylation of UPF1 by SMG-6 and SMG-5/7 heterodimer, in association with protein phosphatase 2A (PP2A), signals for decapping of the mRNA, leading to downstream degradation (G–J). (Chang Y-F et al., The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 2007. 76:51–74.)

in mammalian cells. This rule, the so-called - 55-boundary rule, indicates that only stop codons at least ! 55 nt upstream of the final intron are able to trigger NMD. Most mammalian transcripts that have been tested abide by this - 55-boundary rule (Figure 31.13). Interestingly, these sequence rearrangements alone are not sufficient to trigger NMD. As mentioned above, trans-acting factors are needed to regulate RNA decay by NMD, and the EJC represents the second signal required to trigger NMD. In fact, the proteins of the EJC are deposited 20–24 nt upstream of all exon–exon junctions after RNA splicing, in all mRNAs and not only those containing PTCs. The EJC contains many proteins, including RNPS1, Y14, Magoh, SRm160, REF, and UAP56 (Figure 31.15). Interestingly, many of PININ

SAP18 ACINUS

RNPSI

eIF4A3

5'

AAAA MLN51 UAP56

Y14 MAGOH UPF2

UPF3 SRm160 PYM

UPF1

REF/ALY

EJC core Direct binding important for NMD Possible binding

TAP P15

Involved in NMD

FIGURE 31.15 The exon junction complex (EJC). The EJC contains many proteins, including RNPS1, Y14, Magoh, SRm160, REF, and UAP56. Interestingly, many of these proteins have previously been shown to function in other events, such as splicing and nuclear export. (Chang Y-F et al., The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 2007. 76:51–74.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 31

Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA

these proteins had previously been shown to function in other events, such as RNA splicing (RNPS1 and SRm160), mRNA export (REF and UAP56), and cytoplasmic mRNA localization. It has been shown that only RNPS1, Y14, and Magoh are capable of triggering NMD. Moreover, Y14, REF, and RNPS1 can directly associate with the UPF proteins.

How Do the UPFs and the EJC Work Together to Trigger NMD? First, it is important to consider that the EJC is not a single entity but rather a highly dynamic ensemble of proteins that evolves as an mRNA is processed, exported from the nucleus, and translated in the cytoplasm. In Xenopus laevis oocytes, the first component of the EJC to arrive on the mRNA is REF; it is even found at high levels on unspliced premRNA. After RNA splicing, RNPS1, Y14, Magoh, UAP56, and SRm160 are recruited to the mRNA. This order of recruitment is recapitulated in mammalian cells, except that RNPS1 appears to be recruited before RNA splicing starts, while REF is found mainly on spliced mRNA. After export to the cytoplasm, REF appears to dissociate, while most of or all the other factors remain bound. This sequential association with the target RNA is also true for UPF proteins. UPF proteins typically associate with target mRNAs as follows: UPF3/3B first, then UPF2, and then UPF1. According to this model, UPF3/3B is recruited to be part of the EJC after RNA splicing in the nucleus, UPF2 joins the EJC soon after the mRNA leaves the nucleus, and finally UPF1 is recruited only after the cytoplasmic translation machinery reads a stop codon in the mRNA (Figure 31.14). This model is supported by the locations where these NMD factors reside at highest concentrations during steady state: UPF3/3B is nuclear, UPF2 is perinuclear, and UPF1 is cytoplasmic. However, UPF1 could be recruited earlier to the mRNP, since it has been shown to harbour a nucleocytoplasmic shuttling activity, suggesting that it spends some time in the nucleus.

Translation Dependence of NMD A final question that arises with respect to NMD is timing of the process. It is well-established that both NMD and non-stop decay (NSD) require translation, as use of translation inhibitors, such as cycloheximide, prevent NMD/NSD of aberrant mRNAs. In this regard, a number of observations suggest that NMD occurs during a pioneer round of translation, promoting the idea that aberrant transcripts should be eliminated before mass translation is engaged. First, NMD appears to affect cap-binding complex (CBC)-bound mRNAs, which exist starting from the pre-mRNA stage in the nucleus and end at the stage of bulk translation when CBC is exchanged for the eukaryotic initiation factor 4E (eIF4E), but does not affect eIF4E-bound mRNAs. This observation promotes the idea of preliminary scanning occurring prior to eIF4E-mediated translation for the sake of RNA quality control, as well as involvement of the CBC in NMD. A second piece of evidence lies in the nuclear localization of transcripts affected by NMD, suggesting that the transcripts remain associated with the nucleus during a determinant round of scanning prior to being fully translocated to the cytosol for bulk translation (Figure 31.16).

How Are NMD Substrates Degraded? Most of what we know about the enzymes involved in the terminal stage of NMD—the actual decay of mRNA—comes from studies in yeast. Rather than being degraded by NMD-specific ribonucleases, nonsense codon-bearing transcripts in yeast are degraded by the same ribonucleases that degrade normal mRNAs, such as those targeted by AMD. The major enzyme responsible is Xrn1p, the 5= S 3= exonuclease also responsible for degrading most normal yeast mRNAs. Interestingly, the only difference between the decay of AREcontaining and nonsense codon–bearing transcripts is that the latter does not require deadenylation. The ability of NMD in yeast to bypass this normally rate-limiting step of mRNA decay is probably critical for it to efficiently eliminate nonsense codon-bearing transcripts. Conversely, in mammalian cells, PTC-containing mRNAs are degraded following the removal of the poly(A) tail, suggesting that, unlike in yeast, NMD in mammalian cells could follow the same degradation rules as AMD. It is not yet clear, however, if this NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

1069

1070

Part 5 Information Transfer PTC-Containing mRNA eI F - 4

CBC

G

AAA

2 3b Premature stop codon

eI F - 4

CBC

Normal mRNA

CBC AA

EJC core

AAA

2 3b Normal stop codon

CBC AA

2 3b

AA

EJC core

Normal stop codon

EJC core

mRNA decay

eI F - 4

G

AA AAA 2 3b EJC core

1 eRF

G

Pioneer round

G

AAA

eI F - 4

Dissociation

eIF-4E

eI F - 4

G

AA AAA 2 3b EJC core

Bulk translation

FIGURE 31.16 The pioneer round of translation, or “scanning,” is used as a quality control step to detect aberrant, PTC-containing mRNAs. If a PTC is detected, the mRNA is degraded via NMD. If the mRNA is deemed proper, its CBC is exchanged for eIF4E and bulk translation ensues. (Chang Y-F et al., The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 2007. 76:51–74)

deadenylation precedes 5= or 3= decay. How NMD accomplishes this feat is not known precisely. As mentioned above, the decay of ARE-containing mRNAs by AMD is accomplished through removal of the poly(A) tail by deadenylation, which leads to the loss of PABP, opening the mRNA circle, and exposing the 5=-end to decapping enzymes. However, nonsense codon–bearing mRNAs must get decapped without first being deadenylated. This is accomplished by UPF1, which directly recruits the decapping enzyme complex to target mRNAs, an idea consistent with the fact that UPF1 binds specifically to DCP1. Interestingly, AMD-associated proteins also communicate with the decapping machinery through scavenger decapping enzymes (DcpS) to ensure mRNA decay. Regardless of the precise mechanism, decapping is clearly the rate-limiting step for the decay of nonsense codon-bearing transcripts, as the rate of decapping dictates how the transcript is degraded (5= nonsense codons more strongly elicit NMD than do 3= nonsense codons).

Non-stop Decay Contrarily to NMD, NSD is used to eliminate mRNAs devoid of a stop codon. The end result of translation of this type of mRNA is stalling of the ribosome at the 3=-end of the transcript, leading to recruitment of the cytoplasmic exosome. The exosome, which provides a number of 3= S 5= exonucleases, allows for decay without the need for the slow deadenylation and decapping seen in typical mRNA turnover (Figure 31.17). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 31

Nucleus

Cytoplasm

Ini tia tio n Elo ng ati on Te (E rm F1 ina A) tio n( eR F3

)

Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA

pre-mRNA UTR

AAAAAAAA

UTR UGA

AUG

AAAAAAAA

llin g

Wild type mRNA

Sta

Splicing and polyadenylation

Ini tia tio n Elo ng ati on

AUG

Normal translation

UGA

AAAAAAAA

Non-stop translation

AAAAAAAA

Skl7p recognizes empty A site

Non-stop mRNA AAAAAAAA AUG

A

Ribosome is released Exosome is activated

AAAAA

A Proteolysis

FIGURE 31.17 A model for non-stop decay (NSD). If a transcript does not contain a stop codon, due to mutation, the ribosome will stall at the end of the transcript, recruiting the NSD machinery, which leads to exonucleolytic decay of the mRNA. (Non-stop decay—a new mRNA surveillance pathway. BioEssays. 2002 Sep;24(9):785–8.)

31.5

What Is miRNA-Mediated Gene Regulation?

A major level of post-transcriptional regulation of gene expression is mediated by short, trans-acting, single-stranded RNA (ssRNA) molecules. This form of regulation is known as RNA interference (RNAi). Its discovery not only provided a new tool to conduct biological research but also uncovered a novel and unsuspected regulatory mechanism of gene expression inside the cell. The process of RNAi is conserved in many organisms, from plants to mammals, and is conducted by non-coding RNA, known as micro-RNAs (miRNAs), in collaboration with a variety of RBPs and nucleases. miRNAs are a large family of 21–25 nucleotide (nt)-long single-stranded RNAs that are expressed in many organisms, ranging from plants and worms to humans. miRNAs regulate key homeostatic processes such as cellular proliferation, differentiation, immune response, and cell death. The aberrant expression or malfunction of miRNAs has been associated with many deadly diseases such as various cancers. miRNAs are highly abundant molecules and their number (e.g., greater than 800 in human) is comparable to the number of transcription factors or RNA-binding proteins. In general, miRNAs bind to specific sequences, called “seed elements” located within the 3= or 5=UTR of target mRNAs, to block their translation and, in some cases, to promote their decay (Figure 31.18). It is wellaccepted that the minimal effector complex for miRNA-mediated effects consists of a single-stranded small RNA associated with the AGO2 protein. Since only partial complementarity is required between miRNAs and their target mRNAs, one miRNA can regulate hundreds of mRNAs. For this reason, an individual miRNA can impact several cellular processes at the same time, indicating that a tight regulation of miRNA expression and function is required, specifically in response to extra- and/or intracellular signals. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

1071

1072

Part 5 Information Transfer >15 nucleotides ORF

Bulge NNNNNNNNNN

A UNNNNNNNA

NNNNNNNNNN

NNNNNNNNN

16

13 Bulge 8

3′ complementarity

AAAAAA miRNA

1

‘Seed region’

FIGURE 31.18 miRNAs bind to their target mRNAs at specific sequences known as seed regions. Perfect complementarity at the seed region is required for binding, and complementarity up to the eleventh nucleotide is required for mRNA slicing and degradation. miRNAs also typically bind at another site toward their 3=-end, but with looser complementarity. (Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 2008 Feb;9(2):102–14. Nature Publishing Group.)

miRNA Biogenesis While a number of black boxes still exist with respect to miRNA-mediated effects, numerous aspects of the process have been elucidated over the past decade. The majority of miRNA genes are found in non–protein-coding regions of the genome, while a sizeable minority are found in the introns of protein-coding genes (Figure 31.19). Transcription of miRNA gives rise to a stem-loop structured RNA molecule of roughly 70 nucleotides, known as pri-miRNA. Pri-miRNAs can be transcribed from an individual gene by RNA polymerase II or can be carried by spliced introns of protein-coding genes. The newly synthesized pri-mRNA folds into a hairpin to become a substrate for 2 members of the RNAse III family of endoribonucleases, Drosha and Dicer (Figure 31.20 on page 1074). For complete maturation, the miRNA undergoes 2 cleavage reactions during biogenesis. The first reaction occurs within the nucleus and is mediated by Drosha, an RNase III endonuclease, which cleaves the RNA at the base of the stem-loop. To execute this step, Drosha associates with the dsRNA-binding protein DGCR8 (DiGeorge critical region 8) to process the pri-miRNA into a precursor stem-loop structure known as pre-miRNA (Figure 31.19). Pre-miRNA consists of a hairpin stem of 33 base-pairs, a terminal loop, and 2 single-stranded flanking regions located upstream and downstream of the hairpin (Figure 31.21). Evidence has indicated that some pre-miRNAs are not produced by Drosha– DGCR8-mediated processing but are the direct product of the splicing and debranching of introns (mirtrons) (Figure 31.19). Once exported from the nucleus to the cytoplasm by the exportin-5 pathway, the pre-miRNA is cleaved by a cytosolic RNase III endonuclease known as Dicer, which cleaves the RNA at the level of the loop, yielding a 22-nt doublestranded RNA (dsRNA) molecule (miRNA/miRNA* duplex). The miRNA duplex contains 1 guide strand (miRNA), which harbours complementarity for its target mRNA, and 1   passenger strand (miRNA*) (Figure 31.19), which is degraded following entry of the miRNA duplex into the pocket of an Argonaute family protein, forming a large complex known as the RNA-induced silencing complex (RISC) (Figure 31.19). The miRNA, via its ability to base-pair with its seed element within the target mRNA, promotes the miRISCmediated translational silencing or deadenylation and degradation activity (Figure 31.19). The core components of miRISC are endonucleases belonging to the Argonaute (AGO) family of proteins that directly associate with the miRNA. The AGO proteins are composed of 3 well-conserved domains, with a fourth, less conserved, N-terminal domain. The 3 domains, named MID, PIWI, and PAZ, perform crucial individual functions for silencing (Figure 31.22 on page 1075). The MID domain is responsible for binding to the 5=-end of the mature miRNA, with the PAZ domain binding the 3=-end, while the PIWI domain, which folds as an RNaseH domain, is responsible for the slicer activity of the complex (Figure 31.22). While mammalian cells express 4 AGO proteins, AGO1–4, only AGO2, via its RNAse-H-like endonuclease activity, participates directly in the miRNAmediated mRNA silencing (Figure 31.23 on page 1075). Another protein that is essential for miRNA-mediated repression is the glycine-tryptophan family of proteins of 182 kD (GW182). There are 3 human GW182 paralogs that play a crucial role in miRNA-induced repression by acting downstream of the AGOs with which they bind directly (Figure 31.21 on page 1074). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 31

Canonical Processing

Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA

Mirtrons Exon 1

RNAPII

Exon 2

RNAPII Transcription

Transcription m 7G

Exon 1 Exon 2 AAAA Mature mRNA

Drosha DGCR8 m 7G

m 7G AAAA pri-mRNA Processing

Exon 1 Exon 2 AAAA Spliceosome

Splicing

pre-miRNA

Exportin 5 Nucleus AGO2

Cytoplasm

Dicer

ac-pre-miRNA TRBP pre-miRNA

miRNA /miRNA* duplex

Passenger strand degradation

pre-miR-451

miRNA guide strand-based target mRNA binding

miRISC GW182

CAF1 CCR4

PABP AAAAAA

m 7G

Translational repression and /or deadenylation AGO2 m 7G

(A)n

Endonucleolytic cleavage and mRNA degradation

FIGURE 31.19 miRNA biogenesis. miRNAs can be transcribed either by a canonical pathway or as introns in proteincoding genes. In the former case, the resulting pri-miRNA is processed by Drosha in the nucleus, resulting in pre-miRNA, which is then exported to the cytoplasm. In the cytoplasm, the hairpin-structured pre-miRNA is processed by Dicer to give rise to a mature miRNA, which interacts with Argonaute family proteins (AGO) to form the RNA-induced silencing complex (RISC). Once the passenger miRNA strand is eliminated, the RISC is prepared to scan surrounding mRNAs for complementarity to the loaded miRNA. (The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 2010 Sep;11(9):597–610.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

1073

1074

Part 5 Information Transfer (a) Bridging domain

(b) S5´ RNAse IIIa

S3 S4 S5

S4´ S1

S3´

S2

C Platform domain

N

RNAse IIIb

H2 H2´

H1´

H3 H4

Connector helix

H1

dsRBD2

H5

N

C

dsRBD1

PAZ

FIGURE 31.20 The crystal structures of Dicer (left) and DGCR8 (right), a cofactor of Drosha. ((a) Image of PDB ID: 2ffl (I.J. MacRae, K. Zhou, F. Li, A. Repic, A.N. Brooks, W.Z. Cande, P.D. Adams, J.A. Doudna (2006) Structural basis for double-stranded RNA processing by Dicer. Science 311: 195–198) created with ePMV: G.T. Johnson and L. Autin, D.S. Goodsell, M.F. Sanner, A.J. Olson (2011). ePMV Embeds Molecular Modeling into Professional Animation Software Environments. Structure 19, 293–303. (b) Image of PDB ID: 2yt4 (S.Y. Sohn, W.J. Bae, J.J. Kim, K.H. Yeom, V.N. Kim, Y. Cho (2007) Crystal structure of human DGCR8 core. Nat.Struct.Mol.Biol. 14: 847–853) created with ePMV: G.T. Johnson and L. Autin, D.S. Goodsell, M.F. Sanner, A.J. Olson (2011). ePMV Embeds Molecular Modeling into Professional Animation Software Environments. Structure 19, 293–303.) Pre-miRNA

Pre-miRNA Drosha

m7G

AAAA

FIGURE 31.21 Pri-miRNA processing. Upon transcription, a pri-miRNA is a 70-nucleotide-long, hairpinstructured, double-stranded RNA molecule. In order to remove the single-stranded RNA overhangs, pri-miRNA is processed in the nucleus by Drosha, which cleaves the pri-miRNA such that only the double-stranded hairpin structure remains, known as pre-miRNA.

NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 31

Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA

1075

PAZ

C

N

MID

N

N

PIWI

PAZ

PAZ

MID

PIWI

MID

C

C

PIWI

Anchors the 3' end of small RNAs

Folds as an RNaseH domain and performs ‘slicer’ activity

Binds the 5' phosphate end of small RNAs FIGURE 31.22 The AGO proteins are composed of 3 well-conserved domains, with a fourth, less conserved, N-terminal domain. The MID domain is responsible for binding to the 5=-end of the mature miRNA, with the PAZ domain binding the 3=-end, while the PIWI domain, which folds as an RNaseH domain, is responsible for the slicer activity of the complex. Image of PDB ID: 4ei3 (N.T. Schirle, I.J. MacRae (2012) The Crystal Structure of Human Argonaute2. Science 336: 1037–1040) created with ePMV: G.T. Johnson and L. Autin, D.S. Goodsell, M.F. Sanner, A.J. Olson (2011). ePMV Embeds Molecular Modeling into Professional Animation Software Environments. Structure 19, 293–303.

GW182 Dicer

RRM DEADc

HELICc

PAZ

Drosha

RIBOc

PIWI1

PAZ

PIWI Domain

Argonaute 4

PAZ

PIWI Domain

Argonaute 2

PAZ

RIBOc RIBOc

DSRM

RRM RNA recognition motif DEADc

DEAD-like helicase domain

HELICc Helicase domain PAZ

PIWI Domain

RIBOc DSRM

RIBOc

ssRNA-binding domain Ribonuclease III c-terminal domain

WWP WWP (WW) domain (proline-binding) Pasha (DGCR8)

WWP

DSRM

DSRM

DSRM Double-stranded RNA binding motif PIWI Domain

RNase H-like PIWI domain

FIGURE 31.23 The domains of various miRNA-associated proteins. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

1076

Part 5 Information Transfer

Mechanisms of miRNA-Mediated Repression The typical miRNA-binding site is found in the 3=UTR of target mRNAs, but in some rare cases may also be found in the 5=UTR. In order for miRNAs to interact with their mRNA targets, they require near-perfect complementarity in a region near their 5=-end known as the seed region, and looser complementarity toward the 3=-end of the miRNA. The part of the miRNA between these 2 regions plays a determinant role in deciding whether the target mRNA is sliced and degraded, or if it undergoes miRNA-mediated repression. If the middle part is complementary and interacts with the target mRNA, slicing of the mRNA can ensue, leading to degradation. If this middle part bulges out due to non-complementarity, specifically including nucleotide 11 of the miRNA, then the target mRNA is not sliced by the RISC, but is translationally repressed or degraded in a mechanism that involves the P-body. It is postulated, and scientific evidence suggests, that there are multiple methods by which miRNAs can mediate repression, both at the level of translational modulation and during mRNA turnover.

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

siRNA and shRNA Aside from the fascinating involvement of RNAi in cellular processes, its discovery also led to critical advances in biotechnology associated with cell biology. Whereas gene knockout experiments in mice, which were expensive, lengthy, and tedious, were once the only method for knocking down gene expression in order to observe a phenotype, exogenous forms of RNAi now allow for manipulation of gene expression in tissue culture, as well as in mice, and require far less time and effort. In fact, a cell biology study that does not make use of RNAi has become a rarity considering the convenience of the technique. Exogenous RNAi makes use of either small interfering RNAs (siRNA) duplexes

TABLE 31.4

or short hairpin RNAs (shRNA). siRNAs are chemically synthesized 22-nucleotide dsRNA molecules that can be introduced into tissue culture cells via transfection (see figure). These molecules enter the endogenous miRNA processing pathway at the level of Dicer/RISC (they do not enter the nucleus). As with endogenous miRNAs, the guide strand is retained and the passenger strand eliminated, and targeted mRNA repression ensues. siRNAs typically exhibit maximal RNAi activity roughly 24 hours after transfection, and are almost completely removed from the cells after 48–72 hours. As such, siRNA treatment is ideal for transient depletion of target gene expression.

For stable knockdown of gene expression, either in tissue culture or in mice, shRNA is considerably more effective. shRNA is introduced into cells as an expression vector, transcribed by the host cellular machinery, and processed very similarly to endogenous miRNAs (in the nucleus by Drosha and in the cytoplasm by Dicer) (see figure). Since shRNA vectors can be introduced stably into the genome, they allow for the creation of stable cell lines, where a particular gene of interest is continuously knocked down by RNAi. Table 31.4 highlights several techniques used for expression of siRNA or shRNA in tissue culture cells.

Examples of Vectors Used for Delivery of siRNA and Hairpin-Encoding DNA for In Vivo Experiments

Carrier

Features

Potential Problems

Retrovirus

High transfection efficiency

Random integration into genome; Difficult to transfect into primary cells cultured in vitro

Lentivirus

Can be transfected with efficiency into dividing and non-dividing cells

Its use still limited

Adenovirus

Transient expression with fewer risk of mutation due to DNA integration

It could be toxic if high doses are transfected into the liver

Chemically synthesized siRNA

Straight-forward transfection protocol

Transfection efficiency is lower than other carriers Easily taken up by the cell

Adapted from Li C., et al., 2005. Delivery of RNA interference. Cell Cycle 5(18):2103–9.

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miRNA-Mediated Translation Repression When acting as repressors of translation, miRNAs may exert their effect at the level of translation initiation as well as elongation. By disrupting interactions between the mRNA 5= GTP cap and cap-binding factor eIF4E, miRNAs, it is possible miRNAs may effectively prevent assembly of the 40S ribosome, as well as prevent circularization of the mRNA (which occurs through interactions between PABP and eIF4G; see Chapter 30). This mechanism is supported by the observation that mRNAs containing an internal ribosome entry site (IRES; eIF4E-independent translation) do not undergo miRNA-mediated repression. Alternatively, or in conjunction, miRNAs may also repress translation initiation by preventing joining of the 60S ribosomal subunit to the 40S. At the level of elongation, it is postulated that miRNAs may mediate either an elongation block or may catalyze proteolysis of the nascent polypeptide strand by recruitment of proteases (Figure 31.24).

# Transfection (or infection) of a cell with shRNA. The shRNA is introduced into the cell using a carrier, such as a lentivirus. Upon random integration into the genome, the shRNA is transcribed by host cellular machinery, processed by Drosha, and exported, in the same manner as endogenous miRNA. In the cytoplasm, the processed shRNA is cleaved by Dicer, and then loaded into the RISC, as with miRNA. The red arrow demonstrates at which point transfection of siRNA would enter the pathway. As you can see, siRNA transfection is transient, as the siRNA remains in the cytoplasm and is not generated by the cell.

Reverse transcription complex Pre-integration complex

Protein coat: GAG

Viral integrated shDNA insert

VSVg Pol II Integration

Lentivirus (shRNA)

Dro

sha

m 7G

TTTT

s leu Nuc

Pre-shRNA

sm pla Cyto

Exportin 5

siRNA Antisense (blue)

Dicer

5' ADP

Ran-GTP dependent export

5' 2ADP 2ATP

ATP

RISC AGO2

7

mG

mRNA

RISC AGO2

RISC AGO2 3' UTR

RISC-complex turnover

m 7G AAAA

mRNA CDS

m 7G Bypass mechanism

AAAA

AAAA Cleavage mechanism

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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FIGURE 31.24 Mechanisms of translational repression by miRNAs. (a) By disrupting interactions between the mRNA 5= GTP cap and cap-binding factor eIF4E, miRNAs may effectively prevent assembly of the 40S ribosome, as well as prevent circularization of the mRNA by preventing cap recognition. Alternatively, or in conjunction, miRNAs may also repress translation initiation by preventing joining of the 60S ribosomal subunit to the 40S. (b) At the level of elongation, it is postulated that miRNAs may mediate either an elongation block or may catalyze proteolysis of the nascent polypeptide strand by recruitment of proteases. (Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem. 2010;79:351–79.)

(a) Initiation block Repressed Cap recognition Repressed 60S subunit joining

40S

m 7G

AUG

eIF4E

miRISC ? ? ? GW182 PABP AGO A(n)

Open reading frame

60S

(b) Post-initiation block Elongation block 40S

Nascent polypeptide

Proteolysis miRISC ? ? ? GW182 PABP AGO A(n)

Ribosome drop-off

eIF4E m7G

miRNA-Mediated mRNA Decay Translational repression is not the only way by which miRNAs mediate gene silencing, as numerous mRNAs undergo marked decreases in transcript levels upon miRNA expression. As such, miRNAs also mediate an effect at the level of mRNA stability and turnover. mRNA degradation typically involves first deadenylation of the poly(A) tail by deadenylases, leading to shortening of the poly(A) tail. This event leads to the 3= S 5= degradation of the mRNA target by the exosome and/or decapping of the mRNA 5= cap (by decapping enzymes such as DCP1/DCP2) that trigger the 5= S 3= degradation by cellular exonucleases. The component of the miRNA machinery that mediates deadenylation is the Argonaute family of proteins, which can recruit GW182, which in turn activates the deadenylase CCR4-NOT (Figure 31.25). Deadenylation is then followed by decapping, through DCP1/DCP2 activity, leading to degradation of the target mRNA. Involvement of GW182 and DCP1/DCP2 proteins, largely localized to P-bodies, in miRNA-mediated repression is consistent with the observation that repressed mRNAs typically accumulate in P-bodies. In fact, P-bodies not only provide the proteins necessary for decapping/deadenylation of miRNA-targeted mRNAs, but also act as a site for storing mRNAs (repressed, but not degraded), as well as aid in translational repression of target mRNAs (Figure 31.26).

FIGURE 31.25 Schematic for miRNA-mediated mRNA decay. miRNAs may recruit the deadenylase CAF1/CCR4/NOT1 through interactions with AGO/ GW182, leading to deadenylation and 3= S 5= exonucleolytic decay by the exosome, as well as recruit decapping factors Dcp1/2 for 5= S 3= decay by Xrn1 exonuclease. (Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem. 2010;79:351–79.)

2 Decapping (and subsequent decay) DCP1/ m7G DCP2

AUG

1 Deadenylation CAF1 CCR4 NOT1 ?

PABP

A(n)

? ? GW182 AGO

Open reading frame miRISC NEL

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Chapter 31 Deadenylation (followed by decapping and degradation)

Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA Proteolysis (degradation of nascent peptide) X

miRNPs ORF

AAAAA

P-body (mRNA storage or degradation)

miRNPs

CCR4– NOT

AAAAA

miRNPs ORF

AAAAA miRNP binding

Initiation block (repressed cap recognition or 60S joining)

Elongation block (slow elongation or ribosome ‘drop-off’)

miRNPs elF4E

ORF

miRNPs AAAAA

AAAAA

FIGURE 31.26 Overview of the methods of action of miRNAs. miRNAs, in conjunction with P-bodies, may target mRNAs for translational repression or P-body–associated mRNA decay. In terms of post-initiation, miRNAs may recruit proteases to eliminate nascent polypeptide chains, as well as prevent translation elongation by the ribosome.

miRNAs and AUBPs: A Potential Link? Mounting evidence suggests that the function of miRNAs can be modulated by RBPs that bind to the same target mRNA, such as AUBPs and miRNAs interacting with AREcontaining mRNAs. The effectiveness of miRNA-mediated repression is modulated, in many cases, by AREs in the 3=UTR. The effect of these AREs is dependent upon their distance to the poly(A) tail; AREs closer to the poly(A) tail tend to have a greater effect on miRNA activity. Since the presence of AREs in mRNAs render them more labile, due to activation of AMD, it is possible that this decay process is linked to miRNA-mediated decay. The presence of AREs in proximity to miRNA-binding sites in the 3=UTR of target mRNAs could serve as sites of synergy or competition between AUBPs and miRNAs. In fact, this theory is supported by a number of experimental observations, such as the fact that the AUBP tristetraprolin (TTP), which typically promotes AMD, requires the action of miR16, a miRNA, in order to mediate the AMD of TNFa mRNA. In other cases, and depending on the mRNA and/or the growth conditions of a cell, the same RBP can either inhibit or promote miRISC repression. The RNA-binding protein HuR, one of the wellcharacterized post-transcriptional regulators of gene expression, is one of the best examples to illustrate this notion. It was demonstrated that HuR relieves the miR-122–mediated translation inhibition of CAT-1 mRNA. In human hepatoma cells, translation of CAT-1 mRNA is repressed in a miR-122–dependent manner. Yet in response to stress, HuR translocates from the nucleus to the cytoplasm to promote the translation of CAT-1. In contrast, the translation inhibition of c-Myc mRNA by the let7 miRNA is enhanced by having HuR bind to an ARE that is adjacent to the let7 seed element (Figure 31.27).

31.6

Can We Propose a Unified Theory of Gene Expression?

The stages of eukaryotic gene expression—from transcriptional activation, transcription, transcript processing, nuclear export of mRNA, to translation—have traditionally been presented as a linear series of events, that is, as a pathway of discrete, independent steps. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 5 Information Transfer Positive modulators

Negative modulators

(a) GW182 AGO m 7G

CAF1/ CCR4 AAAAAA

Ribosome

(b)

(c)

AGO AAAAAA

m 7G

(d) ? GW182 AGO

m 7G

GW182

? GW182 AGO AAAAAA

m 7G

AAAAAA

FIGURE 31.27 RBPs and miRNAs can act synergistically or antagonistically if there binding sites within close proximity to one another. (a, b) A nearby, mRNA-bound RBP may serve to either upregulate RISC activity or increase the rate of recruitment of degradation enzymes. (c, d) Alternatively, RBPs can compete for binding sites with miRNAs, as well as downregulate the activity of RISC. (Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Genetics, The widespread regulation of microRNA biogenesis, function and decay. 2010 Sep;11(9):597–610. Epub 2010 Jul 27.)

However, it is now clear that each stage is part of a continuous process, with physical and functional connections between the various transcriptional and post-transcriptional machineries. This realization led George Orphanides and Danny Reinberg to propose in 2002 a “unified theory of gene expression.” The principal tenet of this theory is that eukaryotic gene expression is a continuous process, from transcription through processing and protein synthesis: DNA S RNA S protein (Figure 31.28). Furthermore, regulation occurs at multiple levels in this continuous process, and in a coordinated fashion. Tom Maniatis and Robin Reed (1997) provide additional support for this theory by pointing out that eukaryotic gene expression depends on an interacting network of multicomponent protein machines: nucleosomes, histone acetyltransferases (HATs), and the remodelling apparatus; RNA polymerase II and its associated factors, which include capping, splicing, and polyadenylation enzymes; and the proteins involved in mRNA export to the cytoplasm for translation on ribosomes. As described above, RBPs that associate with mRNA as soon as its transcription begins play a crucial role during all the post-transcriptional events. The collaboration with miRNAs adds another regulatory layer that, in coordination RBPs and transcription factors, ensures proteins are produced only when they are needed (Figure 31.28).

RNA Regulation Is Coordinated: RNA Operons and Regulons For a “unified” theory to be truly unified, it must account for all events from the DNA to the final protein, meaning that post-transcriptional events are relevant. Transcription of mRNAs is an incredibly intricate process, involving cis- and trans-elements, such as upstream/downstream enhancers, and DNA-binding protein activators. Considering the size of the human genome, it is common for genes encoding functionally related proteins to be distant from one another on a genomic scale. However, for the sake of efficiency, one would expect for these genes to be regulated in a concerted manner, so that all of their gene products are available when necessary, and none are available when they are no longer needed. As such, the question is, since transcription of related genes can occur over large distances, how are these genes co-regulated? It has been accepted, for half a century, that transcription of mRNA from the genome is coordinated. The best-known example is that of operons and regulons in bacteria. However, in most eukaryotes, operons and regulons do not exist as they do in prokaryotic bacteria. So it has been assumed that eukaryotic transcription is coordinated by specific transcription factors that activate or inhibit the synthesis of functionally related genes. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 31

Protein

Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA

RISC miRNA

Protein folding

AAA 200

siRNA

RNAi

3"

GpppX5"

5"

Translation

5"

3" AAA 200

Gp

5"

pX5"

p Gp

Gene silencing

pp 5"

X

CYTOPLASM

Nuclear pore

mRNA export

NUCLEUS

X pp

5"

*

Chromatin decompaction Chromatin

Gp

5"

IIF

Mediator IIB TBP RNAPII TATA IIEIIH

RNAPII

X pp 5"

RNAPII

mRNA packaging

G/ L

Splicing

Coupled transcription and mRNA processing Transcription factor

RNAPII

AA AAU

RNAPII

Cleavage and 3" polyadenylation

Gp5"

X"

pp G5p"

5

Coupled initiation and 5" capping

FIGURE 31.28 A unified theory of gene expression. Each step in gene expression, from transcription to translation, is but a stage within a continuous process. Each stage is physically and functionally connected to the next, ensuring that all steps proceed in an appropriate manner, and that overall regulation of gene expression is tightly integrated. (Adapted from Cell, 108, Orphanides, G., and Reinberg, D., A unified theory of gene expression, 439–451, (2002), with permission from Elsevier.)

Nevertheless, there are actually few data to rigorously prove this idea because most of the experiments assessing the implication of transcription factors in gene expression are based on measuring the levels of mRNAs produced from those genes. As described in this chapter, each mRNA has a different stability (half-life), and thus their levels in the cell are affected by both synthesis (transcription) and degradation. This means that measuring the levels of mRNAs in the cell is not a way to assess the coordination of transcription. In fact, measuring mRNA levels may be a better measurement of its degradation than it is a measurement of its synthesis. One solution to this question is to inhibit transcription while we measure RNA degradation, or vice versa. When global changes of messenger RNAs were measured, it was found that mRNA decay is coordinated, and that the proteins that control the decay can also be used to isolate those mRNAs. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Based on these and other observations, in 2002 Jack D. Keene of Duke University was the first to provide experimental evidence supporting his hypothesis that the co-regulation of the expression of various genes occurs at the post-transcriptional level. In essence, mRNAs encoding functionally related proteins are grouped by specific interactions with 1  or more sequence-specific RNA-binding proteins, allowing these mRNAs to be regulated in a concerted manner as post-transcriptional RNA operons (PTRO) or as higherorder RNA regulons. Since a RBP can associate with more than one mRNA, PTRO is also used to describe the combinatorial use of the mRNAs themselves independently of the proteins they interact with. One of the major consequences of these observations is the establishment that posttranscriptional events play a major role in the coordinated expression of a variety of genes. From the moment transcription is completed, related mRNAs are acted upon by a series of RBPs. These RBPs work in combinations to coordinate passage of this group of mRNAs through all the interconnected steps of mRNA processing, from splicing to nuclear export, and from stability to localization. The end goal of this coordinated effort is to make a group of mRNAs available to the translation machinery in the cytoplasm to produce the factors needed for a specific pathway or in response to a specific signal. As described above in this chapter, these RBPs not only serve to group the mRNAs, but are also involved in the actual steps of mRNA processing, such as HuR working at the level of nuclear splicing, export, and stability (Figure 31.29). Consequences of dysregulation of PTROs are currently under study, and may be implicated in a number of human diseases. If an mRNA is typically co-regulated with a number of other messages, but loses its ability to bind to a specific RBP that is required for its recruitment to its appropriate RNA regulon because of a mutation or other defect, then the protein encoded by the RNA may be expressed in an uncontrolled manner. For example, the 2 RBPs, TDP-43 and FUS/TLS, are known to play important roles in neurodegenerative diseases, and mutations in them are considered to cause amyotrophic Genomic DNA

Transcription/Splicing/Export AAAAA

AAAAA mRNA

AAAAA

RBPA

AAAAA

RBPC RBPB

RBPB

RBPA

RBPC

RBPB

AAAAA

RNPA

RBPC

AAAAA RBPC

RBPB

RBPA

RBPC

AAAAA

AAAAA

AAAAA

RNPB

AAAAA

RNPC

Grouping of mRNA via interaction with RBPs to generate various RNPs FIGURE 31.29 Post-transcriptional coordination is dynamic because each mRNA has multiple lives. Upon transcription, functionally related mRNAs are grouped together through specific interactions with 1 or more RBPs, which feed the mRNAs in a coordinated manner through all the mRNA processing steps, leading up to translation. As such, expression of functionally related genes is tightly controlled, such that all genes, of a particular pathway for example, are expressed when necessary.

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Chapter 31

Post-transcriptional Regulation of Gene Expression: mRNA Turnover and Micro-RNA

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lateral sclerosis, also known as Lou Gehrig’s disease. Several studies have analyzed the co-regulated populations of these RBPs and identified key functional relationships per the PTRO model. In addition, the RBP TDRD7 is a cause of cataracts and glaucoma, and its mRNA targets that encode proteins critical to normal lens development can suffer mutations in such patients. Many other examples, including gene polymorphisms found in fibroblast growth factor 20 (FGF20) mRNA, are located in the 3=UTR, and therefore may prevent interaction with RBPs; they are strongly associated with Parkinson’s disease.

Can mRNA Manipulation Serve as a Therapeutic Approach? In the final analysis, the continued discovery of mRNA turnover mechanisms, as well as associated regulatory factors and proteins, serves as evidence to promote the importance of mRNA turnover in gene expression. Continued study in this field will serve to better understand how proto-oncogene levels are tightly regulated at the cellular level, and may perhaps provide new openings for manipulation of gene expression through biotechnology. For the moment, therapeutic approaches acting at the level of RNA are being developed in hopes of targeting a wide variety of gene-related diseases. RNA interference-based therapeutics, for example, can currently be found in clinical trials for targeting various forms of cancer. The major drawback of RNA interference-based therapeutics is the cellular delivery system. However, advances in nano-engineering have allowed for the development of non-viral, biodegradable carrier systems for siRNA delivery.

SUMMARY 31.1 What Is mRNA Turnover? mRNA turnover represents the balance between the rates of stabilization and decay of an mRNA. The length of time for which mRNA is stable is known as its halflife, and can be increased or decreased in the presence of specific RNA-binding proteins. When a transcript is no longer needed, or its translation must be terminated, it is targeted to cytoplasmic foci known as P-bodies, which represent accumulations of translationally expressed mRNAs as well as mRNA degradation enzymes. 31.2 How Is mRNA Decay Carried Out? General mRNA decay begins with shortening of the poly(A) tail by deadenylases. When the poly(A) tail is shortened to 25 nucleotides it can no longer interact with PABP, leading to opening of the circularized mRNA. Further deadenylation can lead to 3= S 5= degradation by exonucleases such as the exosome, or decapping by DCP1/2 can lead to 5= S 3= degradation by Xrn1 exonuclease. Alternative methods of mRNA decay exist, such as deadenylation-independent mRNA decay, which directly recruits decapping enzymes, and endonuclease-mediated decay. cis- and trans-elements involved in mRNA stability or decay serve to accelerate or slow the recruitment of deadenylases and decapping enzymes. 31.3 What Is AU-Rich–Mediated Decay? One of the best studied mRNA cis-elements is the AU-rich element (ARE). AREs are present in 5%–8% of human mRNAs, including many oncogenes. AREcontaining messages are very unstable, as the AREs serve as binding sites for destabilizing RBPs, which recruit mRNA degradation factors and the exosome. AREs can also, however, recruit stabilizing RBPs, which act to increase the limited half-life of the target mRNA. Interactions between RBPs and target mRNAs can be identified using biochemical techniques such as RNA-EMSA or RIP-Chip. In addition to AREs, other cis-elements, such as GU-rich elements, have been shown to contribute to mRNA instability and decay. 31.4 What Cellular Processes Serve to Maintain RNA Integrity? Genomic mutations can lead to transcripts harbouring premature stop codons as a result of frameshifts or deletions. These transcripts can give rise to truncated proteins, which can be dysfunctional or

may have toxic dominant-negative effects. As such, the cell contains a mechanism for eliminating aberrant mRNA, known as nonsensemediated decay (NMD). NMD involves a number of factors, including UPF1–3 and the Ski proteins, as well as the exon–junction complex (EJC). The EJC is deposited on the mRNA during splicing, and plays a major role in triggering NMD upon discovery of a premature stop codon. NMD is subjected to 2 positional rules: the EJC is always placed 20–24 nucleotides upstream of the exon–exon junction post-splicing, and NMD is not triggered if the premature stop codon is within 55 nucleotides of the junction. Finally, for removal of aberrant transcripts lacking any stop codon, the cell uses its nonstop decay pathway. 31.5 What Is miRNA-Mediated Gene Regulation? miRNA-mediated gene regulation involves the action of short, trans-acting, 22-nucleotide, single-stranded RNA molecules. These molecules are originally transcribed as hairpin-structured double-stranded RNA, which is then processed by the nuclear endoribonuclease Drosha and the cytoplasmic endoribonuclease Dicer to give rise to the mature miRNA. The mature miRNA then interacts with Argonaute proteins as part of a larger complex known as the RISC, which carries out the gene silencing activity. miRNAs are capable of triggering both translational repression of target mRNAs and mRNA degradation by binding to specific target mRNAs at regions known as seed elements. 31.6 Can We Propose a Unified Theory of Gene Expression? In analyzing regulation of mRNAs from their introduction at the level of transcription to their degradation, we can propose a unified theory of gene expression in which the different steps of the mRNA life cycle, from transcription to processing and from export to translation and decay, are interconnected and co-regulated. This concept is supported by observations in the field of ribonomics, in which functionally related mRNAs are co-regulated as RNA operons or regulons through the action of RNA-binding proteins. Finally, taking into account all the notions in this chapter, we can see how RNA-based therapies can serve potentially to treat a number of human diseases.

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PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. The stability of 1 mRNA is typically stated by its half-life. What types of mRNAs do you expect to have short half-lives? What types of mRNAs do you expect to have longer half-lives? 2. What is a processing body (P-body)? In what cell compartment may they be found? What types of proteins are typically found in P-bodies? 3. If the poly(A) tail of an mRNA is too short, why is the message no longer translated? 4. Is the shortening of the poly(A) tail reversible? Explain. 5. Fill in the blanks: A mature mRNA typically contains a ____________ of roughly 150–200 nucleotides at its 3=-end. This part of the mRNA is typically bound by several _____________ molecules, which in turn interact with ______________ at the level of the mRNA 5=-GTP cap to render the mRNA circularized. A group of enzymes known as _________________ serve to shorten the 3=-end of the mRNA to about 20–25 nucleotides, known as the ______________ length, leading to downstream mRNA decay. 6. Are decapping enzymes mandatory for mRNA decay? Explain. 7. Name 2 cis-elements that influence mRNA stability. Do these elements enhance or inhibit mRNA turnover? 8. If the 3=UTR of a message containing an AU-rich element is added to a message that is usually stable, what effect do you expect to see on the half-life of the stable message? 9. Three transcripts exhibit the following mRNA sequences:

Transcript A: AGCGAUGGCACCCUGGUAGCCAGUGCUAGAA AUGCCCUGUA Transcript B: AGUCGAUCGAUCGAUCGAUGAUUUAGUCGAU GCCGUAGCUG Transcript C: AGUACGUAGUUAUUUAUAGCUGAUUUAUUUA UUUAGCUAG Which transcript will likely have the shortest half-life? The longest halflife? How might overexpression of a stabilizing AUBP affect each of these transcripts (in terms of half-life)? 10. Would it be expected that cancerous cells, with unregulated growth, have high or low levels of TTP and KSRP (destabilizing RBPs)? Explain. 11. What type of error in a messenger RNA triggers nonsense-mediated decay (NMD)? 12. Why is there typically no exon–junction complex (EJC) downstream of a proper “stop” codon? 13. Where in the cell does NMD occur? 14. In which direction (5= S 3= or 3= S 5=) does non-stop decay (NSD) occur? 15. miRNAs are 21–25 nt long. How is it possible that 1 miRNA can bind and affect multiple mRNAs? 16. If a cell has reduced levels of Drosha, would you expect to find accumulation of pri-miRNA or pre-miRNA? 17. If a miRNA binds to the mRNA of ZAZI with near perfect complementarity, would you expect to see a decrease in ZAZI protein, ZAZI mRNA, or both? 18. What type of ribonuclease is Argonaute? Which domain in Argonaute is responsible for its “slicer” activity?

FURTHER READING mRNA Turnover and Decay Carey, M. F., Peterson, C. L., and Smale, S. T., 2009. Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 2nd ed. Woodbury, NY: Cold Harbor Laboratory Press. Garneau, N. L., Jeffrey Wilusz, J., and Wilusz, C. J. 2007. The highways and byways of mRNA decay. Nature Reviews Molecular Cell Biology 8:113–126. Newbury, S.F., 2006. Control of mRNA stability in eukaryotes. Biochemical Society Transactions 34:30–34. Parker, R., and Sheth, U., 2007. P bodies and the control of mRNA translation and degradation. Molecular Cell 25:635–646. Shyu, A. B., Belasco, J. G., and Greenberg, M. E., 1991. Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Genes and Development 5:221–231. Wilusz, C. J., Wormington, M., and Peltz, S. W., 2001. The cap-to-tail guide to mRNA turnover. Nature Reviews Molecular Cell Biology 2:237–246. AU- and GU-Rich Element–Mediated Decay and AUBPs Bakheet, T., Frevel, M., et al., 2001. ARED: Human AU-rich elementcontaining mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins. Nucleic Acids Research 29:246–254. Barreau, C., Paillard, L., and Osborne, H. B., 2005. AU-rich elements and associated factors: Are there unifying principles? Nucleic Acids Research 33:7138–7150.

Benoit, R., M., Meisner, N. C., et al., 2010. The x-ray crystal structure of the first RNA recognition motif and site-directed mutagenesis suggest a possible HuR redox sensing mechanism. Journal of Molecular Biology 397:1231–1244. Braddock, D. T., Levens, D., and Clore, G. M., 2002. Molecular basis of sequence-specific single-stranded DNA recognition by KH domains: Solution structure of a complex between hnRNP K KH3 and singlestranded DNA. The EMBO Journal 21:3476–3485. Shaw, G., and Kamen, R., 1986. A conserved AU sequence from the 3= untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659–667. Vlasova, I. A., Tahoe, N. M., et al., 2008. Conserved GU-rich elements mediate mRNA decay by binding to CUG-binding protein 1. Molecular Cell 29:263–270. Vlasova-St. Louis, I., and Bohjanen, P. R., 2011. Coordinate regulation of mRNA decay networks by GU-rich elements and CELF1. Current Opinion in Genetic Development 21:444–451. von Roretz, C., and Gallouzi, I-E., 2008. Decoding ARE-mediated decay: Is microRNA part of the equation? Journal of Cell Biology 181:189–194. NMD and NSD Chang, Y.-F., Imam, J. S., and Wilkinson, M. F., 2007. The nonsensemediated decay RNA surveillance pathway. Annual Review of Biochemistry 76:51–74.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Cheng, Z., Muhlrad, D., et al., 2007. Structural and functional insights into the human Upf1 helicase core. The EMBO Journal 26:253–264.

Rao, D. D., Vorhies, J. S., et al., 2009. siRNA vs. shRNA: Similarities and differences. Advanced Drug Delivery Reviews 61:746–759.

Kadlec, J., Izaurralde, E., and Cusack, S., 2004. The structural basis for the interaction between nonsense-mediated mRNA decay factors UPF2 and UPF3. Nature Structural & Molecular Biology 11:330–337.

Sohn, S. Y., Bae, W. J., et al., 2007. Crystal structure of human DGCR8 core. Nature Structural & Molecular Biology 14:847–853. Yuan, X., Naguib, S., and Wu, Z., 2011. Recent advances of siRNA delivery by nanoparticles. Expert Opinion in Drug Delivery 8:521–536.

Vasudevan, S., Peltz, S. W., and Wilusz, C. J., 2002. Non-stop decay—A new mRNA surveillance pathway. BioEssays 24:785–788.

Unifying Theory of Gene Expression and Ribonomics

miRNA-Mediated Gene Regulation

Keene, J. D., Komisarow, J. M., et al., 2006. RIP-Chip: The isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nature Protocols 1:302–307.

Bartel, D. P., 2004. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116:281–297. Cullen, B. R., 2005. RNAi the natural way. Nature Genetics 37:1163–1165. Davis-Dusenbery, B. N., and Hata, A., 2010. Mechanisms of control of microRNA biogenesis. The Journal of Biochemistry 148:381–392. Djuranovic, S., Nahvi, A., and Green, R., 2011. A parsimonious model for gene regulation by miRNAs. Science 331:550–553. Fabian, M. R., Sonenberg, N., and Filipowicz, W., 2010. Regulation of mRNA translation and stability by microRNAs. Annual Review of Biochemistry 79:351–379. Filipowicz, W., Bhattacharyya, S. U., and Sonenberg, N., 2008. Mechanisms of post-transcriptional regulation by microRNAs: Are the answers in sight? Nature Reviews Genetics 9:102–114. Krol, J., Loedige, I., and Filipowicz, W., 2010. The widespread regulation of microRNA biogenesis, function and decay. Nature Reviews Genetics 11:597–610.

Keene, J. D., 2007. Biological clocks and the coordination theory of RNA operons and regulons. Cold Spring Harbor Symposia on Quantitative Biology 72:157–165. Lachke, S. A., Alkuraya, F. S., et al., 2011. Mutations in the RNA granule component TDRD7 cause cataract and glaucoma. Science 331:1571–1576. Polymenidou, M., Lagier-Tourenne, C., et al., 2011. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nature Neuroscience 14:459–468. Sephton, C. F., Cenik, C., et al., 2011. Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes. Journal of Biological Chemistry 286:1204–1215. Tenenbaum, S. A., Lager, P. J., et al., 2002. Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNAbinding proteins and genomic arrays. Methods 26:191–198.

Li, C. X., Parker, A., et al., 2006. Delivery of RNA interference. Cell Cycle 5:2103–2109.

Tollervey, J. R., Tomaž C., et al., 2011. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nature Neuroscience 14:452–458.

MacRae, I. J., Zhou, K., et al., 2006. Structural basis for double-stranded RNA processing by Dicer. Science 311:195–198.

Ule, J., Jensen, K., et al., 2005. CLIP: a method for identifying proteinRNA interaction sites in living cells. Methods 37:376–386.

Mallory, A. C., Reinhart, B. J., et al., 2004. MicroRNA control of PHABULOSA in leaf development: Importance of pairing to the microRNA 5= region. The EMBO Journal 23:3356–3364.

Xiao, S., Sanelli, T., et al., 2011. RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS. Molecular Cell Neuroscience 47:167–180.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

32

Completing the Protein Life Cycle: Folding, Processing, and Degradation

! Vong’s Flying Crane. Origami—the Asian art of paper folding—arose in China almost 2000 years ago when paper was rare and expensive and the folded shape added special meaning. Protein folding, like origami, takes a functionless form and creates a structure with unique identity and purpose.

Life is a process of becoming, a combination of states we have to go through. Anais Nin (1903–1977)

32.1

How Do Newly Synthesized Proteins Fold?

32.2

How Are Proteins Processed Following Translation?

32.3

How Do Proteins Find Their Proper Place in the Cell?

32.4

How Does Protein Degradation Regulate Cellular Levels of Specific Proteins?

Gabriel Vong

KEY QUESTIONS

ESSENTIAL QUESTIONS Proteins are the agents of biological function. Protein turnover (synthesis and decay) is a fundamental aspect of each protein’s natural history. How are newly synthesized polypeptide chains transformed into mature, active proteins, and how are undesired proteins removed from cells?

WHY IT MATTERS

Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

Before we begin our discussion of protein folding and degradation, we pose the question: Why do a protein’s thermodynamic stability and its subsequent native conformation matter? Knowing how proteins fold has become a highly critical field of study due to the association of protein folding with several neurological and hereditary diseases, such as mad cow, Alzheimer’s, and sickle cell anemia. In fact, the outbreak of mad cow disease in Canada in 2003 was the driving force behind the creation of the Centre for Prions and Protein Folding Diseases (CPPFD), located at the University of Alberta. The highly regarded work being conducted at the CPPFD may one day provide insights into the molecular dynamics of protein folding that can eventually lead to the prevention of the aforementioned neurological diseases. From a simple egg being denatured upon frying to a single point mutation in the Hb subunit resulting in the clumping of red blood cells, protein folding is, and will always be, of intense medical interest in today’s society. The human genome apparently contains about 20 500 genes, but some estimates suggest that the total number of proteins in the human proteome may approach 1 million. What processes introduce such dramatically increased variation into the products of protein-encoding genes? We’ve reviewed (or will soon cover) many of these processes; a partial list (with examples) includes 1. Gene rearrangements (immunoglobulin G) 2. Alternative splicing (fast skeletal muscle troponin T) NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 32

Completing the Protein Life Cycle: Folding, Processing, and Degradation

1087

Courtesy Prion Centre, University of Alberta

" The Centre for Prions and Protein Folding Diseases (CPPFD) at the University of Alberta.

3. 4. 5. 6. 7.

RNA editing (apolipoprotein B) Proteolytic processing (chymotrypsinogen or prepro-opiomelanocortin; see Chapter 11) Isozymes (lactate dehydrogenase) Protein sharing (the glycolytic enzyme enolase is identical to t-crystallin in the eye) Protein–protein interactions at many levels (oligomerization, supramolecular complexes, assembly of signalling pathway protein complexes upon scaffold proteins)

8. Covalent modifications of many kinds (phosphorylation or glycosylation, with multisite phosphorylation or variable degrees of glycosylation, to name just 2 of the dozens of possibilities) Thus, the nascent polypeptide emerging from a ribosome is not yet the agent of biological function that is its destiny. First, the polypeptide must fold into its native tertiary structure. Even then, seldom is the nascent, folded protein in its final functional state. Proteins often undergo various proteolytic processing reactions and covalent modifications as steps in their maturation to functional molecules. Finally, at the end of their usefulness, damaged by chemical reactions or denatured due to partial unfolding, they are degraded. In addition, some proteins are targeted for early destruction as part of regulatory programs that carefully control available amounts of particular proteins. Damaged or misfolded proteins are a serious hazard; accumulation of protein aggregates can be a cause of human disease, including the prion diseases (see Chapter 1 and Chapter 28) and diseases of amyloid accumulation, such as Alzheimer’s, Parkinson’s, and Huntington disease.

32.1

Nascent: “Undergoing the process of being born” or, in the molecular sense,“newly synthesized.”

How Do Newly Synthesized Proteins Fold?

As Christian Anfinsen pointed out 40 years ago, the information for folding each protein into its unique 3-dimensional architecture resides within its amino acid sequence or primary structure (see Chapter 6). Proteins begin to fold even before their synthesis by ribosomes is completed (Figure 32.1a on page 1089). However, the cytosolic environment is a very crowded place, with effective protein concentrations as high as 0.3 g/mL. Macromolecular crowding enhances the likelihood of non-specific protein association and aggregation. The primary driving force for protein folding is the burial of hydrophobic side chains away from the aqueous solvent and reduction in solvent-accessible surface area (see Chapter 6). The folded protein typically has a buried hydrophobic core and a hydrophilic surface. Protein aggregation is typically driven by hydrophobic interactions, so burial of hydrophobic regions through folding is a crucial factor in preventing aggregation. To evade NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 5 Information Transfer

HUMAN BIOCHEMISTRY

Alzheimer’s, Parkinson’s, and Huntington Disease Are Late-Onset Neurodegenerative Disorders Caused by the Accumulation of Protein Deposits As noted in Chapter 6, protein-misfolding problems can cause disease by a variety of mechanisms. For example, protein aggregates can impair cell function. Amyloid plaques (so named because they resemble the intracellular starch, or amyloid, deposits found in plant cells) and neurofibrillary tangles (NFTs) are proteinaceous deposits found in the brains of individuals suffering from any of several neurogenerative diseases. In each case, the protein is different. In Alzheimer’s, disease is caused both by extracellular amyloid deposits composed of proteolytic fragments of the amyloid precursor protein (APP) termed amyloid@B (AB) and by intracellular NFTs composed of the microtubule-binding protein tau (T). Ab is a peptide 39–43 amino acids long that polymerizes to form long, highly ordered, insoluble fibrils consisting of a hydrogen-bonded parallel b-sheet structure in which identical residues on adjacent chains are aligned directly, in register (see accompanying figure). Why Ab aggregates in some people but not others is not clear. In Parkinson’s, the culprit is NFTs composed of polymeric t; no amyloid plaques are evident. In Huntington disease, the protein deposits occur as nuclear inclusions composed of polyglutamine (polyQ) aggregates. PolyQ aggregates arise from mutant forms of huntingtin, a protein that characteristically has a stretch of glutamine residues close to its N-terminus. Huntingtin is a 3144-residue protein encoded by the ITI5 gene, which has 67 exons. Exon 1 encodes the polyglutamine region. Individuals whose huntingtin gene has fewer than 35 CAG (glutamine codon) repeats never develop the disease; those with 40 or more always develop the disease within a normal lifetime. The nuclear inclusions in Huntington disease are huntingtin-derived polyglutamine fragments that have aggregated to form b-sheet–containing amyloid fibrils. Impairment of cellular function by proteinaceous deposits may be a general phenomenon. In vitro experiments have demonstrated that aggregates of proteins not associated with disease can be cytotoxic, and the ability to form amyloid deposits is a general property

of proteins. The evolution of chaperones to assist protein folding and proteasomes to destroy improperly folded proteins may have been driven by the necessity to prevent protein aggregation.

40

30 xis

ril a

Fib

12 24

# A model for the Ab1 - 40 structural unit in b@amyloid fibrils. Fibrils contain b-strands perpendicular to the fibril axis, with interstrand hydrogen bonding parallel to the fibre axis. The top face of the b@sheet is hydrophobic and presumably interacts with neighbouring Ab molecules in fibril formation. (Figure adapted from Figure 1 in Thompson, L. K., 2003. Unraveling the secrets of Alzheimer’s b-amyloid fibrils. Proceedings of the National Academy of Sciences U.S.A. 100:383–385.)

such problems, nascent proteins are often assisted in folding by a family of helper proteins known as molecular chaperones (see Chapter 6), because, like the chaperones at a dance, their purpose is to prevent inappropriate liaisons. Chaperones also serve to shepherd proteins to their ultimate cellular destinations. Also, mature proteins that have become partially unfolded may be rescued by chaperone-assisted refolding.

Chaperones Help Some Proteins Fold A number of chaperone systems are found in all cells. Many of the proteins in these systems are designated by the acronym Hsp (for heat shock protein) and a number indicating their relative mass in kilodaltons (as in Hsp60). Hsps were originally observed as abundant proteins in cells given brief exposure to high temperature (42° C or so). The principal Hsp chaperones are Hsp70, Hsp60 (the chaperonins), and Hsp90. In general, proteins whose folding is chaperone dependent pass down a pathway in which Hsp70 first acts on the newly synthesized protein and then passes the partially folded intermediate to a chaperonin for completion of folding. Nascent polypeptide chains exiting the large ribosomal subunits are met by ribosomeassociated chaperones [TF, or trigger factor, in Escherichia coli; NAC (nascent chainassociated complex) in eukaryotes]. In E. coli, the 50S ribosomal protein L23, which is situated at the peptide exit tunnel, serves as the docking site for TF, directly linking protein synthesis with chaperone-assisted protein folding. TF and NAC mediate transfer of the emerging nascent polypeptide chain to the Hsp70 class of chaperones, although many proteins do not require this step for proper folding. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 32

Hsp70 Chaperones Bind to Hydrophobic Regions of Extended Polypeptides

(a)

In Hsp70-assisted folding, proteins of the Hsp70 class bind to nascent polypeptide chains while they are still on ribosomes (Figure 32.1b). Hsp70 (known as DnaK in E. coli) recognizes exposed, extended regions of polypeptides that are rich in hydrophobic residues. By interacting with these regions, Hsp70 prevents non-productive associations and keeps the polypeptide in an unfolded (or partially folded) state until productive folding interactions can occur. Completion of folding requires release of the protein from Hsp70; release is energy dependent and is driven by ATP hydrolysis. Hsp70 proteins such as DnaK consist of 2 domains: a 44-kD N-terminal ATP-binding domain and an 18-kD central domain that binds polypeptides with exposed hydrophobic regions (Figure 32.2a). The DnaK:ATP complex receives an unfolded (or partially folded) polypeptide chain from DnaJ (Figure 32.2b). DnaJ is an Hsp40 family member. Interaction of DnaK with DnaJ triggers the ATPase activity of DnaK; the DnaK:ADP complex forms a stable complex with the unfolded polypeptide, preventing its aggregation with other proteins. A third protein, GrpE, catalyzes nucleotide exchange on DnaK, replacing ADP with ATP, which converts DnaK back to a conformational form having low affinity for its polypeptide substrate. Release of the polypeptide gives it the opportunity to fold. Multiple cycles of interaction with DnaK (or Hsp70) give rise to partially folded intermediates or, in some cases, completely folded proteins. The partially folded intermediates may be passed along to the Hsp60/chaperonin system for completion of folding (Figure 32.1c).

(b)

ATPase domain

GroEL

~85%

385 393

FIGURE 32.1 Protein folding pathways. (a) Chaperone-independent folding. The protein folds as it is synthesized on the ribosome (green) (or shortly thereafter). (b) Hsp70-assisted protein folding. Hsp70 (grey) binds to nascent polypeptide chains as they are synthesized and assists their folding. (c) Folding assisted by Hsp70 and chaperonin complexes. The chaperonin complex in E. coli is GroES–GroEL. The majority of proteins fold by pathway (a) or (b). (Adapted from Figure 2 in Netzer, W. J., and Hartl, F. U., 1998. Protein folding in the cytosol: Chaperonin-dependent and-independent mechanisms. Trends in Biochemical Sciences 23:68–73; Figure 2 in Hartl, F. U., and Hayer-Hartl, M., 2002. Molecular chaperones in the cytosol: From nascent chain to folded protein, Science 295:1852–1858.)

DnaJ-U

N

J

638

DnaK I

B L 1,2

1 4

L 5,6

7

6 8

J

DnaJ

D

C L 3,4

C

U, I

537

C E

Pi

J

ADP ATP

N L 4,5

2

~15%

DnaK mechanism of action

Peptide-binding domain

1

(c)

GroES

The Hsp60 class of chaperones, also known as chaperonins, assists some partially folded proteins to complete folding after their release from ribosomes. Chaperonins sequester partially folded molecules from one another (and from extraneous interactions), allowing folding to proceed in a protected environment. This protected environment is sometimes referred to as an Anfinsen cage because it provides an enclosed space where proteins fold

Domain organization and structure of the Hsp70 family member, DnaK

(b)

Hsp70

The GroES–GroEL Complex of E. coli Is an Hsp60 Chaperonin

(a)

A

GrpE

GroEL

5 3

1089

Completing the Protein Life Cycle: Folding, Processing, and Degradation

N

ATP

ADP, DnaJ

FIGURE 32.2 Structure and function of DnaK. (a) Domain organization and structure of the Hsp70 family member, DnaK. The ribbon diagram on the lower left is the ATP-binding domain of the DnaK analog, bovine Hsc70; bound ADP is shown as a stick diagram (purple). The ribbon diagram on the lower right is the polypeptide-binding domain of DnaK. The small blue ovals highlight the position of the polypeptide substrate; the protein regions that bind the polypeptide substrate are blue-green. (b) DnaK mechanism of action. DnaJ binds an unfolded protein (U) or partially folded intermediate (I) and delivers it to the DnaK:ATP complex. The nucleotide exchange protein GrpE replaces ADP with ATP on DnaK, and the partially folded intermediate (I) is released. I has several possible fates: It may fold into the native state, N; it may undergo another cycle of interaction with DnaJ and DnaK; or it may be become a substrate for folding by the GroEL chaperonin system. (Adapted from Figures 1a and 2a in Frydman, J., 2001. Folding of newly translated proteins in vivo: The role of molecular chaperones. Annual Review of Biochemistry 70:603–647.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

1090

Part 5 Information Transfer

spontaneously, free from the possibility of aggregation with other proteins. Chaperonins are large, cylindrical protein complexes formed from 2 stacked rings of subunits. The chaperonins have been organized into 2 groups, I and II, on the basis of their source and structure. Group I chaperonins are found in bacteria; group II in archaea and eukaryotes. The group I chaperonin in E. coli is the GroES–GroEL complex (Figure 32.1c). GroEL is made of 2 stacked 7-membered rings of 60-kD subunits that form a cylindrical a14 oligomer 15 nm high and 14 nm wide (Figure 32.3). Each GroEL ring has a 5-nm central cavity where folding can take place. This cavity can accommodate proteins up to 60 kD in size. GroES, sometimes referred to as a co-chaperonin, consists of a single 7-membered ring of 10-kD subunits that sits like a dome on one end of GroEL (Figure 32.3). The end of GroEL where GroES is sitting is referred to as the apical end. Each GroEL subunit has 2 structural domains: an equatorial domain that binds ATP and interacts with neighbours in the other a7 ring and an apical domain with hydrophobic residues that can interact with hydrophobic regions on partially folded proteins. The apical domain hydrophobic patches face the interior of the central cavity. An unfolded (or partially folded) protein binds to the apical patches and is delivered to the central cavity of the upper a7 ring (Figure 32.3c). ATP binding to the subunits of the upper a7 ring causes rapid (6 100 ms), forced unfolding of the substrate protein, followed by 2 events that occur on a slower time scale (!1 s): (1) GroES is recruited to GroEL, and (2) the a-subunits undergo a conformational change that buries their hydrophobic patches. The a-subunits now present a hydrophilic surface to the central cavity. This change displaces the bound partially folded polypeptide into the sheltered hydrophilic environment of the central cavity, where it can fold, free from danger of aggregation with other (a)

°

140 A

(b)

°

33 A

°

80 A °

10 A

°

80 A °

184 A

°

71 A

(c) U, I N 7 ATP

GroES

7 ATP ATP

< 100 ms

ATP

~15 sec

ADP

ADP

~1 sec

ATP

7 Pi

ATP

7 ADP, GroES

FIGURE 32.3 Structure and function of the GroES–GroEL complex. (a) Structure and overall dimensions of GroEL–GroES (top view, left; side view, right) (pdb id = 1AON). (b) Section through the centre of the complex to reveal the central cavity. (c) Model of the GroEL cylinder (blue) in action. An unfolded (U) or partially folded (I) polypeptide binds to hydrophobic patches on the apical ring of a7-subunits, followed by ATP binding, forced protein unfolding, and GroES (red) association. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 32

Completing the Protein Life Cycle: Folding, Processing, and Degradation

proteins. GroES also promotes ATP hydrolysis (Figure 32.3c). The GroEL:ADP:GroES complex dissociates when ATP binds to the subunits of the other (lower) a7 ring. Dissociation of GroES allows the partially folded (or folded) protein to escape from GroEL. If the protein has achieved its native conformation, its hydrophobic residues will be buried in its core and the hydrophobic patches on the a7 rings will have no affinity for it. On the other hand, if the protein is only partially folded, it may be bound again, gaining access to the Anfinsen cage of the a7 ring and another cycle of folding. The folding of rhodanese, a 33-kD protein, requires the hydrolysis of about 130 equivalents of ATP. The group II chaperonin and eukaryotic analog of GroEL, CCT (also called TriC) is also a double-ring structure, but each ring consists of 8 different subunits that vary in size from 50 to 60 kD. Furthermore, group II chaperonins lack a GroES counterpart. Prefoldin (also known as GimC), a hexameric protein composed of 6 subunits from 2 related classes (2 a and 4 b), can serve as a co-chaperone for CCT, much as GroES does for GroEL. However, prefoldin also acts like an Hsp70 protein, because it binds unfolded polypeptide chains emerging from ribosomes and delivers them to CCT. Prefoldin resembles a jellyfish, with 6 tentacle-like coils extending from a barrel-shaped body. The ends of the tentacles have hydrophobic patches for binding unfolded proteins. Prior to substrate protein binding, CCT exists in a partly open state. ATP binding opens the ring even more, a state in which prefoldin delivers the substrate protein. ATP hydrolysis closes the chamber and drives the folding process. ATP-induced conformation changes that promote protein folding propagate from one subunit to the next around the ring structure.

The Eukaryotic Hsp90 Chaperone System Acts on Proteins of Signal Transduction Pathways Hsp 90 constitutes 1–2% of the total cytosolic proteins of eukaryotes, its abundance reflecting its importance. Like other Hsp chaperones, its action depends on cyclic binding and hydrolysis of ATP. Conformational regulation of signal transduction molecules seems to be a major purpose of Hsp90. Receptor tyrosine kinases, soluble tyrosine kinases, and steroid hormone receptors are some of the signal transduction molecules (see Chapter 11) that must associate with Hsp90 in order to become fully competent; proteins fitting this description are called Hsp90 “client proteins.” The maturation of Hsp70 client proteins requires other proteins as well, and together with Hsp90, these proteins come together to form an assembly that has been called a foldosome. CFTR (cystic fibrosis transmembrane regulator), telomerase, and nitric oxide synthase are also Hsp90-dependent. Association of nascent polypeptide chains with proteins of the various chaperone systems commits them to a folding pathway, redirecting them away from degradation pathways that would otherwise eliminate them from the cell. However, if these protein chains fail to fold, they are recognized as non-native and targeted for destruction.

32.2

How Are Proteins Processed Following Translation?

Aside from these folding events, release of the completed polypeptide from the ribosome is not necessarily the final step in the covalent construction of a protein. Many proteins must undergo covalent alterations before they become functional. In the course of these post-translational modifications, the primary structure of a protein may be altered, and/or novel derivations may be introduced into its amino acid side chains. Hundreds of different amino acid variations have been described in proteins, virtually all arising post-translationally. The list of such modifications is very large; some are rather common, whereas others are peculiar to a single protein. The diphthamide moiety in elongation factor eEF-2 is one example of an amino acid modification (see the Human Biochemistry box on page 1041 in Chapter  30); the fluorescent group of green fluorescent protein (GFP; see Chapter 4) is another. In addition, common chemical groups such as carbohydrates (see Chapter 7, Section 7.5, and A Deeper Look box on page 218) and lipids may be covalently attached to a protein during its maturation). Phosphorylation, acetylation, and methylation of proteins NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

1091

1092

Part 5 Information Transfer

A DEEPER LOOK

How Does ATP Drive Chaperone-Mediated Protein Folding? The chaperones that mediate protein folding do so in an ATPdependent manner, as illustrated in Figures 32.2 and 32.3. The affinity of chaperones for their unfolded or misfolded protein substrates is determined by the nature of the nucleotide bound by the ATP-binding domain of these proteins, which functions as an ATPase. If ATP is bound, the chaperone adopts a conformation with an open substrate-binding pocket. ATP increases the rate of association of the chaperone Hsp70 (DnaK) with an unfolded peptide or protein substrate by 100-fold, but it increases the rate of dissociation of the unfolded protein from the chaperone even more, by a factor of 1000. Overall, the chaperone’s affinity for an unfolded protein substrate decreases 10-fold (or more) when it binds ATP. On the other hand, if the substrate-binding site on the peptidebinding domain of DnaK is occupied by an unfolded protein substrate in conjunction with binding of the co-chaperone (DnaJ in Figure 32.2), ATP hydrolysis by the ATPase domain is triggered. The presence of ADP in the ATP-binding (ATPase) domain causes a shift in the substrate-binding site of the peptide-binding domain to a closed-conformation, high-affinity state. Thus, ATP-dependent chaperones cycle between 2 stable conformational states, just like allosteric proteins. Bound ATP favours the open conformation for the protein substrate–binding site, and ADP favours the closed conformation. (When ADP is released and no nucleotide occupies the ATP-binding site of the ATPase domain, the peptide-binding site remains in the closed, high-affinity conformation; see Figure 32.2.) What is the underlying mechanism that controls these ATP-regulated conformational changes? The ATP-Dependent Allosteric Regulation of Hsp70 Chaperones Is Controlled by a Proline Switch Clearly, the 2 domains of Hsp70 (DnaK)—the peptide-binding domain and the ATP-binding (or ATPase) domain—communicate with each other, because the nature of the nucleotide bound to the ATP domain determines the affinity of the peptide-binding domain for unfolded substrates. Markus Vogel, Bernd Bukau, and Matthais Mayer of the Center for Molecular Biology at the University of Heidelberg (Germany) argue that 4 distinct elements are needed for communication between these separate domains: an ATP sensor (which must include residues within the ATP-binding site),

a transducer (to communicate the presence of ATP to the distant peptide-binding site), a lever (operated by the ATP-binding domain to exert its effect on the distant peptide-binding domain), and a switch that controls the lever (the switch is needed to lock the protein in either the open conformation or the closed conformation so that either alternative conformation is stable). The switch that controls the conformational transitions of Hsp70 involves 2 universally conserved residues in the ATPase domain of Hsp70 family members, a proline (Pro143) and a surface-exposed arginine [Arg151; panel (a) of the figure]. Pro143 is the switch, and Arg151 is a relay for the lever. Replacement of either of these residues by amino acid substitutions disrupts and/or destabilizes the switch. Other nearby residues, Glu171 and Lys70, function as ATP sensors. It is believed that Lys70 also serves as the nucleophilethatinitiatesATPhydrolysisthroughattackontheg-phosphateof ATP. Arg151 acts as a relay between Pro143, events occurring during ATP hydrolysis, and the peptide-binding domain [panels (b)–(d) of the figure]. When ATP binding is sensed by Lys70 and Glu171 [panel (b)], Pro143 is shifted, which causes Arg151 to move in the direction of the peptide-binding domain of DnaK [panel (c)]. In turn, this protein-binding domain assumes the open, low-affinity conformation. The interaction of DnaK with an unfolded protein substrate and co-chaperone DnaJ moves Arg151 back toward Pro143, which causes Lys70 and Glu171 to initiate ATP hydrolysis [panel (d)]. ADP now occupies the nucleotide-binding site of the ATPase domain, and the protein-binding domain of DnaK is locked in the closed, highaffinity conformation. The consequence of these events is that DnaK cycles between binding and releasing unfolded (or partially folded) proteins, fuelled by ATP hydrolysis within the ATPase domain. In effect, ATP binding and hydrolysis drive DnaK from an open, low-affinity conformational state to a closed, high-affinity conformational state. When unfolded (or partially folded) proteins are not held by DnaK, they have the opportunity to fold so that any solvent-accessible hydrophobic surfaces they might still retain are buried. Once a protein has adopted a stable folded state, it lacks exposed hydrophobic surfaces and thus escapes the cycle of binding and release by DnaK. More generally, Hsp70 provides an elegant example of protein conformational transitions based on the binding of ATP versus ADP at an effector site, with the added dimension that the effector site in this case is also an ATPase.

are common mechanisms for regulating protein function. Interestingly, many proteins are modified in multiple ways, and many post-translational modifications act in combinations—a phenomenon termed cross-talk. (The majority of proteins in cells can be phosphorylated on one or more residues. A survey of some of the more prominent chemical groups conjugated to proteins is given in Chapter 5.) To quantify the significance of posttranslational modifications, recall that the number of human proteins is estimated to exceed the number of human genes (20 000 or so) by more than an order of magnitude.

Proteolytic Cleavage Is the Most Common Form of Post-translational Processing Proteolytic cleavage, as the most prevalent form of protein post-translational modification, merits special attention. The very occurrence of proteolysis as a processing mechanism seems strange: Why join a number of amino acids in sequence and then eliminate some of them? Three reasons can be cited. First, diversity can be introduced where none exists. For example, a simple form of proteolysis, enzymatic removal of N-terminal Met residues, NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 32

Completing the Protein Life Cycle: Folding, Processing, and Degradation

(b)

(a)

O O

O

P!

O

P"

O

P#

O

O

Mg w

w

w

K70 P143

E171 ATP sensors

w

R151 Relay

Switch

(d) O

O O

O

O

w

(c)

O

P!

O

O

P" O

w

Mg w

w

O

O O

P#

O

O K70 K70 P143 R151 w P143 R151 E171 Relay E171 ATP sensors Switch w

O

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P!

O

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P" O

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P#

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w

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w E171 E171 ATPase catalysts

P143 P143 R151 R151 Relay Switch

# (a) Side chains involved in the ATP- and ADP-dependent allosteric regulation of the Hsp70 family member DnaK. Lower numbers (in italics) indicate DnaK amino acid residues; upper numbers indicate the corresponding residues in Hsc70 (the human counterpart to DnaK). This illustration highlights residues in or near the ATP-binding site of the ATPase domain. The site is occupied by ADP, Pi, 1 Mg2+, and 2 K+ ions. See Figure 32.2a for the location of this site within the ATPase domain. Carbon atoms are grey; oxygen, red; nitrogen, blue; and phosphorus, yellow. P143 sits at the centre of an H-bonded network of residues, which includes K70, Y145, F146, R151, and E171. (b–d) The mechanism of ATP- and ADPdependent allosteric transitions in Hsp70. Events in the ATP-binding site of the ATPase domain are communicated to the protein-binding site of the peptide-binding domain through K70 and E171, which act as ATP sensors; P143, which acts as the switch; and R151, which is part of the lever that relays the events from the ATPase domain to the other domain. [(a) Adapted from Figure 1 in Vogel, M., Bukau, B., et al., 2006. Allosteric regulation of Hsp70 chaperones by a proline switch. Molecular Cell 21:359–367. Courtesy of Bernd Bukau and Matthias Mayer. (b–d) Adapted from Figure 6 in Vogel, M., Bukau, B., et al., 2006. Allosteric regulation of Hsp70 chaperones by a proline switch. Molecular Cell 21:359–367.)

occurs in many proteins. Met-aminopeptidase, by removing the invariant Met initiating all polypeptide chains, introduces diversity at N-termini. Second, proteolysis serves as an activation mechanism so that expression of the biological activity of a protein can be delayed until appropriate. A number of metabolically active proteins, including digestive enzymes and hormones, are synthesized as larger inactive precursors termed pro-proteins that are activated through proteolysis (see zymogens, Chapter 14). The N-terminal pro-sequence on such proteins may act as an intramolecular chaperone to ensure correct folding of the active site. Third, proteolysis is involved in the targeting of proteins to their proper destinations in the cell, a process known as protein translocation.

32.3

How Do Proteins Find Their Proper Place in the Cell?

Proteins are targeted to their proper cellular locations by signal sequences: Proteins destined for service in membranous organelles or for export from the cell are synthesized in precursor form carrying an N-terminal stretch of amino acid residues, or leader peptide, that serves as a signal sequence. In effect, signal sequences serve as “postal codes” for NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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sorting and dispatching proteins to their proper compartments. Thus, the information specifying the correct cellular localization of a protein is found within its structural gene. Once the protein is routed to its destination, the signal sequence is often, but not always, proteolytically clipped from the protein by a signal sequence–specific endopeptidase called a signal peptidase.

Proteins Are Delivered to the Proper Cellular Compartment by Translocation Protein translocation is the name given to the process whereby proteins are inserted into membranes or delivered across membranes. Protein translocation occurs in all cells. Newly synthesized chains of membrane proteins or secretory proteins are targeted to the plasma membrane (in prokaryotes) or the endoplasmic reticulum (in eukaryotes) by their signal sequences. In addition to the ER, a number of eukaryotic membrane systems are competent in protein translocation, including the membranes of the nucleus, mitochondria, chloroplasts, and peroxisomes. Several common features characterize protein translocation systems: 1. Proteins to be translocated are made as preproteins containing contiguous blocks of amino acid sequence that act as organelle-specific sorting signals. 2. Signal recognition particles (SRPs) recognize the presence of a nascent protein chain in the ribosomal exit tunnel and, together with signal receptors (SRs), deliver the nascent chain to the membrane. If the nascent sequence emerging from the ribosome is a signal sequence, it is delivered to a specific membrane protein complex, the translocon, that mediates protein integration into the membrane or protein translocation across the membrane. 3. Translocons are selectively permeable protein-conducting channels that catalyze movement of the proteins across the membrane, and metabolic energy in the form of ATP, GTP, or a membrane potential is essential. In eukaryotes, ATP-dependent chaperone proteins within the membrane compartment usually associate with the entering polypeptide and provide the energy for translocation. Proteins destined for membrane integration contain amino acid sequences that act as stop-transfer signals, allowing diffusion of transmembrane segments into the bilayer. 4. Preproteins are maintained in a loosely folded, translocation-competent conformation through interaction with molecular chaperones.

Prokaryotic Proteins Destined for Translocation Are Synthesized as Preproteins Gram-negative bacteria typically have 4 compartments: cytoplasm, plasma (or inner) membrane, periplasmic space (or periplasm), and outer membrane. Most proteins destined for any location other than the cytoplasm are synthesized with amino-terminal leader sequences 16–26 amino acid residues long. These leader sequences, or signal sequences, consist of a basic N-terminal region, a central domain of 7–13 hydrophobic residues, and a non-helical C-terminal region (Figure 32.4). The conserved features of the last part of the leader, the C-terminal region, include a helix-breaking Gly or Pro residue and amino acids with small side chains located 1 and 3 residues before the proteolytic cleavage site. Unlike the basic N-terminal and non-polar central regions, the C-terminal features are not essential for translocation but instead serve as recognition signals for the leader peptidase, which removes the leader sequence. The exact amino acid sequence of the leader peptide is FIGURE 32.4 General features of the N-terminal signal sequences on E. coli proteins destined for translocation: a basic N-terminal region, a central apolar domain, and a non-helical C-terminal region.

Leader sequence 16–26 residues N

Basic region

7–13 hydrophobic residues

Non-helical C-terminal region

0

Cleavage site

Gly or Pro NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 32

Completing the Protein Life Cycle: Folding, Processing, and Degradation

unimportant. Non-polar residues in the centre and a few Lys residues at the amino terminus are sufficient for successful translocation. The functions of leader peptides are to retard the folding of the preprotein so that molecular chaperones have a chance to interact with it and to provide recognition signals for the translocation machinery and leader peptidase.

Eukaryotic Proteins Are Routed to Their Proper Destinations by Protein Sorting and Translocation Eukaryotic cells are characterized by many membrane-bound compartments. In general, signal sequences targeting proteins to their appropriate compartments are located at the N-terminus as cleavable presequences, although many proteins have N-terminal localization signals that are not cleaved and others have internal targeting sequences that may or may not be cleaved. Proteolytic removal of the leader sequences is also catalyzed by specialized proteases, but removal is not essential to translocation. No sequence similarity is found among the targeting signals for each compartment. Thus, the targeting information resides in more generalized features of the leader sequences, such as charge distribution, relative polarity, and secondary structure. For example, proteins destined for secretion enter the lumen of the endoplasmic reticulum (ER) and reach the plasma membrane via a series of vesicles that traverse the endomembrane system. Recognition by the ER depends on an N-terminal amino acid sequence that contains one or more basic amino acids followed by a run of 6–12 hydrophobic amino acids. An example is serum albumin, which is synthesized in precursor form (preproalbumin) having a MKWVTFLLLLFISGSAFSR N-terminal signal sequence. The italicized K highlights the basic residue in the sequence, and the bold residues denote a continuous stretch of (mostly) hydrophobic residues. A signal peptidase in the ER removes the signal sequence by cleaving the preproprotein between the S and R.

The Synthesis of Secretory Proteins and Many Membrane Proteins Is Coupled to Translocation Across the ER Membrane The signals recognized by the ER translocation system are virtually indistinguishable from bacterial signal sequences; indeed, the 2 are interchangeable in vitro. In addition, the translocon systems in prokaryotes and eukaryotes are highly analogous. In higher eukaryotes, translation and translocation of many proteins destined for processing via the ER are tightly coupled. Translocation across the ER occurs co-translationally (i.e., as the protein is being translated on the ribosome). As the N-terminal sequence of a protein undergoing synthesis enters the exit tunnel of the ribosome, it is detected by a signal recognition particle (SRP; Figure 32.5). SRP is a 325-kD nucleoprotein assembly that contains 6 polypeptides and a 300-nucleotide 7S RNA. SRP54, a 54-kD subunit of SRP and a G-protein family member, recognizes the nascent protein’s signal sequence, and SRP binding of the signal sequence causes the ribosome to cease translation. This arrest prevents release of the growing protein into the cytosol before it reaches the ER and its intended translocation. The SRP–ribosome complex is referred to as the RNC–SRP (ribosome nascent chain:SRP complex).

Interaction between the RNC-SRP and the SR Delivers the RNC to the Membrane The RNC-SRP is then directed to the cytosolic face of the ER, where it binds to the signal receptor (SR), an ab heterodimeric protein. The 70-kD a-subunit is anchored to the membrane by the transmembrane b-subunit; both subunits are G-protein family members, and both have bound GTP. When SRP54 docks with SRa, the RNC-SRP becomes membrane associated (Figure 32.5). If the nascent chain emerging from the ribosome is not a signal sequence, the RNC is released from the SRP and the membrane. If the nascent chain emerging from the ribosome is a signal sequence, the complex remains intact, and SRP54 and SRa function together as reciprocal GTPase-activating proteins. GTP hydrolysis causes the dissociation of SRP from SR and transfer of the RNC to the translocon. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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FIGURE 32.5 Synthesis of a eukaryotic secretory protein and its translocation into the endoplasmic reticulum. (1) The signal recognition particle (SRP, red) recognizes the signal sequence emerging from a translating ribosome [ribosome nascent complex (RNC), grey]. (2) The RNC-SRP interacts with the signal receptor (SR, purple) and is transferred to the translocon (pink). (3) Release of the SRP and alignment of the peptide exit tunnel of the RNC with the protein-conducting channel of the translocon stimulates the ribosome to resume translation. (4) The membrane-associated signal peptidase (purple circle) clips off the N-terminal signal sequence, and BiP (the ER lumen Hsp70 chaperone, blue) binds the nascent chain mediating its folding into its native conformation. (5) Following dissociation of the ribosome, BiP plugs the translocon channel. Not shown are subsequent secretory protein maturation events, such as glycosylation. (Adapted from Figures 1a and 2a in Frydman, J., 2001. Folding of newly translated proteins in vivo: The role of molecular chaperones. Annual Review of Biochemistry 70:603–647.)

Translating ribosome

Signal recognition particle (SRP)

Cytosol Signal sequence

1 2

Signal receptor (SR)

BiP

3

Signal peptidase

ER lumen

Translocon 4

5

Clipped signal sequence

Secretory protein

The Ribosome and the Translocon Form a Common Conduit for Transfer of the Nascent Protein through the ER Membrane and into the Lumen Through interactions with the translocon, the ribosome resumes protein synthesis, delivering its growing polypeptide through the ER membrane. The peptide exit tunnel of the large ribosomal subunit and the protein-conducting channel of the translocon are aligned with one another, forming a continuous conduit from the peptidyl transferase centre of the ribosome to the ER lumen. The mammalian translocon is a complex, multifunctional entity that has as its core the Sec61 complex, a heterotrimeric complex of membrane proteins, and a unique fourth subunit, TRAM, that is required for insertion of nascent integral membrane proteins into the membrane. The 53-kD a-subunit of Sec61p has 10 membrane-spanning segments, whereas the b- and g-subunits are single TMS proteins. Sec61a forms the transmembrane protein– conducting channel through which the nascent polypeptide is transported into the ER lumen (Figure 32.5). The pore size of Sec61p is very dynamic, ranging from about 0.6 to 6 nm in diameter. Thus, a great variety of protein structures could be accommodated easily within the translocon. This flexibility allows the Sec61p translocon complex to function in post-translational translocation (translocation of completely formed proteins) as well as co-translational translocation. As the protein is threaded through the Sec61p channel into the lumen, an Hsp70 chaperone family member called BiP binds to it and mediates proper folding. BiP function, like that of other Hsp70 proteins, is ATP-dependent, and ATP-dependent protein folding provides the driving force for translocation of the polypeptide into the lumen. When the ribosome dissociates from the translocon, BiP serves as a plug to block the protein-conducting channel, preventing ions and other substances from moving between the ER lumen and the cytosol. A Signal Peptidase within the ER Lumen Clips Off the Signal Peptide Soon after it enters the ER lumen, the signal peptide is clipped off by membrane-bound signal peptidase (also called leader peptidase), which is a complex of 5 proteins. Other modifying enzymes within the lumen introduce additional post-translational alterations into the polypeptide, such as glycosylation with specific carbohydrate residues. ER-processed proteins destined for secretion from the cell or inclusion in vesicles such as lysosomes end up contained within the soluble phase of the ER lumen. On the other hand, polypeptides destined to become membrane proteins carry stop-transfer sequences within their mature domains. The stop-transfer sequence is typically a 20-residue stretch of hydrophobic amino acids that arrests the passage across the ER membrane. Proteins with stop-transfer sequences NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 32

Completing the Protein Life Cycle: Folding, Processing, and Degradation

1097

remain embedded in the ER membrane with their C-termini on the cytosolic face of the ER. Such membrane proteins arrive at their intended destinations via subsequent processing of the ER. Retrograde Translocation Prevents Secretion of Damaged Proteins and Recycles Old ER Proteins To prevent secretion of inappropriate proteins, fragmented or misfolded secretory proteins are passed from the ER back into the cytosol via Sec61p. Thus, Sec61p also serves as a channel for aberrant secretory proteins to be returned to the cytosol so that they can be destroyed by the proteasome degradation apparatus (see Section 32.4). Among these proteins are ER membrane proteins that are damaged or no longer needed. Mitochondrial Protein Import Most mitochondrial proteins are encoded by the nuclear genome and synthesized on cytosolic ribosomes. Mitochondria consist of 4 principal subcompartments: the outer membrane, the intermembrane space, the inner membrane, and the matrix. Thus, mitochondrial proteins must not only find mitochondria, they must also gain access to the proper subcompartment; once there, they must attain a functionally active conformation. As a consequence, mitochondria possess multiple preprotein translocons and chaperones. Similar considerations apply to protein import to chloroplasts, organelles with 5 principal subcompartments (outer membrane, intermembrane space, inner/thylakoid membrane, stroma, and thylakoid lumen; see Chapter 20). Signal sequences on nuclear-encoded proteins destined for the mitochondria are N-terminal cleavable presequences 10–70 residues long. These mitochondrial presequences lack contiguous hydrophobic regions. Instead, they have positively charged and hydroxy amino acid residues spread along their entire length. These sequences form amphipathic A@helices (Figure 32.6) with basic residues on one side of the helix and uncharged and hydrophobic residues on the other; that is, mitochondrial presequences are positively charged amphipathic sequences. In general, mitochondrial targeting sequences share no sequence homology. Once synthesized, mitochondrial preproteins are retained in an unfolded state with their target sequences exposed, through association with Hsp70 molecular chaperones. Import involves binding of a preprotein to the mitochondrial outer membrane translocon (TOM) (Figure 32.7). If the protein is destined to be an outer mitochondrial membrane protein, it is transferred from the TOM to the sorting and assembly complex (SAM) and inserted in the outer membrane. If it is an integral protein of the inner mitochondrial membrane, it traverses the TOM complex, enters the intermembrane space, and is taken up by the inner mitochondrial membrane translocon (TIM22) and inserted into the inner membrane. On the other hand, if it is destined to be a mitochondrial matrix protein, a different TIM complex, TIM23, binds the preprotein and threads it across the inner mitochondrial membrane into the matrix. Chloroplasts have TOCs (translocon outer chloroplast membranes) and TICs (translocon inner chloroplast membranes) for these purposes.

+ + +

TOM complex

Mas 37

Outer membrane SAM complex

Outer-membrane protein

Intermembrane space Inner membrane

TIM23 complex

Matrix protein

TIM22 complex

Innermembrane protein

R

FIGURE 32.6 Structure of an amphipathic a-helix having basic (+) residues on one side and uncharged and hydrophobic (R) residues on the other.

Cytosol TOM 40

R

R

Mitochondrial precursor protein

20

R

FIGURE 32.7 Translocation of mitochondrial preproteins involves distinct translocons. All mitochondrial proteins must interact with the outer mitochondrial membrane (TOM). From there, depending on their destiny, they (1) are passed to the SAM complex if they are integral proteins of the outer mitochondrial membrane or (2) traverse the TOM and enter the intermembrane space, where they are taken up by either TIM22 or TIM23, depending on whether they are integral membrane proteins of the inner mitochondrial membrane (TIM22) or mitochondrial matrix proteins (TIM23). (Adapted from Figure 1 in Mihara, K., 2003. Moving inside membranes. Nature 424:505–506.)

Matrix

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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32.4

How Does Protein Degradation Regulate Cellular Levels of Specific Proteins?

Cellular proteins are in a dynamic state of turnover, with the relative rates of protein synthesis and protein degradation ultimately determining the amount of protein present at any time. In many instances, transcriptional regulation determines the concentrations of specific proteins expressed within cells, with protein degradation playing a minor role. In other instances, the amounts of key enzymes and regulatory proteins, such as cyclins and transcription factors, are controlled via selective protein degradation. In addition, abnormal proteins arising from biosynthetic errors or postsynthetic damage must be destroyed to prevent the deleterious consequences of their buildup. The elimination of proteins typically follows first-order kinetics, with half-lives (t1/2) of different proteins ranging from several minutes to many days. A single random proteolytic break introduced into the polypeptide backbone of a protein is believed sufficient to trigger its rapid disappearance because no partially degraded proteins are normally observed in cells. Protein degradation poses a real hazard to cellular processes. To control this hazard, protein degradation is compartmentalized, either in macromolecular structures known as proteasomes or in degradative organelles such as lysosomes. Protein degradation within lysosomes is largely non-selective; selection occurs during lysosomal uptake. Proteasomes are found in eukaryotic as well as prokaryotic cells. The proteasome is a functionally and structurally sophisticated counterpart to the ribosome. Regulation of protein levels via degradation is an essential cellular mechanism. Regulation by degradation is both rapid and irreversible.

Eukaryotic Proteins Are Targeted for Proteasome Destruction by the Ubiquitin Pathway Ubiquitination is the most common mechanism to label a protein for proteasome degradation in eukaryotes. Ubiquitin is a highly conserved, 76-residue (8.5-kD) polypeptide widespread in eukaryotes. Proteins are condemned to degradation through ligation to ubiquitin. Three proteins in addition to ubiquitin are involved in the ligation process: E1, E2, and E3 (Figure 32.8). E1 is the ubiquitin-activating enzyme (105-kD dimer). It becomes attached via a thioester bond to the C-terminal Gly residue of ubiquitin through ATP-driven formation of an activated (Top) A ubiquitin : E1 heterodimer complex (pdb id = 1R4N; ubiquitin is shown in blue). (Middle) A ubiquitin : E2 complex (pdb id = 1FXT; E2 is shown in orange; ubiquitin in blue). (Bottom) The clamp-shaped E3 heteromultimer (pdb id = 1LDK and 1FQV). The target protein is bound between the jaws of the clamp.

E1

E2

E3

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 32 E1 : Ubiquitin-activating enzyme O 1 Ubiquitin C + – O (C-term. Gly)

O

E1

ATP

Completing the Protein Life Cycle: Folding, Processing, and Degradation

+

P P

Ubiquitin

Ubiquitin

AMP

Ubiquitinyl-acyladenylate

O 2

C

1099

FIGURE 32.8 Enzymatic reactions in the ligation of ubiquitin to proteins. Ubiquitin is attached to selected proteins via isopeptide bonds formed between the ubiquitin carboxy-terminus and free amino groups (a-NH2 terminus, Lys e-NH2 side chains) on the protein.

O

C AMP

+

E1

SH

AMP

+

E1

S

C

Ubiquitin

(Thioester)

E2 : Ubiquitin-carrier protein O E1

S

C

O

Ubiquitin

+

E2

SH

E1

SH

+

E2

S

C

Ubiquitin

E3 : Ligase E3

+

E3 : Protein

Protein (substrate)

E3 : Protein

+

E2

O S

Ubiquitin

C

E2

SH

+

E3

+

Protein

Ubiquitin

ubiquitin-adenylate intermediate. Ubiquitin is then transferred from E1 to an SH group on E2, the ubiquitin-carrier protein. (E2 is actually a family of at least 7 different small proteins, several of which are heat shock proteins; there is also a variety of E3 proteins.) In protein degradation, E2-S ! ubiquitin transfers ubiquitin to free amino groups on proteins selected by E3 (180 kD), the ubiquitin-protein ligase. Upon binding a protein substrate, E3 catalyzes the transfer of ubiquitin from E2-S ! ubiquitin to free amino groups (usually Lys e-NH2) on the protein. More than one ubiquitin may be attached to a protein substrate, and tandemly linked chains of ubiquitin also occur via isopeptide bonds between the C-terminal glycine residue of one ubiquitin and the e-amino of Lys residues in another. Ubiquitin has 7 lysine residues, at positions 6, 11, 27, 29, 33, 48, and 63. Only isopeptide linkages to K11, K29, K48, and K63 have been found, with the K48-type being most common as a degradation signal. E3 plays a central role in recognizing and selecting proteins for degradation. E3 selects proteins by the nature of the N-terminal amino acid. Proteins must have a free a-amino terminus to be susceptible. Proteins having Met, Ser, Ala, Thr, Val, Gly, or Cys at the amino terminus are resistant to the ubiquitin-mediated degradation pathway. However, proteins having Arg, Lys, His, Phe, Tyr, Trp, Leu, Asn, Gln, Asp, or Glu N-termini have half-lives of only 2–30 minutes. Interestingly, proteins with acidic N-termini (Asp or Glu) show a tRNA requirement for degradation (Figure 32.9). Transfer of Arg from Arg-tRNA to the N-terminus of these Arg

FIGURE 32.9 Proteins with acidic N-termini show a tRNA requirement for degradation. Arginyl-tRNAArg:protein transferase catalyzes the transfer of Arg to the free a-NH2 of proteins with Asp or Glu N-terminal residues. Arg-tRNAArg:protein transferase serves as part of the protein degradation recognition system.

Arg-tRNAArg Arg-tRNAArg

(Asp, Glu)

Protein transferase

Protein with acidic N-terminal residue (Asp or Glu)

Arg

(Asp, Glu) Protein with N-terminal Arg

tRNAArg NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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proteins alters their N-terminus from acidic to basic, rendering the protein susceptible to E3. It is also interesting that Met is less likely to be cleaved from the N-terminus if the next amino acid in the chain is one particularly susceptible to ubiquitin-mediated degradation. Most proteins with susceptible N-terminal residues are not normal intracellular proteins but tend to be secreted proteins in which the susceptible residue has been exposed by action of a signal peptidase. Perhaps part of the function of the N-terminal recognition system is to recognize and remove from the cytosol any invading “foreign” or secreted proteins. Other proteins targeted for ubiquitin ligation and proteasome degradation contain PEST sequences—short, highly conserved sequence elements rich in proline (P), glutamate (E), serine (S), and threonine (T) residues.

Proteins Targeted for Destruction Are Degraded by Proteasomes Proteasomes are large oligomeric structures enclosing a central cavity where proteolysis takes place. The 20S proteasome from the archaeon Thermoplasma acidophilum is a 700-kD barrel-shaped structure composed of 2 different kinds of polypeptide chains, a and b, arranged to form 4 stacked rings of a7b7b7a7-subunit organization. The barrel is about 15 nm in height and 11 nm in diameter, and it contains a 3-part central cavity (Figure 32.10a). The proteolytic sites of the 20S proteasome are found within this cavity. Access to the cavity is controlled through a 1.3-nm opening formed by the outer a7 rings. These rings are believed to unfold proteins destined for degradation and transport them into the central cavity. The b-subunits possess the proteolytic activity. Proteolysis occurs when the b-subunit N-terminal threonine side-chain O atom makes nucleophilic attack on the carbonyl-C of a peptide bond in the target protein. The products of proteasome degradation are oligopeptides 7–9 residues long. Eukaryotic cells contain 2 forms of proteasomes: the 20S proteasome and its larger counterpart, the 26S proteasome. The eukaryotic 26S proteasome is a 45-nm-long structure composed of a 20S proteasome plus 2 additional substructures known as 19S regulators (a)

(b) 19S 890 kD

20S 720 kD

19S 890 kD

base lid

lid base

15 nm

FIGURE 32.10 The structure of the 26S proteasome. (a) The yeast (Saccharomyces cerevisiae) 20S proteasome core with bortezomib bound (red) (pdb id = 2F16). Bortezomib is the first therapeutic proteasome inhibitor used in humans. It was approved in Canada in 2008 for treatment of relapsed multiple myeloma and mantle cell lymphoma. (b) Composite model of the 26S proteasome. The 20S proteasome core is shown in yellow; the 19S regulator (19S cap) structures are in blue. (Adapted from Figure 5 in Voges, D., Zwickl, P., and Baumeister, W., 1999. The 26S proteasome: A molecular machine designed for controlled proteolysis. Annual Review of Biochemistry 68:1015–1068.) NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 32

Completing the Protein Life Cycle: Folding, Processing, and Degradation

[also called 19S caps or PA700 (for proteasome activator-700 kD)] (Figure 32.10b). Overall, the 26S proteasome (approximately 2.5 megadaltons) has 2 copies each of 32–34 distinct subunits, 14 in the 20S core and 18–20 in the cap structures. Unlike the archaeal 20S proteasome, the eukaryotic 20S core structure contains 7 different kinds of a-subunits and 7 different kinds of b-subunits. Interestingly, only 3 of the 7 different b-subunits have protease active sites. The 26S proteasome forms when the 19S regulators dock to the 2 outer a7 rings of the 20S proteasome cylinder. Many of the 19S regulator subunits have ATPase activity. Replacement of certain 19S regulator subunits with others changes the specificity of the proteasome. The 19S regulators cause the proteolytic function of the 20S proteasome to become ATP-dependent and specific for ubiquitinylated proteins as substrates. That is, these 19S caps act as regulatory complexes for the recognition and selection of ubiquitinylated proteins for degradation by the 20S proteasome core (Figure 32.11). The 26S proteasome shows a preference for proteins having 4 or more ubiquitin molecules attached to them. The 19S regulators also carry out the unfolding and transport of ubiquitinylated protein substrates into the proteolytic central cavity. The 19S regulators consist of 2 parts: the base and the lid. The base subcomplex connects to the 20S proteasome and contains the 6 ATPase subunits that unfold proteasome substrates. These subunits are members of the AAA family of ATPases (ATPases associated with various cellular activities); AAA–ATPases are an evolutionarily ancient family of proteins involved in a variety of cellular functions requiring energy-dependent unfolding, disassembly, and remodelling of proteins. The lid subcomplex acts as a cap on the base subcomplex, and 1 of its subunits functions in recognition and ubiquitin-chain processing of proteasome protein substrates.

ATPase Modules Mediate the Unfolding of Proteins in the Proteasome The base of the 19S regulators that cap the 26S proteasome consists of a hexameric ring of AAA–ATPases that mediate the ATP-dependent unfolding of ubiquitinated proteins targeted for destruction in the proteasome. Structural studies have revealed the presence of loops extending from these AAA–ATPase subunits; these loops face the central channel of the hexameric ring. The loops are in an “up” position when an AAA–ATPase subunit has ATP bound in its active site, but they shift to a “down” position when ADP occupies the active site. Apparently, these loops bind protein substrates and act like the levers of a machine, using the energy of ATP hydrolysis to tug the protein into an unfolded state and thread it down through the narrow central channel. This channel leads into the cavity of the 20S proteasome, where proteolytic degradation takes place. The AAA–ATPase cycle of ATP binding, protein substrate binding, ATP hydrolysis, and protein unfolding is reminiscent of the ATP-dependent action of Hsp70 chaperones.

1101

E2

E1

Ubiquitin

E2 E3 Substrate

Substrate

26S proteasome

FIGURE 32.11 Diagram of the ubiquitin-proteasome degradation pathway. Pink “lollipop” structures symbolize ubiquitin molecules. (Adapted from Figure 1 in Hilt, W., and Wolf, D. H., 1996. Proteasomes: Destruction as a program. Trends in Biochemical Sciences 21:96–102.)

Ubiquitination Is a General Regulatory Protein Modification Protein ubiquitination is a signal for protein degradation, as described on page 1098. Ubiquitin conjugation to proteins is also used for other purposes in cells. Non-degradative functions for ubiquitination include roles in chromatin remodelling, DNA repair, transcription, signal transduction, endocytosis, spliceosome assembly, and sorting of proteins to specific organelles and cell structures. In addition, cells possess a variety of protein modifiers attached to target proteins by processes similar to the ubiquitin pathway, as described in the following section.

Small Ubiquitin-Like Protein Modifiers Are Post-transcriptional Regulators Small ubiquitin-like protein modifiers (SUMOs) are a highly conserved family of proteins found in all eukaryotic cells. Like ubiquitin, SUMO family members are covalently ligated to lysine residues in target proteins by a 3-enzyme conjugating system (Figure 32.12). NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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FIGURE 32.12 The mechanism of reversible sumoylation. Before conjugation, small ubiquitin-like protein modifiers (SUMOs) need to be proteolytically processed, removing anywhere from 2 to 11 amino acids to reveal the C-terminal Gly–Gly motif. SUMOs are then activated by the E1 enzyme in an ATP-dependent reaction to form a thioester bond between SUMO and E1. SUMO is then transferred to the catalytic Cys residue of the E2 enzyme, Ubc9. Finally, an “isopeptide bond” is formed between the C-terminal Gly of SUMO and a Lys residue on the substrate protein through the action of an E3 enzyme. The SUMO-specific protease SENP can deconjugate SUMO from target proteins.

AMP + PPi

SUMO GG

ATP

SENP SUMO GGXXXX...

E1

C173 UBA2 AOS1

SUMO GG C UBC9

SUMO GG

SENP

SUMO GG

E2

E3

K Target

SUMO proteins share only limited homology to ubiquitin, and sumoylated proteins are not targeted for destruction. Instead, sumoylation alters the ability of the modified protein to interact with other proteins. This ability to change protein–protein interactions is believed to be the biological purpose of SUMO proteins. Sumoylation can have 3 general consequences for modified proteins (Figure 32.13): (1) Sumoylation can interfere with the interactions between the target and its partner so that the interaction can only occur in the absence of sumoylation; (2) sumoylation can create a binding site for an interacting partner protein; and (3) sumoylation can induce a conformational change in the modified target protein, altering its interactions with partner proteins. The regulatory opportunities associated with sumoylation are significant for many cellular functions, including transcriptional regulation, chromosome organization, nuclear transport, and signal transduction (see Chapter 11). Many E2 enzymes participate in ubiquitination processes, but the only known SUMO E2 enzyme is Ubc9 (Figure 32.14a). Ubc9 recognizes a cKXD/E consensus sequence in proteins destined for sumoylation. In this sequence, c is an aliphatic branched amino acid (such as Leu), K is the lysine to which SUMO is conjugated, and X is any amino acid, with an acidic D or E completing the sequence. Recognition by Ubc9 is only possible if the consensus sequence is in a relatively unstructured part of a target protein or if it is part of an extended loop, as in RanGAP1 (Figure 32.14a). In the Ubc9–RanGAP1 complex, Leu525 of RanGAP1 is in van der Waals contact with several non-polar Ubc9 residues, whereas RanGAP1 Lys526 lies in a hydrophobic groove of Ubc9, juxtaposed with the catalytic Cys93 (Figure 32.14b). The pKa of Lys526 is lowered in this complex, activating the lysine amino group for nucleophilic attack to form the SUMO–target protein conjugate.

FIGURE 32.13 The molecular consequences of sumoylation. The process can affect a modified protein in 3 ways: (a) Sumoylation can interfere with the interaction between a target protein and its binding partner. (b) Sumoylation can provide a new binding site for an interacting partner. (c) Sumoylation can induce a conformational change in the modified target protein.

SUMO

E1, E2, E3 enzymes, ATP

Target

SUMO Target

Isopeptidases

(a)

(b)

Target

B

Target

r tne

SUMO

Par

Partner A

(c) SUMO Target

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Chapter 32 (a)

Completing the Protein Life Cycle: Folding, Processing, and Degradation

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(b)

C

Leu525 C

Cys93

Ser527

E A

F

D

B

N

Asp127

Lys526

Ser89

Glu528 C D B

3 N

2

Thr81

Glu88

4

Lys74

C 1

A FIGURE 32.14 (a) The complex formed by the E2 enzyme, Ubc9 (yellow), and a target protein, RanGAP1 (blue). (b) In the Ubc9–RanGAP1 complex, the exposed loop of RanGAP1 lies in the binding pocket of Ubc9. The exposed loop contains the consensus sequence for sumoylation (cKXD/E), including Leu525, which is surrounded by hydrophobic residues from Ubc9; Lys526, which is coordinated by Asp127 and Cys93 of Ubc9; and Glu528, which is coordinated by Ubc9 Thr81.

HtrA Proteases Also Function in Protein Quality Control The discussion thus far has stressed the importance of protein quality control to cellular health. HtrA proteases are a class of proteins involved in quality control that combine the dual functions of chaperones and proteasomes. (The acronym Htr comes from “high-temperature requirement” because E. coli strains bearing mutations in HtrA genes do not grow at elevated temperatures.) In addition to their novel ability to be either chaperones or proteases, HtrA proteases are the only known protein quality control factor that is not ATPdependent. Prokaryotic HtrA proteases act as chaperones at low temperatures (20 °C), where they have negligible protease activity. However, as the temperature increases, these proteins switch from a chaperone function to a protease function to remove misfolded or unfolded proteins from the cell. With this functional duality, HtrA proteases have the potential to mediate quality control through protein triage (see A Deeper Look box on page 1104). The E. coli HtrA protein DegP is the best characterized HtrA protease. DegP is localized in the E. coli periplasmic space, where it oversees quality control of proteins involved in the cell envelope. It is a 448-residue protein containing a central protease domain with a classic Ser protease Asp-His-Ser catalytic triad (see Chapter 13) and 2 C-terminal PDZ domains. These domains are structural modules involved in protein–protein interactions, and they recognize and bind selectively 3 or 4 residues of target proteins to the C-terminal. Like other quality control systems, HtrA proteases have a central cavity (see Figure 32.15)

HUMAN BIOCHEMISTRY

Proteasome Inhibitors in Cancer Chemotherapy Proteasome inhibition offers a promising approach to treating cancer. The counterintuitive rationale goes like this: The proteasome is responsible for the regulated destruction of proteins involved in cell cycle progression and the control of apoptosis (programmed cell death). Inhibition of proteasome function leads to cell cycle arrest and apoptosis. In clinical trials, proteasome inhibitors have retarded cancer progression by interfering with the programmed degradation of regulatory proteins, causing cancer cells to self-destruct.

Bortezomib (Brand name: Velcade), a small-molecule proteasome inhibitor developed by Millennium Pharmaceuticals, Inc., has received FDA approval for the treatment of multiple myeloma, a cancer of plasma cells that accounts for 10% of all cancers of the blood (see Figure 32.10a). Source: Adams, J., 2003. The proteasome: Structure, function, and role in the cell. Cancer Treatment Reviews Supplement 1:3–9.

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Part 5 Information Transfer (a) (b)

PDZ

PDZ

90°

FIGURE 32.15 The HtrA protease structure. (a) A trimer of DegP subunits represents the HtrA functional unit. The different domains are colour-coded: The protease domain is green, PDZ domain 1 (PDZ1) is yellow, and PDZ domain 2 (PDZ2) is orange. Protease active sites are highlighted in blue. The trimer has somewhat of a funnel shape, with the protease in the centre and the PDZ domains on the rim. (b) Two HtrA trimers come together to form a hexameric structure in which the 2 protease domains form a rigid molecular cage (blue) and the 6 PDZ domains are like tentacles (red) that both bind protein substrate targets and control lateral access into the protease cavity. (Adapted from Figure 3 in Clausen, T., Southan, C., et al., 2002. The HtrA family of proteases: Implications for protein composition and cell fate. Molecular Cell 0:443–455.)

A DEEPER LOOK

Protein Triage—A Model for Quality Control Triage is a medical term for the sorting of patients according to their need for (and their likelihood to benefit from) medical treatment. Sue Wickner, Michael Maurizi, and Susan Gottesman have pointed out that cells control the quality of their proteins through a system of triage based on chaperones and the ubiquitination-proteasome degradation pathway. These systems recognize non-native proteins (proteins that are only partially folded, misfolded, incorrectly modified, damaged, or in an inappropriate compartment). Depending on the severity of its damage, a non-native protein is directed to chaperones for refolding or targeted for destruction by a proteasome (see accompanying figure).

Unfolded and misfolded proteins Eukaryotic chaperones

Ubiquitination system Ubiquitin and ATP

Source: Adapted from figures 2 and 3 in Wickner, S., Maurizi, M., and Gottesman, S., 1999. Posttranslational quality control: Folding, refolding, and degrading proteins. Science 286:1888–1893.

Bound protein

Ubiquitinated protein

ATP

Protein remodelling Protein bound to proteasome Native protein

ATP

Degraded protein

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Chapter 32

Completing the Protein Life Cycle: Folding, Processing, and Degradation

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A DEEPER LOOK

Methods for Examining Protein Folding Isothermal titration calorimetry (ITC) and differential scanning calorimety (DSC) have become powerful techniques for studying the thermodynamic properties of protein and nucleic acid folding/unfolding processes.*,**,† Although ITC was initially used to characterize protein–protein interactions, the combination of thermal with structural data, such as X-ray crystallography, allows for a better grasp of the underlying complexities associated with protein folding.*,**,† For example, ITC has been used to thermodynamically study amyloid formation and the denaturing process of muscle creatine kinase.* The thermodynamics of protein folding can essentially be measured as changes in heat capacity (i.e., the production of a heat signal upon protein–protein complex formation), which can be used as an indicator of altered protein folding (which is usually determined by non-covalent interactions and the hydrophobic effect).*,** Both ITC and DSC instrumentation require a high sensitivity for the simple detection of the very minute energy changes occurring with these

processes.† For DNA unfolding, DSC can give clear indications and details of unfolding in addition to the presence/existence of intermediate states.† Finally, the technique of dynamic fluorescence depolarization is particularly useful in providing information about the dynamic processes associated with newly synthesized proteins that are still ribosomally bound.‡ *Liang,

Y., 2008. Applications of isothermal titration calorimetry in protein science. Acta Biochimica et Biophysica Sinica 40:565–576. **Pierce, M., Raman, C. S., et al., 1999. Isothermal titration calorimetry of protein-protein interactions. Methods 19:213–221. †Spink, C.H., 2008. Differential scanning calorimetry. Methods in Cell Biology 84:115–141. ‡Weinreis, S.A., Ellis, J. P., et al., 2010. Dynamic flurorescence depolarization: A powerful tool to explore protein folding on the ribosome. Methods 52:57–73.

where proteolysis occurs; the height of this cavity is 1.5 nm, which excludes folded proteins from access to the proteolytic sites. Thus, HtrA proteases can act only on misfolded proteins. As we have seen, limited access to proteolytic sites is an important regulatory feature of quality control proteases; only proteins targeted for destruction have access to such sites. The PDZ domains of the HtrA proteases act as gatekeepers, determining access of protein substrates to the proteolytic centres. Human HtrA proteases are implicated in stress response pathways. Human HtrA1 is expressed at higher levels in osteoarthritis and aging and lower levels in ovarian cancer and melanoma. Secreted human Htr1 may be involved in degradation of extracellular matrix proteins involved in arthritis as well as tumour progression and invasion.

SUMMARY 32.1 How Do Newly Synthesized Proteins Fold? Most proteins fold spontaneously, as anticipated by Anfinsen, whose experiments suggested that all the information necessary for a polypeptide chain to assume its active, folded conformation resides in its amino acid sequence. However, some proteins depend on molecular chaperones to achieve their folded conformation within the crowded intracellular environment. Hsp70 chaperones are ATP-dependent proteins that bind to exposed hydrophobic regions of polypeptides, preventing non-productive associations with other proteins and keeping the protein in an unfolded state until productive folding steps can take place. Hsp60 chaperones such as GroEL–GroES are ATPdependent cylindrical chaperonins that provide a protected central cavity or “Anfinsen cage,” where partially folded proteins can fold spontaneously, free from the danger of non-specific hydrophobic interactions with other unfolded protein chains. Hsp90 chaperones assist a subset of “client proteins” involved in signal transduction pathways in assuming their active conformations. 32.2 How Are Proteins Processed Following Translation? Nascent proteins are seldom produced in their final, functional form. Maturation typically involves proteolytic cleavage and may require post-translational modification, such as phosphorylation, glycosylation, or other covalent substitutions. Removal of nascent N-terminal methionine residues is a common form of protein processing by proteolysis. The number of human proteins is believed to exceed the number of human genes (20 000 or so) by an order of magnitude or

more. The great number of proteins available from a fixed set of genes is attributed to a variety of processes, including alternative splicing of mRNAs and post-translational modification of proteins. 32.3 How Do Proteins Find Their Proper Place in the Cell? Most proteins destined for compartments other than the cytosol are synthesized with N-terminal signal sequences that target them to their proper destinations. These signal sequences are recognized by signal recognition particles as they emerge from translating ribosomes. The signal recognition particle interacts with a membrane-bound signal receptor, delivering the translating ribosome to a translocon, a multimeric integral membrane protein structure having at its core the Sec61p complex. The translocon transfers the growing protein chain across the membrane (or in the case of nascent integral membrane proteins, inserts the protein into the membrane). Signal peptidases within the membrane compartment clip off the signal sequence. Other post-translational modifications may follow. Mitochondrial protein import and membrane insertion are mediated by specific translocon complexes in the outer mitochondrial membrane called TOM and SAM. Proteins destined for the inner mitochondrial membrane or mitochondrial matrix must interact with inner mitochondrial translocons (either TIM23 or TIM22) as well as TOM. 32.4 How Does Protein Degradation Regulate Cellular Levels of Specific Proteins? Protein degradation is potentially hazardous to cells, because cell function depends on active proteins. Therefore, protein degradation is compartmentalized in lysosomes or in

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proteasomes. Proteins targeted for destruction are selected by ubiquitination. A set of 3 enzymes (E1, E2, and E3) mediate transfer of ubiquitin to free —NH2 groups in targeted proteins. Proteins with charged or hydrophobic residues at their N-termini are particularly susceptible to ubiquitination and destruction. The ubiquitin moieties are recognized by 19S cap structures found at either end of 26S proteasomes. Protein degradation occurs when, in an ATP-dependent process, the ubiquitinated protein is unfolded and threaded into the central cavity of the cylindrical a7b7b7a7 20S part of the

26S proteasome. The b-subunits possess protease active sites that chop the protein substrate into short (7- to 9-residue) fragments; the ubiquitin moieties are recycled. Linkage of SUMO (small ubiquitinlike protein modifiers) to target proteins can alter their protein– protein interactions. HtrA proteases also function in protein quality control. HtrA proteins are novel in 2 aspects: Unlike other chaperones and proteasomes, they are ATP-independent, and, unlike the others, they have dual chaperone and protease activities and switch between these 2 functions in response to stress conditions.

PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. (Integrates with Chapter 30) Human rhodanese (33 kD) consists of 296 amino acid residues. Approximately how many ATP equivalents are consumed in the synthesis of the rhodanese polypeptide chain from its constituent amino acids and the folding of this chain into an active tertiary structure? 2. A single proteolytic break in a polypeptide chain of a native protein is often sufficient to initiate its total degradation. What does this fact suggest to you regarding the structural consequences of proteolytic nicks in proteins? 3. Protein molecules, like all molecules, can be characterized in terms of general properties such as size, shape, charge, and solubility/hydrophobicity. Consider the influence of each of these general features on the likelihood of whether folding of a particular protein will require chaperone assistance or not. Be specific regarding just Hsp70 chaperones or both Hsp70 chaperones and Hsp60 chaperonins. 4. Many multidomain proteins apparently do not require chaperones to attain the fully folded conformations. Suggest a rational scenario for chaperone-independent folding of such proteins. 5. The GroEL ring has a 5-nm central cavity. Calculate the maximum molecular weight for a spherical protein that can just fit in this cavity, assuming the density of the protein is 1.25 g/mL. 6. (Integrates with Chapter 9) Acetyl-CoA carboxylase has at least 7 possible phosphorylation sites (residues 23, 25, 29, 76, 77, 95, and 1200) in its 2,345-residue polypeptide (see Figure 9.4). How many different covalently modified forms of acetyl-CoA carboxylase protein are possible if there are 7 phosphorylation sites? 7. (Integrates with Chapter 30) In what ways are the mechanisms of action of EF-Tu/EF-Ts and DnaK/GrpE similar? What mechanistic functions do the ribosome A-site and DnaJ have in common? 8. The amino acid sequence deduced from the nucleotide sequence of a newly discovered human gene begins MRSLLILVLCFLPLAALGK…. Is this a signal sequence? If so, where does the signal peptidase act on it? What can you surmise about the intended destination of this protein? 9. The Sec61p translocon complex is not only essential for translocation of proteins into the ER lumen, but it also mediates the incorporation of integral membrane proteins into the ER membrane. The mechanism for integration is triggered by stop-transfer signals that cause a pause in translocation. Figure 32.5 shows the translocon as a closed cylinder spanning the membrane. Suggest a mechanism for lateral transfer of an integral membrane protein from the protein-conducting channel of the translocon into the hydrophobic phase of the ER membrane.

10. The Sec61p core complex of the translocon has a highly dynamic pore whose internal diameter varies from 0.6 to 6 nm. In post-translational translocation, folded proteins can move across the ER membrane through this pore. What is the molecular weight of a spherical protein that would just fit through a 6-nm pore? (Adopt the same assumptions used in Problem 5.) 11. (Integrates with Chapters 6, 10, and 30) During co-translational translocation, the peptide tunnel running from the peptidyl transferase centre of the large ribosomal subunit and the protein-conducting channel are aligned. If the tunnel through the ribosomal subunit is 10 nm and the translocon channel has the same length as the thickness of a phospholipid bilayer, what is the minimum number of amino acid residues sequestered in this common conduit? 12. Draw the structure of the isopeptide bond formed between Gly76 of one ubiquitin molecule and Lys48 of another ubiquitin molecule. 13. Assign the 20 amino acids to either of 2 groups based on their susceptibility to ubiquitin ligation by E3 ubiquitin protein ligase. Can you discern any common attributes among the amino acids in the less susceptible versus the more susceptible group? 14. Lactacystin is a Streptomyces natural product that acts as an irreversible inhibitor of 26S proteasome b-subunit catalytic activity by covalent attachment to N-terminal threonine —OH groups. Predict the effects of lactacystin on cell cycle progression. 15. HtrA proteases are dual-function chaperone-protease protein quality control systems. The protease activity of HtrA proteases depends on a proper spatial relationship between the Asp-His-Ser catalytic triad. Propose a mechanism for the temperature-induced switch of HtrA proteases from chaperone function to protease function. 16. As described in this chapter, the most common post-translational modifications of proteins are proteolysis, phosphorylation, methylation, acetylation, and linkage with ubiquitin and SUMO proteins. Carry out a Web search to identify at least 8 other post-translational modifications and the amino acid residues involved in these modifications. 17. Fluorescence resonance energy transfer (FRET) is a spectroscopic technique that can be used to provide certain details of the conformation of biomolecules. Look up FRET on the Web or in an introductory text on FRET uses in biochemistry, and explain how FRET could be used to observe conformational changes in proteins bound to chaperonins such as GroEL. A good article on FRET in protein folding and dynamics is Haas, E., 2005. The study of protein folding and dynamics by determination of intramolecular distance distributions and their fluctuations using ensemble and single-molecule FRET measurements. ChemPhysChem 6:858–870. Studies of GroEL using FRET analysis include the following: Sharma, S., Chakraborty, K., et al., 2008. Monitoring protein conformation along the pathway of chaperonin-assisted folding. Cell 133:142–153; and Lin, Z., Madan, D., et al., 2008. GroEL stimulates protein folding through NEL

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Chapter 32

forced unfolding. Nature Structural and Molecular Biology 15:303–311. 18. The cross-talk between phosphorylation and ubiquitination in protein degradation processes is encapsulated in the concept of the “phosphodegron.” What is a phosphodegron, and how does

Completing the Protein Life Cycle: Folding, Processing, and Degradation

1107

phosphorylation serve as a recognition signal for protein degradation? (A good reference on the phosphodegron and cross-talk between phosphorylation and ubiquitination is Hunter, T., 2007. The age of crosstalk: Phosphorylation, ubiquitination, and beyond. Molecular Cell 28:730–738.)

FURTHER READING Protein-Folding Diseases Bates, G., 2003. Huntingtin aggregation and toxicity in Huntington’s disease. Lancet 361:1642–1644. Bucciantini, M., Giannoni, E., et al., 2002. Inherent toxicity of aggregates implies a common mechanism for protein misfolding. Nature 416:507–511. Gamblin, T. C., Chen, F., et al., 2003. Caspase cleavage of tau: Linking amyloid and neurofibrillary tangles in Alzheimer’s disease. Proceedings of the National Academy of Sciences U.S.A. 100: 10032–10037. Herczenik, E., and Gebbink, M. F., 2008. Molecular and cellular aspects of protein misfolding and disease. FASEB Journal 22:2115–2133. Soto, C., Estrada, L., et al., 2006. Amyloids, prions, and the inherent infectious nature of misfolded protein aggregates. Trends in Biochemical Sciences 31:150–156. Winklhofer, K. F., Tatzelt, J., et al., 2008. The two faces of protein misfolding: Gain- and loss-of-function in neurodegenerative diseases. EMBO Journal 27:336–349. Chaperone-Assisted Protein Folding Bigotti, M. G., and Clarke, A. R., 2008. Chaperonins: The hunt for the group II mechanism. Archives of Biochemistry and Biophysics 474: 331–339. Bukau, B., Deuerling, E., et al., 2000. Getting newly synthesized proteins into shape. Cell 101:119–122. Bukau, B., Weissman, J., et al., 2006. Molecular chaperones and protein quality control. Cell 125:443–451. Ellis, J. R., 2001. Molecular chaperones: Inside and outside the Anfinsen cage. Current Biology 11:R1038–R1040. Frydman, J., 2001. Folding of newly translated proteins in vivo: The role of molecular chaperones. Annual Review of Biochemistry 70:603–647. Hartl, F. U., and Hayer-Hartl, H., 2002. Molecular chaperones in the cytosol: From nascent chain to folded chain. Science 295:1852–1858. Jahn, T., and Radford, S. E., 2007. Folding versus aggregation: Polypeptide conformations on competing pathways. Archives of Biochemistry and Biophysics 469:100–117. Kramer, G., Rauch, T., et al., 2002. L23 protein functions as a chaperone docking site on the ribosome. Nature 419:171–174. Lin, Z., Madan, D., et al., 2008. GroEL stimulates protein folding through forced unfolding. Nature Structural and Molecular Biology 15:303–311. Luheshi, L. M., Crowther, D. C., et al., 2008. Protein misfolding and disease: From the test tube to the organism. Current Opinion in Chemical Biology 12:25–31. Sharma, S., Chakraborty, K., et al., 2008. Monitoring protein conformation along the pathway of chaperonin-assisted folding. Cell 133:142–153. Vogel, M., Bukau, B., et al., 2006. Allosteric regulation of Hsp70 chaperones by a proline switch. Molecular Cell 21:359–367. Protein Translocation Bolender, N., Sickmann, A., et al., 2008. Multiple pathways for sorting mitochondrial precursor proteins. EMBO Reports 9:42–49.

Bornemann, T., Jockel, J., et al., 2008. Signal sequence-independent membrane targeting of ribosomes containing short nascent peptides within the exit tunnel. Nature Structural and Molecular Biology 15:494–499. Brodersen, D. E., and Nissen, P., 2005. The social life of ribosomal proteins. FEBS Journal 272:2098–2108. Kutik, S., Guiard, B., et al., 2007. Cooperation of translocase complexes in mitochondrial protein import. Journal of Cell Biology 179:585–591. Mihara, K., 2003. Moving inside membranes. Nature 424:505–506. Rapoport, T. A., 2007. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450:663–669. Schnell, D. J., and Hebert, D. N., 2003. Protein translocons: Multifunctional mediators of protein translocation across membranes. Cell 112:491–505. Schwartz, S., and Blobel, G., 2003. Structural basis for the function of the b-subunit of the eukaryotic signal recognition particle. Cell 112: 793–803. Skach, W., 2007. The expanding role of the ER translocon in membrane protein folding. Journal of Cell Biology 179:1333–1335. Wirth, A., Jung, M., et al., 2003. The Sec61p complex is a dynamic precursor activated channel. Molecular Cell 12:261–268. Ubiquitin Selection of Proteins for Degradation Bellare, P., Small, E. C., et al., 2008. A role for ubiquitin in the spliceosome assembly pathway. Nature Structural and Molecular Biology 15:444–451. Hershko, A., 1996. Lessons from the discovery of ubiquitin system. Trends in Biochemical Sciences 21:445–449. Hochstrasser, M., 1996. Ubiquitin-dependent protein degradation. Annual Review of Genetics 30:405–439. Hunter, T., 2007. The age of crosstalk: Phosphorylation, ubiquitination, and beyond. Molecular Cell 28:730–738. Madsen, L., Schulze, A., et al., 2007. Ubiquitin domain proteins in disease. BMC Biochemistry 8:1–8. Varshvsky, A., 1997. The ubiquitin system. Trends in Biochemical Sciences 22:383–387. Proteasome-Mediated Protein Degradation Borissenko, L., and Groll, M., 2007. 20S proteasome and its inhibitors: Crystallographic knowledge for drug development. Chemical Reviews 107:687–717. Breusing, N., and Grune, T., 2008. Regulation of proteasome-mediated protein degradation during oxidative stress and aging. Biological Chemistry 389:203–209. Dahlmann, B., 2007. Role of proteasomes in disease. BMC Biochemistry 8:1–12. Ferrel, K., Wilkinson, C. R., et al., 2000. Regulatory subunit interactions of the 26S proteasome, a complex problem. Trends in Biochemical Sciences 25:83–88. Groll, M., Berkers, C. R., et al., 2006. Crystal structure of the boronic acid-based proteasome inhibitor Bortezomib in complex with the yeast 20S proteasome. Structure 14:451–456.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Zhang, F., Paterson, A. J., et al., 2007. Metabolic control of proteasome function. Physiology 22:373–379. HtrA Proteases Clausen, T., Southan, C., et al., 2002. The HtrA family of proteases: Implications for protein composition and cell fate. Molecular Cell 10:443–455. Kim, D. Y., and Kim, K. K., 2005. Structure and function of HtrA family proteins, the key players in protein quality control. Journal of Biochemistry and Molecular Biology 38:266–274. Sohn, J., Grant, R. A., et al., 2007. Allosteric activation of DegS, a stress sensor PDZ protease. Cell 131:572–583. Walle, L. V., Lamkanfi, M., et al., 2008. The mitochondrial serine protease HtrA2/Omi: An overview. Cell Death and Differentiation 15:453–460. Post-translational Modification by Sumoylation Anckar, J., and Sistonen, L., 2007. SUMO: Getting it on. Biochemical Society Transactions 35:1409–1413.

Bernier-Villamor, V., Sampson, D. A., et al., 2002. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108:345–356. Geiss-Friedlander, R., and Melchior, F., 2007. Concepts in sumoylation: A decade on. Nature Reviews Molecular Cell Biology 8:947–956. Hay, R. T., 2005. SUMO: A history of modification. Molecular Cell 18:1–12. Johnson, E. S., 2004. Protein modification by SUMO. Annual Review of Biochemistry 73:355–382. Ulrich, H. D., 2005. Mutual interactions between the SUMO and ubiquitin systems: A plea of no contest. Trends in Cell Biology 15:525–532. Vertegaal, A. C. O., 2007. Small ubiquitin-related modifiers in chains. Biochemical Society Transactions 35:1422–1423. Yunus, A. A., and Lima, D. D., 2006. Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway. Nature Structural and Molecular Biology 13:491–499.

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33 Copyright © 2012 Toronto Transit Commission

Metabolic Integration and Organ Specialization

! The Toronto Transit Commission (TTC)

ESSENTIAL QUESTION Cells are systems in a dynamic steady state maintained by a constant flux of nutrients that serve as energy sources or as raw material for the maintenance of cellular structures. Catabolism and anabolism are ongoing, concomitant processes. What principles underlie the integration of catabolism and energy production with anabolism and energy consumption? How is metabolism integrated in complex organisms with multiple organ systems?

Subway/RT Route Map. The coordinated flow of passengers along different transit lines is an apt metaphor for metabolic regulation.

Study of an enzyme, a reaction, or a sequence can be biologically relevant only if its position in the hierarchy of function is kept in mind. Daniel E. Atkinson

WHY IT MATTERS In the preceding chapters, we have explored the major metabolic pathways: glycolysis, the citric acid cycle, electron transport and oxidative phosphorylation, photosynthesis, gluconeogenesis, fatty acid oxidation, lipid biosynthesis, amino acid metabolism, and nucleotide metabolism. Several of these pathways are catabolic and serve to generate chemical energy useful to the cell; others are anabolic and use this energy to drive the synthesis of essential biomolecules. Despite their opposing purposes, these reactions typically occur at the same time as nutrient molecules are broken down to provide the building blocks and energy for ongoing biosynthesis. Cells maintain a dynamic steady state through processes that involve considerable metabolic flux. The metabolism that takes place in just a single cell is so complex that it defies detailed quantitative description. However, an appreciation of overall relationships can be achieved by stepping back and considering intermediary metabolism at a systems level of organization.

33.1

Can Systems Analysis Simplify the Complexity of Metabolism?

The metabolism of a typical heterotrophic cell can be portrayed by a schematic diagram consisting of just 3 interconnected functional blocks: (1) catabolism, (2) anabolism, and (3) macromolecular synthesis and growth (Figure 33.1 on page 1111). 1. Catabolism Energy-yielding nutrients are oxidized to CO2 and H2O in catabolism, and most of the electrons liberated are passed to oxygen via an electron-transport pathway

Cellular Energy Metabolism and Its Regulation (1977)

KEY QUESTIONS 33.1

Can Systems Analysis Simplify the Complexity of Metabolism?

33.2

What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism?

33.3

Is There a Good Index of Cellular Energy Status?

33.4

How Is Overall Energy Balance Regulated in Cells?

33.5

How Is Metabolism Integrated in a Multicellular Organism?

33.6

What Regulates Our Eating Behaviour?

33.7

Can You Really Live Longer by Eating Less? Online homework and a Student Self-Assessment for this chapter may be assigned in OWL.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 5 Information Transfer

BIOCHEMISTRY: A CANADIAN CONTEXT

In 1923, Canadian scientist Sir Frederick Banting, along with his collaborator John Macleod, received the Nobel Prize in Physiology or Medicine for their discovery of insulin. Banting, along with his co-worker Charles Best, was able to extract intact insulin from pancreases in which the pancreatic duct had been surgically closed. It had previously been proposed that a substance produced by the pancreas was responsible for the control of sugar metabolism. However, all attempts to isolate this unknown substance for therapeutic purposes had failed because, it was believed, the proteolytic enzymes secreted by the pancreas would digest it. Banting, having read an article demonstrating that experimental closure of the pancreatic duct lead to the degeneration of the cells secreting proteolytic enzymes, hypothesized that by closing the duct it would be possible to extract the metabolic regulating protein intact from the pancreas. In the summer of 1921, Banting and Best began testing their extract on diabetic dogs and were able to show that the treatment ameliorated diabetic symptoms. With the help of the biochemist James Collip, they were able to extract relatively pure insulin from ox pancreas, and, in the winter of 1922, a 14-year-old boy dying of diabetes in Toronto General Hospital was given the first injection of insulin, saving his life. In later years, the development of new genetic and biosynthetic techniques allowed for the production of large quantities of synthetic human insulin, which is used today to treat diabetic patients around the world. The role of insulin in diabetes provides a clear example of the importance of proper metabolic integration and regulation in multicellular organisms.

Arthur S. goss/Library and Archives Canada/PA-123481

Dr. Frederick Banting

coupled to oxidative phosphorylation, resulting in the formation of ATP. Some electrons go on to reduce NADP+ to NADPH, the source of reducing power for anabolism. Glycolysis, the citric acid cycle, electron transport and oxidative phosphorylation, and the pentose phosphate pathway are the principal pathways within this block. The metabolic intermediates in these pathways also serve as substrates for processes within the anabolic block. 2. Anabolism The biosynthetic reactions that form the many cellular molecules collectively comprise anabolism. For thermodynamic reasons, the chemistry of anabolism is more complex than that of catabolism [i.e., it takes more energy (and often more steps) to synthesize a molecule than can be produced from its degradation]. Metabolic intermediates derived from glycolysis and the citric acid cycle are the precursors for this synthesis, with NADPH supplying the reducing power and ATP the coupling energy. 3. Macromolecular Synthesis and Growth The organic molecules produced in anabolism are the building blocks for creation of macromolecules. Like anabolism, macromolecular synthesis is driven by energy from ATP, although indirectly in some cases: GTP is the principal energy source for protein synthesis, CTP for phospholipid synthesis, and UTP for polysaccharide synthesis. However, keep in mind that ATP is the principal phosphoryl donor for formation of GTP, CTP, and UTP from GDP, CDP, and UDP, respectively. Macromolecules are the agents of biological function and information—proteins, nucleic acids, lipids that self-assemble into membranes, and so on. Growth can be represented as cellular accumulation of macromolecules and the partitioning of these materials of function and information into daughter cells in the process of cell division.

Only a Few Intermediates Interconnect the Major Metabolic Systems Despite the complexity of processes taking place within each block, the connections between blocks involve only a limited number of substances. Only 10 or so kinds of NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 33

h"

Metabolic Integration and Organ Specialization

CO2

ADP H2O

PHOTOSYNTHESIS

O2

ATP NADP+

CARBON DIOXIDE FIXATION

NADPH Carbohydrates Amino acids Nucleotides Fatty acids, etc.

ADP Fat

CATABOLISM

Protein

ATP NADP+

ANABOLISM

NTP NDP

MACROMOLECULAR SYNTHESIS AND ACCUMULATION (GROWTH)

NADPH CO2

+

H2O

Triose-P Tetrose-P Pentose-P Hexose-P PEP Pyruvate Acetyl-CoA !-KG Succinyl-CoA Oxaloacetate

Proteins Nucleic acids Complex lipids

Membranes Organelles Cell walls, etc.

FIGURE 33.1 Block diagram of intermediary metabolism.

catabolic intermediates from glycolysis, the pentose phosphate pathway, and the citric acid cycle serve as the raw material for most of anabolism: 4 kinds of sugar phosphates (triose-P, tetrose-P, pentose-P, and hexose-P), 3 a-keto acids (pyruvate, oxaloacetate, and a-ketoglutarate), 2 coenzyme A derivatives (acetyl-CoA and succinyl-CoA), and PEP (phosphoenolpyruvate).

ATP and NADPH Couple Anabolism and Catabolism Metabolic intermediates are consumed by anabolic reactions and must be continuously replaced by catabolic processes. In contrast, the energy-rich compounds ATP and NADPH are recycled rather than replaced. When these substances are used in biosynthesis, the products are ADP and NADP+, and ATP and NADPH are regenerated during catabolism. ATP and NADPH are unique in that they are the only compounds whose purpose is to couple the energy-yielding processes of catabolism to the energy-consuming reactions of anabolism. Certainly, other coupling agents serve essential roles in metabolism. For example, NADH and [FADH2] participate in the transfer of electrons from substrates to O2 during oxidative phosphorylation. However, these reactions are solely catabolic, and the functions of NADH and [FADH2] are fulfilled within the block called catabolism. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 5 Information Transfer

Phototrophs Have an Additional Metabolic System: The Photochemical Apparatus The systems in Figure 33.1 reviewed thus far are representative only of metabolism as it exists in aerobic heterotrophs. The photosynthetic production of ATP and NADPH in photoautotrophic organisms entails a fourth block, the photochemical system (Figure 33.1). This block consumes H2O and releases O2. When this fourth block operates, energy production within the catabolic block can be largely eliminated. Yet another block, one to account for the fixation of carbon dioxide into carbohydrates, is also required for photoautotrophs. The inputs to this fifth block are the products of the photochemical system (ATP and NADPH) and CO2 derived from the environment. The carbohydrate products of this block may enter catabolism, but not primarily for energy production. In photoautotrophs, carbohydrates are fed into catabolism to generate the metabolic intermediates needed to supply the block of anabolism. Although these diagrams are oversimplifications of the total metabolic processes in heterotrophic or phototrophic cells, they are useful illustrations of functional relationships between the major metabolic subdivisions. This general pattern provides an overall perspective on metabolism, making its purpose easier to understand.

33.2

Stoichiometry: The measurement of the amounts of chemical elements and molecules involved in chemical reactions (from the Greek stoicheion, meaning “element,” and metria, meaning “measure”).

What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism?

Virtually every metabolic pathway either consumes or produces ATP. The amount of ATP involved (i.e., the stoichiometry of ATP synthesis or hydrolysis) lies at the heart of metabolic relationships. The overall thermodynamic efficiency of any metabolic sequence, be it catabolic or anabolic, is determined by ATP coupling. In every case, the overall reaction mediated by any metabolic pathway is energetically favourable because of its particular ATP stoichiometry. In the highly exergonic reactions of catabolism, much of the energy released is captured in ATP synthesis. In turn, the thermodynamically unfavourable reactions of anabolism are driven by energy released upon ATP hydrolysis. To illustrate this principle, we must first consider the 3 types of stoichiometries. The first 2 are fixed by the laws of chemistry, but the third is unique to living systems and reveals a fundamental difference between the inanimate world of chemistry and physics and the world of biological function, as shaped by evolution, that is, the world of living organisms. The fundamental difference is the stoichiometry of ATP coupling. 1. Reaction Stoichiometry This is simple chemical stoichiometry: the number of each kind of atom in any chemical reaction remains the same, and thus equal numbers must be present on both sides of the equation. This requirement holds even for a process as complex as cellular respiration: C6H12O6 + 6 O2 h 6 CO2 + 6 H2O. The 6 carbons in glucose appear as 6 CO2, the 12 H of glucose appear as the 12 H in 6 molecules of water, and the 18 oxygens are distributed between CO2 and H2O. 2. Obligate Coupling Stoichiometry Cellular respiration is an oxidation–reduction process, and the oxidation of glucose is coupled to the reduction of NAD+ and [FAD]. (Brackets here denote that the relevant FAD is covalently linked to succinate dehydrogenase; see Chapter 19.) The NADH and [FADH2] thus formed are oxidized in the electrontransport pathway: (a) C6H12O6 + 10 NAD+ + 2 [FAD] + 6 H2O h 6 CO2 + 10 NADH + 10 H+ + 2 [FADH2]; (b) 10 NADH + 10 H+ + 2 [FADH2] + 6 O2 h 12 H2O + 10 NAD+ + 2 [FAD]. Sequence (a) accounts for the oxidation of glucose via glycolysis and the citric acid cycle. Sequence (b) is the overall equation for electron transport per glucose. The stoichiometry NEL

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Chapter 33

Metabolic Integration and Organ Specialization

of coupling by the biological e- carriers, NAD+ and FAD, is fixed by the chemistry of electron transfer; each of the coenzymes serves as an e--pair acceptor. Reduction of each O atom takes an e- pair. Metabolism must obey these facts of chemistry: Biological oxidation of glucose releases 12 e- pairs, creating a requirement for 12 equivalents of e--pair acceptors, which transfer the electrons to 12 O atoms. By evolutionary design, NAD+/ NADH and FAD/FADH2 carry these electrons, but the stoichiometry is fixed by the chemistry. 3. Evolved Coupling Stoichiometries The participation of ATP is fundamentally different from the role played by pyridine nucleotides and flavins. The stoichiometry of adenine nucleotides in metabolic sequences is not fixed by chemical necessity. Instead, the “stoichiometries” we observe are the consequences of evolutionary design. The overall equation for cellular respiration,1 including the coupled formation of ATP by oxidative phosphorylation, is C6H12O6 + 6 O2 + 38 ADP + 38 Pi h 6 CO2 + 38 ATP + 44 H2O. The “stoichiometry” of ATP formation, 38 ADP + 38 Pi S 38 ATP + 38 H2O, cannot be predicted from any chemical considerations. The value of 38 ATP is an end result of biological adaptation. It is a trait that evolved through interactions between chemistry, heredity, and the environment over the course of evolution. Like any evolved character, ATP stoichiometry is the result of compromise. The final trait is one particularly suited to the fitness of the organism. The number 38 is not magical. Recall that in eukaryotes, the consensus value for the net yield of ATP per glucose is 30 to 32, not 38 (see Table 19.4). Also, the value of 38 was established a long time ago in evolution, when the prevailing atmospheric conditions and the competitive situation were undoubtedly very different from those of today. The significance of this number is that it provides a high yield of ATP for each glucose molecule, yet the yield is still low enough that essentially all the glucose is metabolized.

ATP Coupling Stoichiometry Determines the Keq for Metabolic Sequences The fundamental biological purpose of ATP as an energy-coupling agent is to drive thermodynamically unfavourable reactions. In effect, the energy release accompanying ATP hydrolysis is transmitted to the unfavourable reaction so that the overall free energy change for the coupled process is negative (i.e., favourable). The involvement of ATP serves to alter the free energy change for a reaction; or to put it another way, the role of ATP is to change the equilibrium ratio of [reactants] to [products] for a reaction. (See the A Deeper Look box on page 72.) Another way of viewing these relationships is to note that, at equilibrium, the concentrations of ADP and Pi will be vastly greater than that of ATP because !G "# for ATP hydrolysis is a large negative number.2 However, the cell where this reaction is at equilibrium is a dead cell. Living cells break down energy-yielding nutrient molecules to generate ATP. These catabolic reactions proceed with a very large overall decrease in free energy. Kinetic controls over the rates of the catabolic pathways are designed to ensure that the [ATP]/([ADP][Pi]) ratio is maintained very high. The cell, by employing kinetic controls over the rates of metabolic pathways, maintains a very high [ATP]/ ([ADP][Pi]) ratio so that ATP hydrolysis can serve as the driving force for virtually all biochemical events. 1

This overall equation for cellular respiration is for the reaction within an uncompartmentalized (prokaryotic) cell. In eukaryotes, where much of the cellular respiration is compartmentalized within mitochondria, mitochondrial ADP/ATP exchange imposes a metabolic cost on the proton gradient of 1 H+ per ATP, so the overall yield of ATP per glucose is 32, not 38. 2 Since !G"= = -30.5 kJ/mol, ln K = 12.3. So K = 2.2 $ 105. Choosing starting conditions of [ATP] = 8 mM, [ADP] = 8 mM, and [P ] = 1 mM, we can assume that, at eq eq i equilibrium, [ATP] has fallen to some insignificant value x, [ADP] = approximately 16 mM, and [Pi] = approximately 9 mM. The concentration of ATP at equilibrium, x, then calculates to be about 1 nM. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 5 Information Transfer

ATP Has Two Metabolic Roles The role of ATP in metabolism is 2-fold: 1. It serves in a stoichiometric role to establish large equilibrium constants for metabolic conversions and to render metabolic sequences thermodynamically favourable. This is the role referred to when we call ATP the energy currency of the cell. 2. ATP also serves as an important allosteric effector in the kinetic regulation of metabolism. Its concentration (relative to those of ADP and AMP) is an index of the energy status of the cell and determines the rates of regulatory enzymes situated at key points in metabolism, such as PFK in glycolysis and FBPase in gluconeogenesis.

33.3

Is There a Good Index of Cellular Energy Status?

Energy transduction and energy storage in the adenylate system (ATP, ADP, and AMP) lie at the very heart of metabolism. The amount of ATP a cell uses per minute is roughly equivalent to the steady-state amount of ATP it contains. Thus, the metabolic lifetime of an ATP molecule is brief. ATP, ADP, and AMP are all important effectors in exerting kinetic control on regulatory enzymes situated at key points in metabolism, so uncontrolled changes in their concentrations could have drastic consequences. The regulation of metabolism by adenylates, in turn, requires close control of the relative concentrations of ATP, ADP, and AMP. Some ATP-consuming reactions produce ADP: PFK and hexokinase are examples. Others lead to the formation of AMP, as in fatty acid activation by acyl-CoA synthetases: Fatty acid + ATP + coenzyme A h AMP + PPi + fatty acyl-CoA

Adenylate Kinase Interconverts ATP, ADP, and AMP Adenylate kinase (see Chapter 17), by catalyzing the reversible phosphorylation of AMP by ATP, provides a direct connection among all 3 members of the adenylate pool: ATP + AMP ∆ 2 ADP. The free energy of hydrolysis of a phosphoanhydride bond is essentially the same in ADP and ATP (see Chapter 3), and the standard free energy change for this reaction is close to zero.

ATP

+

AMP

Adenylate kinase

Energy Charge Relates the ATP Levels to the Total Adenine Nucleotide Pool The role of the adenylate system is to provide phosphoryl groups at high group-transfer potential in order to drive thermodynamically unfavourable reactions. The capacity of the adenylate system to fulfill this role depends on how fully charged it is with phosphoric anhydrides. Energy charge is an index of this capacity:

2 ADP

100

% of total

80

ATP

AMP

60

Energy charge =

40

ADP

20 0 0

0.2

0.4 0.6 Energy charge

0.8

1.0

FIGURE 33.2 Relative concentrations of AMP, ADP, and ATP as a function of energy charge. (This graph was constructed assuming that the adenylate kinase reaction is at equilibrium and that !G"# for the reaction is - 473 J/mol; Keq = 1.2.)

2 [ATP] + [ADP] 1 ¢ ≤. 2 [ATP] + [ADP] + [AMP]

The denominator represents the total adenylate pool ([ATP] + [ADP] + [AMP]); the numerator is the number of phosphoric anhydride bonds in the pool, 2 for each ATP and 1 for each ADP. The factor 12 normalizes the equation so that energy charge, or E.C., has the range 0–1.0. If all the adenylate is in the form of ATP, E.C. = 1.0, and the potential for phosphoryl transfer is maximal. At the other extreme, if AMP is the only adenylate form present, E.C. = 0. It is reasonable to assume that the adenylate kinase reaction is never far from equilibrium in the cell. Then the relative amounts of the 3 adenine nucleotides are fixed by the energy charge. Figure 33.2 shows the relative changes in the concentrations of the adenylates as energy charge varies from 0 to 1.0. NEL

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Chapter 33

Metabolic Integration and Organ Specialization

1115

Key Enzymes Are Regulated by Energy Charge R (ATPgenerating) Reaction rate

Regulatory enzymes typically respond in reciprocal fashion to adenine nucleotides. For example, PFK is stimulated by AMP and inhibited by ATP. If the activities of various regulatory enzymes are examined in vitro as a function of energy charge, an interesting relationship appears. Regulatory enzymes in energy-producing catabolic pathways show greater activity at low energy charge, but the activity falls off abruptly as E.C. approaches 1.0. In contrast, regulatory enzymes of anabolic sequences are not very active at low energy charge, but their activities increase exponentially as E.C. nears 1.0. These contrasting responses are termed R, for ATP-regenerating, and U, for ATP-utilizing (Figure 33.3). Regulatory enzymes such as PFK and pyruvate kinase in glycolysis follow the R response curve as E.C. is varied. Note that PFK itself is an ATP-utilizing enzyme, using ATP to phosphorylate fructose-6-phosphate to yield fructose-1,6-bisphosphate. Nevertheless, because PFK acts physiologically as the valve controlling the flux of carbohydrate down the catabolic pathways of cellular respiration that lead to ATP regeneration, it responds as an “R” enzyme to energy charge. Regulatory enzymes in anabolic pathways, such as acetylCoA carboxylase, which initiates fatty acid biosynthesis, respond as “U” enzymes. The overall purposes of the R and U pathways are diametrically opposite in terms of ATP involvement. Note in Figure 33.3 that the R and U curves intersect at a high E.C. value. As E.C. increases past this point, R activities decline precipitously and U activities rise. That is, when E.C. is very high, biosynthesis is accelerated, while catabolism diminishes. The consequence of these effects is that ATP is used up faster than it is regenerated, and so E.C. begins to fall. As E.C. drops below the point of intersection, R processes are favoured over U. Then, ATP is generated faster than it is consumed, and E.C. rises again. The net result is that the value of energy charge oscillates about a point of steady state (Figure 33.3). The experimental results obtained from careful measurement of the relative amounts of AMP, ADP, and ATP in living cells reveals that normal cells have an energy charge in the neighbourhood of 0.85– 0.88. Maintenance of this steady-state value is one criterion of cell health and normalcy.

Point of metabolic steady state U (ATP-utilizing) 0

Energy charge

FIGURE 33.3 Responses of regulatory enzymes to variation in energy charge.

Phosphorylation Potential Is a Measure of Relative ATP Levels Because energy charge is maintained at a relatively constant value in normal cells, it is not an informative index of cellular capacity to carry out phosphorylation reactions. The relative concentrations of ATP, ADP, and Pi do provide such information, and a function called phosphorylation potential has been defined in terms of these concentrations: ADP + Pi ∆ ATP + H2O Phosphorylation potential, ≠, is equal to [ATP]/([ADP][Pi]). Note that this expression includes a term for the concentration of inorganic phosphate. [Pi] has substantial influence on the thermodynamics of ATP hydrolysis. In contrast with energy charge, phosphorylation potential varies over a significant range as the actual proportions of ATP, ADP, and Pi in cells vary in response to metabolic state. ≠ ranges from 200 to 800 M-1, or more, with higher levels signifying more ATP and correspondingly greater phosphorylation potential.

33.4

How Is Overall Energy Balance Regulated in Cells?

AMP-activated protein kinase (AMPK) is the cellular energy sensor. Metabolic inputs to this sensor determine whether its output, protein kinase activity, takes place. When cellular energy levels are high, as signalled by high ATP concentrations, AMPK is inactive. When cellular energy levels are depleted, as signalled by high [AMP], AMPK is allosterically activated and phosphorylates many targets controlling cellular energy production and consumption. Recall that, due to the nature of the adenylate kinase equilibrium (see pages 569–570), AMP levels increase exponentially as ATP levels decrease. AMP is an allosteric activator of AMPK, whereas ATP at high levels acts as an allosteric inhibitor by displacing AMP from the allosteric site. Thus, competition between AMP and ATP for binding to the NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

1

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Part 5 Information Transfer

FIGURE 33.4 Domain structure of the AMP-activated protein kinase subunits. (Adapted from Figure 1 in Hardie, D. G., Hawley, S. A., and Scott, J. W., 2006. AMP-activated protein kinase: Development of the energy sensor concept. Journal of Physiology 574:7–15.)

(a) !-Subunit Upstream kinases

P

N

C Kinase

#$ Binding

(b) #-Subunit N

C !$ Binding

(c) $-Subunit N

CBS1

CBS2

AMP/ATP binding

CBS3

CBS4

C

AMP/ATP binding

AMPK allosteric sites determines the activity of AMPK. Activation of AMPK (1) sets in motion catabolic pathways leading to ATP synthesis, and (2) shuts down pathways that consume ATP energy, such as biosynthesis and cell growth. AMPK is an abg-heterotrimer (Figure 33.4). The a-subunit is the catalytic subunit; it has an N-terminal Ser/Thr protein kinase domain and a C-terminal bg-binding domain. The b-subunit has at its C-terminus an ag-binding domain. The g-subunit is the regulatory subunit; it has a pair of allosteric sites where either AMP or ATP binds. These sites are located toward its C-terminus in the form of 4 CBS domains (so named for their homology to cystathionine-b-synthase). These CBS domains act in pairs to form structures known as Bateman modules. The Bateman modules provide the binding sites for the allosteric ligands AMP and ATP. AMP binding to these sites is highly cooperative, such that binding of AMP to one module markedly enhances AMP-binding at the other. This cooperativity renders AMPK exquisitely sensitive to changes in AMP concentration. AMP binding to AMPK increases its protein kinase activity by more than 1000-fold. The underlying mechanism involves a pseudosubstrate sequence (see the Protein Kinases: Target Recognition and Intrasteric Control section in Chapter 14, page 487) within CBS domain 2 that fits into the a-subunit catalytic site. When AMP binds to the Bateman modules, conformational changes in the g-subunit displace the pseudosubstrate sequence from the kinase catalytic site, freeing it to act. The structural relationships between the AMPK subunits can be seen in the Schizosaccharomyces pombe abg complex (Figure 33.5). Actually, AMP activates AMPK in 2 ways: First, it is an allosteric activator; second, AMP binding favours phosphorylation of Thr172 within the a-subunit kinase domain. Phosphorylation of Thr172 is necessary for a-subunit protein kinase activity. Thr172 lies within the activation loop of the kinase; activation loops are common features of protein kinases whose activation requires phosphorylation by other protein kinases. Both of these favourable actions by AMP are reversed if ATP displaces AMP from the allosteric site.

FIGURE 33.5 Core structure of the Schizosaccharomyces pombe AMPK heterotrimer. The a-subunit is green, the b-subunit is yellow, and the g-subunit is white. A bound AMP (red) is also shown. A second AMP-binding site (vacant) lies directly above this AMP (pdb id = 2OOX).

AMPK Targets Key Enzymes in Energy Production and Consumption Activation of AMPK leads to phosphorylation of many key enzymes in energy metabolism. Those involved in energy production and that are activated upon phosphorylation by AMPK include phosphofructokinase-2 (PFK-2; see Chapter 21). In contrast to protein NEL

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Chapter 33

Metabolic Integration and Organ Specialization

1117

kinase A phosphorylation of PFK-2, AMPK phosphorylation of liver PFK-2 enhances fructose-2,6-bisphosphate synthesis, which in turn stimulates glycolysis. Enzymes involved in energy consumption that are down-regulated upon phosphorylation by AMPK include glycogen synthase (see Chapter 21), acetyl-CoA carboxylase (which catalyzes the committed step in fatty acid biosynthesis; see Chapter 9), and 3-hydroxy-3-methylglutaryl-CoA reductase, which carries out the key regulatory reaction in cholesterol biosynthesis (see Chapter 9). Further, AMPK phosphorylation of various transcription factors leads to diminished expression of genes encoding biosynthetic enzymes and elevated expression of catabolic genes.

AMPK Controls Whole-Body Energy Homeostasis Beyond these cellular effects, AMPK plays a central role in energy balance in multicellular organisms (Figure 33.6). AMPK in skeletal muscle is activated by hormones such as adiponectin and leptin, adipocyte-derived hormones that govern eating behaviour and energy homeostasis (see Section 33.6). Physical activity (exercise) also activates muscle AMPK. In turn, skeletal muscle AMPK activates glucose uptake, fatty acid oxidation, and mitochondrial biogenesis through its phosphorylation of metabolic enzymes and transcription factors that control expression of genes involved in energy production and consumption. AMPK’s actions in the liver lead to lowered ATP (energy) consumption through down-regulation of fatty acid synthesis, cholesterol synthesis, and gluconeogenesis. Metformin, a widely used drug for the treatment of type 2 diabetes (page 701), lowers blood glucose levels through inhibition of liver gluconeogenesis; metformin achieves this result through activation of AMPK. AMPK blocks insulin secretion by pancreatic b-cells; insulin is a hormone that favours energy storage (glycogen and fat synthesis). AMPK is also a master regulator of eating behaviour through its activity in the hypothalamus, the key centre for regulation of food intake. These effects of AMPK are described in Section 33.6. Brain Heart

Skeletal muscle Exercise

Fatty acid uptake and oxidation, glucose uptake, glycolysis

leptin, adiponectin

Hypothalamus Food intake

Fatty acid uptake and oxidation, glucose uptake, mitochondrial biogenesis

AMPK

Insulin secretion

Fatty acid synthesis, lipolysis

Adipose cells

FIGURE 33.6 AMPK regulation of energy production and consumption in mammals. (Adapted from Cell Metabolism, 1, Kahn, B. B., Alquier, T., Carling, D., and Hardie, D. G., AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism,15–25. 2005, with permission from Elsevier.)

Fatty acid synthesis, cholesterol synthesis, gluconeogenesis

Pancreatic #-cells

Liver NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 5 Information Transfer

33.5

How Is Metabolism Integrated in a Multicellular Organism?

In complex multicellular organisms, organ systems have arisen to carry out specific physiological functions. Each organ expresses a repertoire of metabolic pathways that is consistent with its physiological purpose. Such specialization depends on coordination of metabolic responsibilities among organs so that the organism as a whole may thrive. Essentially all cells in animals have the set of enzymes common to the central pathways of intermediary metabolism, especially the enzymes involved in the formation of ATP and the synthesis of glycogen and lipid reserves. Nevertheless, organs differ in the metabolic fuels they prefer as substrates for energy production. Important differences also occur in the ways ATP is used to fulfill the organs’ specialized metabolic functions. To illustrate these relationships, we will consider the metabolic interactions among the major organ systems found in humans: brain, skeletal muscle, heart, adipose tissue, and liver. In particular, the focus will be on energy metabolism in these organs (Figure 33.7). The major fuel depots in animals are glycogen in liver and muscle; triacylglycerols (fats) stored in adipose tissue; and protein, most of which is in skeletal muscle. In general, the order of preference for the use of these fuels is the order given: glycogen > triacylglycerol > protein. Nevertheless, the tissues of the body work together to maintain energy homeostasis (caloric homeostasis), defined as a constant availability of fuels in the blood. Brain

Heart

Ketone bodies Fatty acids

CO2

+

H2O

CO2

Glucose

+

H2O

Glucose

Ketone bodies Liver

Adipose tissue

Ketone bodies

Lactate

Acetyl-CoA

Pyruvate

Amino acids

Glucose

Proteins

Urea

Fatty acids Fatty acids Triacylglycerols

Triacylglycerols

+ Glycerol

Glycerol

Glucose

CO2

+

H2O

Glycogen

Fatty acids

Alanine + glutamine

Lactate

Ketone bodies

Amino acids

Pyruvate

Proteins

CO2

+

H2O

Glucose Glycogen

Muscle Red arrows indicate preferred routes in the well-fed state FIGURE 33.7 Metabolic relationships among the major human organs. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Chapter 33

Metabolic Integration and Organ Specialization

The Major Organ Systems Have Specialized Metabolic Roles Table 33.1 summarizes the energy metabolism of the major human organs. Brain The brain has 2 remarkable metabolic features. First, it has a very high respiratory metabolism. In resting adult humans, 20% of the oxygen consumed is used by the brain, even though it constitutes only 2% or so of body mass. Interestingly, this level of oxygen consumption is independent of mental activity, continuing even during sleep. Second, the brain is an organ with no significant fuel reserves, that is, no glycogen, expendable protein, or fat (even in “fatheads”!). Normally, the brain uses only glucose as a fuel and is totally dependent on the blood for a continuous incoming supply. Interruption of the glucose supply for even brief periods of time (as in a stroke) can lead to irreversible losses in brain function. The brain uses glucose to carry out ATP synthesis via cellular respiration. High rates of ATP production are necessary to power the plasma membrane Na+,K+-ATPase so that the membrane potential essential for transmission of nerve impulses is maintained. During prolonged fasting or starvation, the body’s glycogen reserves are depleted. Under such conditions, the brain adapts to use b-hydroxybutyrate (Figure 33.8) as a source of fuel, converting it to acetyl-CoA for energy production via the citric acid cycle. b-Hydroxybutyrate (see Chapter 22) is formed from fatty acids in the liver. Although the brain cannot use free fatty acids or lipids directly from the blood as fuel, the conversion of these substances to b-hydroxybutyrate in the liver allows the brain to use body fat as a source of energy. The brain’s other potential source of fuel during starvation is glucose obtained from gluconeogenesis in the liver (see Chapter 21), using the carbon skeletons of amino acids derived from muscle protein breakdown. The adaptation of the brain to use b-hydroxybutyrate from fat spares protein from degradation until lipid reserves are exhausted. Muscle Skeletal muscle is responsible for about 30% of the O2 consumed by the human body at rest. During periods of maximal exertion, skeletal muscle can account for more than 90% of the total metabolism. Muscle metabolism is primarily dedicated to the production of ATP as the source of energy for contraction and relaxation. Muscle contraction occurs when a motor nerve impulse causes Ca2+ release from specialized endomembrane compartments (the transverse tubules and sarcoplasmic reticulum). Ca2+ floods the sarcoplasm (the term denoting the cytosolic compartment of muscle cells), where it binds to troponin C, a regulatory protein, initiating a series of events that culminate in the sliding of myosin thick filaments along actin thin filaments. This mechanical movement is driven by energy released upon hydrolysis of ATP (see Chapter 15). The net result is that the muscle shortens. Relaxation occurs when the Ca2+ ions are pumped back into the sarcoplasmic reticulum by the action of a Ca2+-transporting membrane ATPase. Two Ca2+ ions are translocated per ATP hydrolyzed. The amount of ATP used during relaxation is almost as much as that consumed during contraction.

TABLE 33.1

Energy Metabolism in Major Vertebrate Organs

Organ

Energy Reservoir

Brain

None

Glucose (ketone bodies during starvation)

None

Skeletal muscle (resting)

Glycogen

Fatty acids

None

Skeletal muscle (strenuous exercise)

None

Glucose from glycogen

Lactate

Heart muscle

Glycogen

Fatty acids

None

Adipose tissue

Triacylglycerol

Fatty acids

Fatty acids, glycerol

Liver

Glycogen, triacylglycerol

Amino acids, glucose, fatty acids

Fatty acids, glucose, ketone bodies

Preferred Substrate

Energy Sources Exported

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FIGURE 33.8 Ketone bodies such as b-hydroxybutyrate provide the brain with a source of acetyl-CoA when glucose is unavailable.

OH D -#-Hydroxybutyrate

CH3

C

O CH2

C O–

H NAD+

#-Hydroxybutyrate dehydrogenase

+

NADH

H+

O Acetoacetate

CH3

C

O CH2

C O–

Succinyl-CoA

3-Ketoacyl-CoA transferase

Succinate O Acetoacetyl-CoA

CH3

C

O CH2

C

CoA

S

Thiolase

HS- CoA O CH3

C

S

CoA

S

CoA

O

2 Acetyl-CoA CH3

C

Because muscle contraction is an intermittent process that occurs upon demand, muscle metabolism is designed for a demand response. Muscle at rest uses free fatty acids, glucose, or ketone bodies as fuel and produces ATP via oxidative phosphorylation. Resting muscle also contains about 2% glycogen and about 0.08% phosphocreatine by weight (Figure 33.9). When ATP is used to drive muscle contraction, the ADP formed can be reconverted to ATP by creatine kinase at the expense of phosphocreatine. Muscle phosphocreatine can generate enough ATP to power about 4 seconds of exertion. During strenuous exertion, such as a 100 metre sprint, once the phosphocreatine is depleted, muscle relies solely on its glycogen reserves, making the ATP for contraction via glycolysis. In contrast with the citric acid cycle and oxidative phosphorylation pathways, glycolysis is capable of explosive bursts of activity, and the flux of glucose-6-phosphate through this pathway can increase 2000-fold almost instantaneously. The triggers for this activation are Ca2+ and the

FIGURE 33.9 Phosphocreatine serves as a reservoir of ATP-synthesizing potential.

O –O

O–

P

NH2+

C H3C

O

ATP

ADP

NH

N

+

NH2

Mg2+

∆Go' = –13 kJ/mol

NH2+

C

Creatine kinase H3C

N

CH2

CH2

C

C O–

Phosphocreatine

O

O–

Creatine NEL

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HUMAN BIOCHEMISTRY

Athletic Performance Enhancement with Creatine Supplements?

Popperfoto/Getty Images

During the 1996 Olympic Games, Canadian athlete Donovan Bailey set a world record when he ran the 100 m in 9.84 seconds, reaching a top speed of 12.10 m/sec. In order to perform short, intense bursts of exercise such as this, skeletal muscle relies initially on its existing ATP reserves. However, these reserves are used up rapidly (within the first 2 seconds), and so the muscle must then rely on the activity of creatine kinase to maintain ATP reserves. Normally, [phosphocreatine]muscle is sufficient to restore ATP levels for a few extra seconds, but no more. By increasing the creatine pool, however, it is possible to improve one’s performance during short, high intensity exercise (such as sprinting), although there is no benefit in endurance events (such as distance running). The creatine pool in a 70 kg (154 lb) human body is about 120 grams. This pool includes dietary creatine (from meat) and creatine synthesized by the human body from its precursors (arginine, glycine, and methionine). Of this creatine, 95% is stored in the skeletal and smooth muscles, about 70% of which is in the form of phosphocreatine. Supplementing the diet with 20–30 grams of creatine per day for 4–21 days can increase the muscle creatine pool by as much as 50% in someone with a previously low creatine level. Thereafter, supplements of 2 grams per day will maintain elevated creatine stores. As per Health Canada recommendation, consumers should consult with their doctors before using creatine as a dietary supplement.

“fight or flight” hormone epinephrine (see Chapters 11 and 21). Little inter-organ cooperation occurs during strenuous (anaerobic) exercise. Muscle fatigue is the inability of a muscle to maintain power output. During maximum exertion, the onset of fatigue takes only 20 seconds or so. Fatigue is not the result of exhaustion of the glycogen reserves, nor is it a consequence of lactate accumulation in the muscle. Instead, it is caused by a decline in intramuscular pH as protons are generated during glycolysis. (The overall conversion of glucose to 2 lactates in glycolysis is accompanied by the release of 2 H+.) The pH may fall as low as 6.4. It is likely that the decline in PFK activity at low pH leads to a lowered flux of hexose through glycolysis and inadequate ATP levels, causing a feeling of fatigue. One benefit of PFK inhibition is that the ATP remaining is not consumed in the PFK reaction, thereby sparing the cell from the more serious consequences of losing all its ATP. During fasting or excessive activity, skeletal muscle protein is degraded to amino acids so that their carbon skeletons can be used as fuel. Many of the skeletons are converted to pyruvate, which can be transaminated back into alanine for export via the circulation (Figure 33.10). Alanine is carried to the liver, which in turn deaminates it back into pyruvate so that it can serve as a substrate for gluconeogenesis. Although muscle protein can be

O H3C

C

+

NH3

O

O

O–

–O

C

Pyruvate

C

C

O CH2

C

CH2

O–

Glutamate

H

FIGURE 33.10 The transamination of pyruvate to alanine by glutamate:alanine aminotransferase.

Glutamate alanine aminotransferase NH3+ H3C Alanine

C H

O

O

O–

–O

O C

C

C

O CH2

CH2

!-Ketoglutarate

C O–

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mobilized as an energy source, it is not efficient for an organism to consume its muscle and lower its overall fitness for survival. Muscle protein represents a fuel of last resort. Heart In contrast with the intermittent work of skeletal muscle, the activity of heart muscle is constant and rhythmic. The range of activity in heart is also much less than that in muscle. Consequently, the heart functions as a completely aerobic organ and, as such, is very rich in mitochondria. Roughly half the cytoplasmic volume of heart muscle cells is occupied by mitochondria. Under normal working conditions, the heart prefers fatty acids as fuel, oxidizing acetyl-CoA units via the citric acid cycle and producing ATP for contraction via oxidative phosphorylation. Heart tissue has minimal energy reserves: a small amount of phosphocreatine and limited quantities of glycogen. As a result, the heart must be continually nourished with oxygen and free fatty acids, glucose, or ketone bodies as fuel. Adipose Tissue Adipose tissue is an amorphous tissue that is widely distributed about the body: around blood vessels, in the abdominal cavity and mammary glands, and most prevalently, as deposits under the skin. Long considered merely a storage depot for fat, adipose tissue is now appreciated as an endocrine organ responsible for secretion of a variety of hormones that govern eating behaviour and caloric homeostasis. It consists principally of cells known as adipocytes that no longer replicate. However, adipocytes can increase in number as adipocyte precursor cells divide, and obese individuals tend to have more of them. As much as 65% of the weight of adipose tissue is triacylglycerol is stored in adipocytes, essentially as oil droplets. The average 70 kg man has enough caloric reserve stored as fat to sustain a 6000 kJ/day rate of energy production for 3 months, which is adequate for survival, assuming no serious metabolic aberrations (such as nitrogen, mineral, or vitamin deficiencies). Despite their role as energy storage depots, adipocytes have a high rate of metabolic activity, synthesizing and breaking down triacylglycerol so that the average turnover time for a triacylglycerol molecule is just a few days. Adipocytes actively carry out cellular respiration, transforming glucose to energy via glycolysis, the citric acid cycle, and oxidative phosphorylation. If glucose levels in the diet are high, glucose is converted to acetyl-CoA for fatty acid synthesis. However, under most conditions, free fatty acids for triacylglycerol synthesis are obtained from the liver. Because adipocytes lack glycerol kinase, they cannot recycle the glycerol of triacylglycerol but instead depend on glycolytic conversion of glucose to dihydroxyacetone-3-phosphate (DHAP) and the reduction of DHAP to glycerol-3-phosphate for triacylglycerol biosynthesis. Adipocytes also require glucose to feed the pentose phosphate pathway for NADPH production. Glucose plays a pivotal role for adipocytes. If glucose levels are adequate, glycerol-3phosphate is formed in glycolysis and the free fatty acids liberated in triacylglycerol breakdown are re-esterified to glycerol to re-form triacylglycerols. However, if glucose levels are low, [glycerol-3-phosphate] falls and free fatty acids are released to the bloodstream (see Chapter 22).

HUMAN BIOCHEMISTRY

Fat-Free Mice: A Snack Food for Pampered Pets? No, a Model for One Form of Diabetes Scientists at the National Institutes of Health have bred transgenic mice that lack white adipose tissue throughout their lifetimes. These mice were produced by blocking the normal differentiation of stem cells into adipocytes so that essentially no white adipose tissue can be formed in these animals. These “fat-free” mice have double the food intake and 5 times the water intake of normal mice. Fat-free mice also show decreased physical activity and must be kept warm on little heating pads to survive, because they lack insulating fat. They are also diabetic, with 3 times normal blood glucose and triacylglycerol levels and only 5% of normal leptin levels; they die prematurely. Like type 2 diabetic patients, fat-free mice

have markedly elevated insulin levels (50–400 times normal) but are unresponsive to insulin. These mice serve as an excellent model for the disease lipoatrophic diabetes, an inherited disease characterized by the absence of adipose tissue and severe diabetic symptoms. Indeed, transplantation of adipose tissue into these fat-free mice cured their diabetes. As the major organ for triacylglycerol storage, white adipose tissue helps control energy homeostasis (food intake and energy expenditure) via the release of leptin and other hormone-like substances (see the discussion on page 1126). Clearly, absence of adipose tissue has widespread, harmful consequences for metabolism.

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Chapter 33

Metabolic Integration and Organ Specialization

“Brown Fat” A specialized type of adipose tissue, so-called brown fat, is found in newborns and hibernating animals. Brown fat cells contain multiple smaller lipid vacuoles and a large quantity of mitochondria, unlike their white fat cell counterparts, which contain only a single large lipid vacuole and a handful of mitochondria (Figure 33.11). The abundance of mitochondria, which are rich in cytochromes, is responsible for the brown colour of this fat. As usual, these mitochondria are very active in electron transport–driven proton translocation, but these particular mitochondria contain in their inner membranes a protein, thermogenin, also known as uncoupling protein 1 (see Chapter 19), that creates a passive proton channel, permitting the H+ ions to reenter the mitochondrial matrix without generating ATP. Instead, the energy of oxidation is dissipated as heat. Indeed, brown fat is specialized to oxidize fatty acids for heat production rather than ATP synthesis. Liver The liver serves as the major metabolic processing centre in vertebrates. Except for dietary triacylglycerols, which are metabolized principally by adipose tissue, most of the incoming nutrients that pass through the intestinal tract are routed via the portal vein to the liver for processing and distribution. Much of the liver’s activity centres around conversions involving glucose-6-phosphate (Figure 33.12). Glucose-6-phosphate can be converted to glycogen, released as blood glucose, used to generate NADPH and pentoses via the pentose phosphate cycle, or catabolized to acetyl-CoA for fatty acid synthesis or for energy production via oxidative phosphorylation. Most of the liver glucose-6-phosphate arises from dietary carbohydrate, degradation of glycogen reserves, or muscle lactate that enters the gluconeogenic pathway. The liver plays an important regulatory role in metabolism by buffering the level of blood glucose. Liver has 2 enzymes for glucose phosphorylation: hexokinase and glucokinase (type IV hexokinase). Unlike hexokinase, glucokinase has a low affinity for glucose. Its Km for glucose is high, on the order of 10 mM. When blood glucose levels are high, glucokinase activity augments hexokinase in phosphorylating glucose as an initial step leading to its storage in glycogen. All the major metabolic hormones (epinephrine, glucagon, and insulin) influence glucose metabolism in the liver to keep blood glucose levels relatively constant (see Chapters 11 and 21). The liver is a major centre for fatty acid turnover. When the demand for metabolic energy is high, triacylglycerols are broken down and fatty acids are degraded in the liver to acetyl-CoA to form ketone bodies, which are exported to the heart, brain, and other

FIGURE 33.11 The cytological differences between white and brown adipocytes. White adipocytes (left) accumulate a large quantity of fat (yellow) in a single vacuole and harbour only a few mitochondria (red). Conversely, brown adipocytes (right) store a smaller amount of fat in multiple vacuoles but are characterized by an abundance of mitochondria. These cytological differences reflect the different physiological roles of these cells; while white adipocytes are mainly used to store fat, brown adipocytes oxidize fat in mitochondria to generate heat. (Adapted from Richard D, Picard F. Brown Fat biology and thermogenesis. Front Biosci. 2011; 16:1233–1260)

Blood glucose

Glucose-6-phosphate

Lipid synthesis

Pentose phosphate pathway

Glycolysis Cholesterol

ATP

Fatty acids Pyruvate

NADPH and pentose phosphates

Fatty acid synthesis Acetyl-CoA ATP Citric acid Oxidative cycle phosphorylation

CO2

+

Brown adipocyte

FIGURE 33.12 Metabolic conversions of glucose-6phosphate in the liver.

Glycogen

Triacylglycerols, phospholipids

White adipocyte

1123

H2O

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tissues. If energy demands are low, fatty acids are incorporated into triacylglycerols that are carried to adipose tissue for deposition as fat. Cholesterol is also synthesized in the liver from 2-carbon units derived from acetyl-CoA. In addition to these central functions in carbohydrate and fat-based energy metabolism, the liver serves other purposes. For example, the liver can use amino acids as metabolic fuels. Amino acids are first converted to their corresponding a-keto acids by aminotransferases. The amino group is excreted after incorporation into urea in the urea cycle. The carbon skeletons of glucogenic amino acids can be used for glucose synthesis, whereas those of ketogenic amino acids appear in ketone bodies (see Figure 23.41). The liver is also the principal detoxification organ in the body. The endoplasmic reticulum of liver cells is rich in enzymes that convert biologically active substances such as hormones, poisons, and drugs into less harmful by-products. Liver disease leads to serious metabolic derangements, particularly in amino acid metabolism. In cirrhosis, the liver becomes defective in converting NH+4 to urea for excretion, and blood levels of NH+4 rise. Ammonia is toxic to the central nervous system, and coma ensues.

33.6

What Regulates Our Eating Behaviour?

Approximately 36% of Canadians are overweight, and about 1 in 5 Canadians are considered obese (having a BMI of 30 or higher). These numbers are even higher in the United States, where as many as 65% of Americans are overweight, and 1 in 3 Americans is clinically obese (overweight by 20% or more). Obesity is the single most important cause of type 2 (adult-onset insulin-independent) diabetes. Research into the regulatory controls that govern our feeding behaviour has become a medical urgency with great financial incentives, given the epidemic proportions of obesity and widespread preoccupation with dieting and weight loss.

The Hormones That Control Eating Behaviour Come from Many Different Tissues Appetite and weight regulation are determined by a complex neuroendocrine system that involves hormones produced in the stomach, small intestines, pancreas, adipose tissue, and central nervous system. These hormones act in the brain, principally on neurons within the arcuate nucleus region of the hypothalamus. The arcuate nucleus is an anatomically distinct brain area that functions in homeostasis of body weight, body temperature, blood pressure, and other vital functions (Figure 33.13). The neurons respond to these signals by activating, or not, pathways involved in eating (food intake) and energy expenditure. Hormones that regulate eating behaviour can be divided into short-term regulators that determine individual meals and long-term regulators that act to stabilize the levels of body fat deposits. Two subsets of neurons are involved: the NPY/AgRP-producing neurons that release NPY (neuropeptide Y), the protein that stimulates the neurons that trigger eating behaviour; and the melanocortin-producing neurons, whose products inhibit the neurons initiating eating behaviour. AgRP is agouti-related peptide, a protein that blocks the activity of melanocortin-producing neurons. Melanocortins are a group of peptide hormones that includes A-and B-melanocyte–stimulating hormones (A-MSH and B-MSH). Melanocortins act on melanocortin receptors (MCRs), which are members of the 7-TMS G protein– coupled receptor (GPCR) family of membrane receptors; MCRs trigger cellular responses through adenylyl cyclase activation (see Chapters 11 and 14).

Ghrelin and Cholecystokinin Are Short-Term Regulators of Eating Behaviour Short-term regulators of eating include ghrelin and cholecystokinin. Ghrelin is an appetitestimulating peptide hormone produced in the stomach. Production of ghrelin is maximal NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Neuron Hypothalamus

Food intake

Energy expenditure

" Melanocortinproducing neuron

NPY/ AgRP

"

!

Melanocortin receptor (MC4R) (blocked by AgRP) Ghrelin receptor

"

Ghrelin

!

NPY/PYY3-36 receptor Y2R Melanocortin receptor (MC3R) NPY receptor Y1R

PYY3-36 Stomach

Arcuate nucleus

Insulin Leptin

Leptin receptor or insulin receptor Pancreas

Intestines Fat tissue

FIGURE 33.13 The regulatory pathways that control eating. (Adapted by permission from Macmillan Publishers. Schwartz, M. W., and Morton, G. J., 2002. Obesity: Keeping hunger at bay. Nature 418:595–597.)

when the stomach is empty, but ghrelin levels fall quickly once food is consumed. Cholecystokinin is a peptide hormone released from the gastrointestinal tract during eating. In contrast to ghrelin, cholecystokinin signals satiety (the sense of fullness) and tends to curtail further eating. Together, ghrelin and cholecystokinin constitute a meal-tomeal control system that regulates the onset and end of eating behaviour. The activity of this control system is also modulated by the long-term regulators.

HUMAN BIOCHEMISTRY

The Metabolic Effects of Alcohol Consumption Ethanol metabolism alters the NAD+/NADH ratio. Ethanol is metabolized to acetate in the liver by alcohol dehydrogenase and aldehyde dehydrogenase: CH3CH2OH + NAD+ N CH3CHO + NADH + H+ CH3CHO + NAD+ + H2O N CH3COO- + NADH + 2H+. Acetate is then converted to acetyl-CoA. Excessive conversion of available NAD+ to NADH impairs NAD+-requiring reactions, such as the citric acid cycle, gluconeogenesis, and fatty acid oxidation. Accumulation of acetyl-CoA favours fatty acid synthesis, which,

along with blockage of fatty acid oxidation, causes elevated triacylglycerol levels in the liver. Over time, these triacylglycerols accumulate as fatty deposits, which ultimately contribute to cirrhosis of the liver. Impairment of gluconeogenesis leads to buildup of this pathway’s substrate, lactate. Lactic acid accumulation in the blood causes acidosis. A further consequence is that acetaldehyde can form adducts with protein i NH2 groups, which may inhibit protein function. Because gluconeogenesis is limited, alcohol consumption can cause hypoglycemia (low blood sugar) in someone who is undernourished. In turn, hypoglycemia can cause irreversible damage to the central nervous system.

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Insulin and Leptin Are Long-Term Regulators of Eating Behaviour Long-term regulators include insulin and leptin, both of which inhibit eating and promote energy expenditure. Insulin is produced in the b-cells of the pancreas when blood glucose levels rise. A major role of insulin is to stimulate glucose uptake from the blood into muscle, fat, and other tissues. Blood insulin levels correlate with body fat amounts. Insulin also stimulates fat cells to make leptin. Leptin (from the Greek word lepto, meaning “thin”) is a 16 kD, 146–amino acid residue protein produced principally in adipocytes (fat cells). Leptin has a 4-helix bundle tertiary structure similar to that of cytokines (protein hormones involved in cell–cell communication). Normally, as fat deposits accumulate in adipocytes, more and more leptin is produced in these cells and spewed into the bloodstream. Leptin levels in the blood communicate the status of triacylglycerol levels in the adipocytes to the central nervous system so that appropriate changes in appetite take place. If leptin levels are low (“starvation”), appetite increases; if leptin levels are high (“overfeeding”), appetite is suppressed. Leptin also regulates fat metabolism in adipocytes, inhibiting fatty acid biosynthesis and stimulating fat metabolism. In the latter case, leptin induces synthesis of the enzymes in the fatty acid oxidation pathway and increases expression of uncoupling protein 2 (UCP2), a mitochondrial protein that uncouples oxidation from phosphorylation so that the energy of oxidation is lost as heat (thermogenesis). Leptin binding to leptin receptors in the hypothalamus inhibits release of NPY. Because NPY is a potent orexic (appetite stimulating) peptide hormone, leptin is therefore an anorexic (appetite suppressing) agent. Functional leptin receptors are also essential for pituitary function, growth hormone secretion, and normal puberty. When body fat stores decline, the circulating levels of leptin and insulin also decline. Hypothalamic neurons sense this decline and act to increase appetite to restore body fat levels. Intermediate regulation of eating behaviour is accomplished by the gut hormone PYY3-36. PYY3-36 is produced in endocrine cells found in distal regions of the small intestine, areas that receive ingested food some time after a meal is eaten. PYY3-36 inhibits eating for many hours after a meal by acting on the NPY/AgRP-producing neurons in the arcuate nucleus. Clearly, the regulatory controls that govern eating are complex and layered. Some believe that defects in these controls are common and biased in favour of overeating, an advantageous evolutionary strategy that may have unforeseen consequences in these bountiful times.

AMPK Mediates Many of the Hypothalamic Responses to These Hormones The actions of leptin, ghrelin, and NPY converge at AMPK. Leptin inhibits AMPK activity in the arcuate nucleus, and this inhibition underlies the anorexic effects of leptin. Leptin action on AMPK depends on a particular melanocortin receptor type known as the melanocortin-4 receptor (MC4R). On the other hand, ghrelin and NPY activate hypothalamic AMPK, which stimulates food intake and leads, over time, to increased body weight. The effects of AMPK in the hypothalamus that lead to alterations in eating behaviour may be mediated through changes in malonyl-CoA levels. Low [malonyl-CoA] in hypothalamic neurons is associated with increased food intake, and elevated malonyl-CoA levels are associated with suppression of eating. The inhibition of acetyl-CoA carboxylase (and thus, malonyl-CoA synthesis) as a result of phosphorylation by AMPK plays an important part in the regulation of our eating behaviour.

33.7

Can You Really Live Longer by Eating Less?

Caloric Restriction Leads to Longevity Nutritional studies published in 1935 showed that rats fed a low-calorie, but balanced and nutritious diet lived nearly twice as long as rats with unlimited access to food (2.4 years versus 1.3 years). Subsequent research over the ensuing 70 years has shown that the relationship between diet and longevity is a general one for organisms from yeast to mammals. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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To achieve this effect of caloric restriction (CR), animals are given a level of food that amounts to 60% to 70% of what they would eat if they were allowed free access to food. In animals, CR results in lower blood glucose levels, declines in glycogen and fat stores, enhanced responsiveness to insulin, lower body temperature, and diminished reproductive capacity. The extended lifespan given by CR offers a definite evolutionary advantage: Any animal that could slow the aging process and postpone reproduction in times of food scarcity and then resume reproduction when food became available would out-compete animals without such ability. Another remarkable feature of CR is that it diminishes the likelihood for development of many age-related diseases, such as cancer, diabetes, and atherosclerosis. Is this benefit simply the result of lowered caloric intake, or does CR lead to significant regulatory changes that affect many aspects of an organism’s physiology? The answer to this and other questions is emerging from vigorous research efforts toward understanding CR.

Mutations in the SIR2 Gene Decrease Life Span Many important clues came from genetic investigations. Deletion of a gene termed SIR2 (for silent information regulator 2) abolished the ability of CR to lengthen lifespan in yeast and roundworms, implicating the SIR2 gene product in longevity. SIR2 was discovered through its ability to silence the transcription of genes that encode rRNA. SIR2related genes are found in some prokaryotes and virtually all eukaryotes, including yeast, nematodes, fruit flies, and humans. The human gene is designated SIRT1, for sirtuin 1; sirtuin is the generic name for proteins encoded by SIR2 genes. Sirtuins are NAD+-dependent protein deacetylases (Figure 33.14). Cleavage of acetyl groups from proteins is an exergonic reaction, as is cleavage of the N-glycosidic bond in NAD+ (!G "= = - 34 kJ/mol). Thus, involvement of NAD+ in the reaction is not a thermodynamic necessity. However, NAD+ involvement does couple the reaction to an important signal of metabolic status, namely, the NAD+/NADH ratio. Furthermore, both nicotinamide and NADH are potent inhibitors of the deacetylase reaction. Thus, the NAD+/NADH ratio controls sirtuin protein deacetylase activity, so oxidative metabolism, which drives conversion of NADH to NAD+, enhances sirtuin activity. One adaptive response found with CR is increased mitochondrial biogenesis in liver, fat, and muscle, which is a response that would raise the NAD+/NADH ratio. Sirtuin-catalyzed removal of acetyl groups from lysine residues of histones, the core proteins of nucleosomes, allows the nucleosomes to interact more strongly with DNA,

FIGURE 33.14 The NAD+-dependent protein deacetylase reaction of sirtuins. Acetylated peptides include N-acetyl lysine side chains of histone H3 and H4 and acetylated p53 (p53 is the protein product of the p53 tumour suppressor gene).

O C Peptide NH H3C

C

+

NH2 NH2

+ N O–

O

O

Acetyl-peptide

H2C

O

P

N

O– O

P

O

O

O

N

N

O NAD+

HO OH

CH2

N

HO OH NH2 O–

O Peptide NH2 Deacylatedpeptide

+

C

HO NH2

+ N Nicotinamide

O

+ O

H2C

O

P O

C

O OH

N

O– O

P

O

CH2

O

N

N N

O

2#-O-acetyl-ADPribose

HO OH

CH3 NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Part 5 Information Transfer

making transcription more difficult (see Chapter 29 for a discussion of the relationship between histone acetylation and transcriptional activity of genes).

SIRT1 Is a Key Regulator in Caloric Restriction As a key regulator in CR, the human sirtuin protein SIRT1 connects nutrient availability to the expression of metabolic genes. A striking feature of CR is the loss of fat stores and reduction in white adipose tissue. SIRT1 participates in the transcriptional regulation of adipogenesis through interaction with PPARG (peroxisome proliferator-activator receptorg), a nuclear hormone receptor that activates transcription of genes involved in adipogenesis and fat storage. SIRT1 binding to PPARg represses transcription of these genes, leading to loss of fat stores. Because adipose tissue functions as an endocrine organ, this loss of fat has significant hormonal consequences for energy metabolism. In liver, SIRT1 interacts with and deacetylates PGC-1 (peroxisome proliferatoractivator receptor-g coactivator-1), a transcriptional regulator of genes involved in glucose production. Thus, CR leads to increased transcription of the genes encoding the enzymes of gluconeogenesis and repression of genes encoding glycolytic enzymes. Acting in these roles, SIRT1 connects nutrient availability to the regulation of major pathways of energy storage (glycogen and fat) and fuel utilization.

Resveratrol, a Compound Found in Red Wine, Is a Potent Activator of Sirtuin Activity

OH HO

OH Resveratrol (trans-3,4#,5-trihydroxystilbene) FIGURE 33.15 Resveratrol, a phytoalexin, is a member of the polyphenol class of natural products. As a polyphenol, resveratrol is a good free-radical scavenger, which may account for its cancer preventative properties.

Resveratrol (trans-3,4#,5-trihydroxystilbene; Figure 33.15) is a phytoalexin. Phytoalexins are compounds produced by plants in response to stress, injury, or fungal infection. Resveratrol is abundant in wine grape skins as a result of common environmental stresses, such as infection by Botrytis cinerea, a fungus important in making certain wines. Because the skins are retained when grapes are processed to make red wines, red wine is an excellent source of resveratrol. Resveratrol might be the basis of the French paradox—the fact that the French people enjoy longevity and relative freedom from heart disease despite a highfat diet. When resveratrol is given to yeast cultures, roundworms (Caenorhabditis elegans), or fruit flies (Drosophila melanogaster), it has the same life-extending effects as CR. Resveratrol increased the replicative lifespan (the number of times a cell can divide before dying) of yeast by 70%. Resveratrol activates SIRT1 NAD+-dependent deacetylase activity. Furthermore, resveratrol activates AMPK in the brain and in cultured neurons. Because AMPK is a key energy sensor, resveratrol’s influences on longevity may arise through its effects on caloric homeostasis. Because the effects of CR and resveratrol on longevity are not additive, a reasonable conclusion is that they operate via a common mechanism. If this is so, then if you want to enjoy longevity, the appropriate advice would be “Eat less or drink red wine,” not “Eat less and drink red wine”!

SUMMARY 33.1 Can Systems Analysis Simplify the Complexity of Metabolism? Cells are in a dynamic steady state maintained by considerable metabolic flux. The metabolism going on in even a single cell is so complex that it defies meaningful quantitative description. Nevertheless, overall relationships become more obvious by a systems analysis approach to intermediary metabolism. The metabolism of a typical heterotrophic cell can be summarized in 3 interconnected functional blocks: (1) catabolism, (2) anabolism, and (3) macromolecular synthesis and growth. Phototrophic cells require a fourth block: photosynthesis. Only a few metabolic intermediates connect these systems,

and ATP and NADPH serve as the carriers of chemical energy and reducing power, respectively, between these various blocks. 33.2 What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism? ATP coupling determines the thermodynamics of metabolic sequences. The ATP coupling stoichiometry cannot be predicted from chemical considerations; instead it is a quantity selected by evolution and the fundamental need for metabolic sequences to be emphatically favourable from a thermodynamic perspective. Catabolic sequences generate ATP with an overall favourable Keq (and hence, a negative !G), and anabolic NEL

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Chapter 33

sequences consume this energy with an overall favourable Keq, even though such sequences may span the same starting and end points (as in fatty acid oxidation of palmitoyl-CoA to 8 acetyl-CoA versus synthesis of palmitoyl-CoA from 8 acetyl-CoA). ATP has 2 metabolic roles: a stoichiometric role in rendering metabolic sequences thermodynamically favourable and a regulatory role as an allosteric effector. 33.3 Is There a Good Index of Cellular Energy Status? The level of phosphoric anhydride bonds in the adenylate system of ATP, ADP, and AMP can be expressed in terms of the energy charge equation: Energy charge =

2 [ATP] + [ADP] 1 ¢ ≤ 2 [ATP] + [ADP] + [AMP]

More revealing of the potential for an ATP-dependent reaction to occur is ≠, the phosphorylation potential: ≠ = [ATP]/([ADP][Pi]). 33.4 How Is Overall Energy Balance Regulated in Cells? AMPactivated protein kinase (AMPK) is the cellular energy sensor. When cellular energy levels are high, as signalled by high ATP concentrations, AMPK is inactive. When cellular energy levels are depleted, as signalled by high [AMP], AMPK is activated, both allosterically and by phosphorylation at Thr172, and phosphorylates many enzymes involved in cellular energy production and consumption. Competition between AMP and ATP for binding to the AMPK allosteric sites determines AMPK activity. AMPK activation leads to elevated energy production and diminished energy consumption. AMPK is an abg-heterotrimer. The a-subunit is the catalytic subunit. AMP or ATP binding to the g-subunit determines AMPK activity, with AMP binding increasing activity by more than 1000-fold. Activation of AMPK leads to phosphorylation of key enzymes in energy metabolism, activating those involved in ATP production and inactivating those in ATP consumption. In addition, AMPK phosphorylation of various transcription factors leads to elevated expression of catabolic genes and diminished expression of genes encoding biosynthetic enzymes. Beyond these cellular effects, AMPK plays a central role in energy balance in multicellular organisms, because its activity is responsive to hormones that govern eating behaviour and energy homeostasis. 33.5 How Is Metabolism Integrated in a Multicellular Organism? Organ systems in complex multicellular organisms carry out specific physiological functions, with each expressing those metabolic pathways appropriate to its physiological purpose. Essentially all cells in animals carry out the central pathways of intermediary metabolism, especially the reactions of ATP synthesis. Nevertheless, organs differ in the metabolic fuels they prefer as substrates for energy production. The major fuel depots in animals are glycogen in liver and muscle; triacylglycerols (fats) in adipose tissue; and protein, most of which is in skeletal muscle. The order of preference for the use of these fuels is glycogen 7 triacylglycerol 7 protein. Nevertheless, the tissues of the body work together to maintain caloric homeostasis: constant availability of fuels in the blood. The major organ systems have specialized metabolic roles within the organism. The brain has a strong reliance on glucose as fuel. Muscle at rest primarily relies on fatty acids, but under conditions of strenuous contraction when O2 is limiting, muscle shifts to glycogen as its primary fuel. The heart is a completely aerobic organ, rich in mitochondria, with a preference for fatty acids as fuel under normal operating conditions. The liver is the body’s metabolic processing centre, taking in nutrients and sending out products such as glucose, fatty acids, and ketone bodies. Adipose tissue takes up glucose and, to a lesser extent, fatty acids for the synthesis and storage of triacylglycerols. 33.6 What Regulates Our Eating Behaviour? Appetite and weight regulation are governed by hormones produced in the stomach, small intestines, pancreas, adipose tissue, and central nervous system. These

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hormones act on neurons within the arcuate nucleus region of the hypothalamus that control pathways involved in eating (food intake) and energy expenditure. Hormones that regulate eating behaviour can be divided into short-term regulators that determine individual meals and long-term regulators that act to stabilize the levels of body fat deposits. Short-term regulators of eating include ghrelin and cholecystokinin. Ghrelin is produced when the stomach is empty, but ghrelin levels fall quickly once food is consumed. Cholecystokinin is released from the gastrointestinal tract during eating. In contrast to ghrelin, cholecystokinin signals satiety (the sense of fullness) and tends to curtail further eating. Together, ghrelin and cholecystokinin constitute a meal-to-meal control system that regulates the onset and end of eating behaviour. Long-term regulators include insulin and leptin, both of which inhibit eating and promote energy expenditure. Blood insulin levels correlate with body fat amounts. Insulin also stimulates fat cells to make leptin. Leptin is produced principally in adipocytes. As fat accumulates in adipocytes, more leptin is released into the bloodstream to communicate the status of adipocyte fat to the central nervous system. If leptin levels are low (“starvation”), appetite increases; if leptin levels are high (“overfeeding”), appetite is suppressed. Leptin binding to its receptors in the hypothalamus inhibits release of NPY. NPY is a potent orexic hormone; therefore, leptin is an anorexic agent. When body fat stores decline, the circulating levels of leptin and insulin also decline. Hypothalamic neurons sense this decline and act to increase appetite to restore body fat levels. Intermediate regulation of eating behaviour is accomplished by the gut hormone PYY3–36. Produced in distal regions of the intestines, PYY3–36 delays eating for many hours after a meal by inhibiting the NPY/AgRP-producing neurons in the arcuate nucleus. The regulatory controls that govern eating are complex and layered. 33.7 Can You Really Live Longer by Eating Less? Caloric restriction (CR) prolongs the longevity of organisms from yeast to mammals. CR results in lower blood glucose levels, decline in glycogen and fat stores, enhanced responsiveness to insulin, lower body temperature, and diminished reproductive capacity. CR also diminishes the likelihood for development of many age-related diseases, such as cancer, diabetes, and atherosclerosis. Genetic investigations revealed that mutations in the SIR2 gene abolish the extension of life span by CR. The human SIR2 gene equivalent is SIRT1. SIRT genes encode sirtuins, a family of NAD+-dependent protein deacetylases. The NAD+/NADH ratio controls sirtuin protein deacetylase activity, so oxidative metabolism, which drives conversion of NADH to NAD+, enhances sirtuin action. CR increases mitochondrial biogenesis in liver, fat, and muscle, a response that would raise the NAD+/NADH ratio. SIRT1 is a key regulator in CR. The physiological responses caused by CR are the result of a tightly regulated program that connects nutrient availability to the expression of metabolic genes. A striking feature of CR is the loss of fat stores and reduction in white adipose tissue. SIRT1 binding to PPARg, a nuclear hormone receptor that activates transcription of genes involved in adipogenesis and fat storage, represses transcription of these genes, leading to the loss of fat stores. Because adipose tissue functions as an endocrine organ, this loss of fat has significant hormonal consequences for energy metabolism. In liver, SIRT1 interacts with and deacetylates PGC-1, a transcriptional regulator of glucose production. Transcription of genes encoding the enzymes of gluconeogenesis and repression of genes encoding glycolytic enzymes are increased upon PGC-1 deacetylation. Thus, SIRT1 connects nutrient availability to the regulation of major pathways of energy storage and fuel use. Resveratrol, a phytoalexin, has the same life-extending effects as CR. Resveratrol activates both SIRT1 activity and brain AMPK, a key energy sensor. The influences of resveratrol on longevity may arise through its effects on caloric homeostasis.

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PROBLEMS Log in to OWL to develop problem-solving skills and complete online homework assigned by your professor.

1. (Integrates with Chapters 3, 17, and 21) The conversion of PEP to pyruvate by pyruvate kinase (glycolysis) and the reverse reaction to form PEP from pyruvate by pyruvate carboxylase and PEP carboxykinase (gluconeogenesis) represent a so-called substrate cycle. The direction of net conversion is determined by the relative concentrations of allosteric regulators that exert kinetic control over pyruvate kinase, pyruvate carboxylase, and PEP carboxykinase. Recall that the last step in glycolysis is catalyzed by pyruvate kinase: PEP + ADP m pyruvate + ATP. The standard free energy change is -31.7 kJ/mol. a. Calculate the equilibrium constant for this reaction. b. If [ATP] = [ADP], by what factor must [pyruvate] exceed [PEP] for this reaction to proceed in the reverse direction? The reversal of this reaction in eukaryotic cells is essential to gluconeogenesis and proceeds in 2 steps, each requiring an equivalent of nucleoside triphosphate energy: pyruvate carboxylase pyruvate + CO2 + ATP h oxaloacetate + ADP + Pi PEP carboxykinase oxaloacetate + GTP h PEP + CO2 + GDP Net: pyruvate + ATP + GTP h PEP + ADP + GDP + Pi c. The !G "= for the overall reaction is +0.8 kJ/mol. What is the value of Keq? d. Assuming [ATP] = [ADP], [GTP] = [GDP], and Pi = 1 mM when this reaction reaches equilibrium, what is the ratio of [PEP]/ [pyruvate]? e. Are both directions in the substrate cycle likely to be strongly favoured under physiological conditions? 2. (Integrates with Chapter 3) Assume the following intracellular concentrations in muscle tissue: ATP = 8 mM, ADP = 0.9 mM, AMP = 0.04 mM, Pi = 8 mM. What is the energy charge in muscle? What is the phosphorylation potential? 3. Strenuous muscle exertion (as in the 100 metre dash) rapidly depletes ATP levels. How long will 8 mM ATP last if 1 gram of muscle consumes 300 mmol of ATP per minute? (Assume muscle is 70% water.) Muscle contains phosphocreatine as a reserve of phosphorylation potential. Assuming [phosphocreatine] = 40 mM, [creatine] = 4 mM, and !G "= (phosphocreatine + H2O m creatine + Pi) = -43.3 kJ/mol, how low must [ATP] become before it can be replenished by the reaction: phosphocreatine + ADP m ATP + creatine? [Remember, !G "= (ATP hydrolysis) = -30.5 kJ/mol.] 4. (Integrates with Chapter 19) The standard reduction potentials for the (NAD+/NADH) and (NADP+/NADPH) couples are identical, namely, -320 mV. Assuming the in vivo concentration ratios NAD+/NADH = 20 and NADP+/NADPH = 0.1, what is !G for the following reaction? NADPH + NAD+ m NADP+ + NADH Assuming standard state conditions for the reaction, ADP + Pi S ATP + H2O, calculate how many ATP equivalents can be formed from ADP + Pi by the energy released in this reaction. 5. (Integrates with Chapter 3) Assume the total intracellular pool of adenylates (ATP + ADP + AMP) = 8 mM, 90% of which is ATP. What are [ADP] and [AMP] if the adenylate kinase reaction is at equilibrium? Suppose [ATP] drops suddenly by 10%. What are the

concentrations now for ADP and AMP, assuming that the adenylate kinase reaction is at equilibrium? By what factor has the AMP concentration changed? 6. (Integrates with Chapters 17 and 21) The reactions catalyzed by PFK and FBPase constitute another substrate cycle. PFK is AMP activated; FBPase is AMP inhibited. In muscle, the maximal activity of PFK (mmol of substrate transformed per minute) is 10 times greater than FBPase activity. If the increase in [AMP] described in Problem 5 raised PFK activity from 10% to 90% of its maximal value but lowered FBPase activity from 90% to 10% of its maximal value, by what factor is the flux of fructose-6-P through the glycolytic pathway changed? (Hint: Let PFK maximal activity = 10, FBPase maximal activity = 1; calculate the relative activities of the 2 enzymes at low [AMP] and at high [AMP]; let J, the flux of F-6-P through the substrate cycle under any condition, equal the velocity of the PFK reaction minus the velocity of the FBPase reaction.) 7. (Integrates with Chapters 9 and 22) Leptin not only induces synthesis of fatty acid oxidation enzymes and uncoupling protein 2 in adipocytes, but it also causes inhibition of acetyl-CoA carboxylase, resulting in a decline in fatty acid biosynthesis. This effect on acetylCoA carboxylase, as an additional consequence, enhances fatty acid oxidation. Explain how leptin-induced inhibition of acetyl-CoA carboxylase might promote fatty acid oxidation. 8. (Integrates with Chapters 18 and 19) Acetate produced in ethanol metabolism can be transformed into acetyl-CoA by the acetyl thiokinase reaction acetate + ATP + CoASH h acetyl-CoA + AMP + PPi. Acetyl-CoA then can enter the citric acid cycle and undergo oxidation to 2 CO2. How many ATP equivalents can be generated in a liver cell from the oxidation of 1 molecule of ethanol to 2 CO2 by this route, assuming oxidative phosphorylation is part of the process? (Assume all reactions prior to acetyl-CoA entering the citric acid cycle occur outside the mitochondrion.) Per carbon atom, which is a better metabolic fuel, ethanol or glucose? That is, how many ATP equivalents per carbon atom are generated by combustion of glucose versus ethanol to CO2? 9. (Integrates with Chapter 22) Assume each NADH is worth 3 ATP, each FADH2 is worth 2 ATP, and each NADPH is worth 4 ATP. How many ATP equivalents are produced when 1 molecule of palmitoylCoA is oxidized to 8 molecules of acetyl-CoA by the fatty acid b-oxidation pathway? How many ATP equivalents are consumed when 8 molecules of acetyl-CoA are transformed into 1 molecule of palmitoyl-CoA by the fatty acid biosynthetic pathway? Can both of these metabolic sequences be metabolically favourable at the same time if !G for ATP synthesis is +50 kJ/mol? 10. (Integrates with Chapters 17–20) If each NADH is worth 3 ATP, each FADH2 is worth 2 ATP, and each NADPH is worth 4 ATP, calculate the equilibrium constant for cellular respiration, assuming synthesis of each ATP costs 50 kJ/mol of energy. Calculate the equilibrium constant for CO2 fixation under the same conditions, except here ATP will be hydrolyzed to ADP + Pi with the release of 50 kJ/mol. Comment on whether these reactions are thermodynamically favourable under such conditions. 11. (Integrates with Chapter 21) In type 2 diabetics, glucose production in the liver is not appropriately regulated, so glucose is overproduced. One strategy to treat this disease focuses on the development of drugs targeted against regulated steps in glycogenolysis and gluconeogenesis, the pathways by which liver produces glucose for release into the blood. Which enzymes would you select for as potential targets for such drugs? 12. As chief scientist for drug development at PhatFarmaceuticals, Inc., you want to create a series of new diet drugs. You have a NEL

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Chapter 33

grand plan to design drugs that might limit production of some hormones or promote the production of others. Which hormones are on your “limit production” list and which are on your “raise levels” list? 13. The existence of leptin was revealed when the ob/ob genetically obese strain of mice was discovered. These mice have a defective leptin gene. Predict the effects of daily leptin injections into ob/ob mice on food intake, fatty acid oxidation, and body weight. Similar clinical trials have been conducted on humans, with limited success. Suggest a reason why this therapy might not be a miracle cure for overweight individuals. 14. Would it be appropriate to call neuropeptide Y (NPY) the obesitypromoting hormone? What would be the phenotype of a mouse whose melanocortin-producing neurons failed to produce melanocortin? What would be the phenotype of a mouse lacking a functional MC3R gene? What would be the phenotype of a mouse lacking a functional leptin receptor gene? 15. The Human Biochemistry box, The Metabolic Effects of Alcohol Consumption, points out that ethanol is metabolized to acetate in the liver by alcohol dehydrogenase and aldehyde dehydrogenase: CH3CH2OH + NAD+ m CH3CHO + NADH + H+

CH3CHO + NAD+ + H2O m CH3COO- + NADH + 2H+ These reactions alter the NAD+/NADH ratio in liver cells. From your knowledge of glycolysis, gluconeogenesis, and fatty acid oxidation, what might be the effect of an altered NAD+/NADH ratio on these pathways? What is the basis of this effect?

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16. A T172D mutant of the AMPK is locked in a permanently active state. Explain. 17. a. Some scientists support the “malonyl-CoA hypothesis,” which suggests that malonyl-CoA is a key indicator of nutrient availability and the brain uses its abundance to assess whole-body energy homeostasis. Others have pointed out that malonyl-CoA is a significant inhibitor of carnitine acyltransferase-1 (see Figure 9.16). Thus, malonyl-CoA may be influencing the levels of another metabolite whose concentration is more important as a signal of energy status. What metabolite might that be? b. Another test of the malonyl-CoA hypothesis was conducted through the creation of a transgenic strain of mice that lacked functional hypothalamic fatty acid synthase (see Chapter 9). Predict the effect of this genetic modification on cellular malonylCoA levels in the hypothalamus, the eating behaviour of these transgenic mice, their body fat content, and their physical activity levels. Defend your predictions. 18. a. Leptin was discovered when a congenitally obese strain of mice (ob/ob mice) was found to lack both copies of a gene encoding a peptide hormone produced mainly by adipose tissue. The peptide hormone was named leptin. Leptin is an anorexic (appetite suppressing) agent; its absence leads to obesity. Propose an experiment to test these ideas. b. A second strain of obese mice (db/db mice) produces leptin in abundance but fails to respond to it. Assuming the db mutation leads to loss of function in a protein, what protein is likely to be non-functional or absent? How might you test your idea?

FURTHER READING Systems Analysis of Metabolism

Creatine as a Nutritional Supplement

Brand, M. D., and Curtis, R. K., 2002. Simplifying metabolic complexity. Biochemical Society Transactions 30:25–30.

Ekblom, B., 1996. Effects of creatine supplementation on performance. American Journal of Sports Medicine 24(6S):S38–S39.

Atkinson, D. F., 1977. Cellular Energy Metabolism and Its Regulation. New York: Academic Press.

Kreider, R., 1998. Creatine supplementation: Analysis of ergogenic value, medical safety, and concerns. Journal of Exercise Physiology 1: [an international online journal available at http://faculty.css.edu/tboone2/ asep/jan3.htm/]

Newsholme, E. A., and Leech, A. R., 1983. Biochemistry for the Medical Sciences. New York: John Wiley & Sons.

Fat-Free Mice

Newsholme, E. A., Challiss, R. A. J., et al, 1984. Substrate cycles: Their role in improving sensitivity in metabolic control. Trends in Biochemical Sciences 9:277–280.

Gavrilova, O., Marcus-Samuels, B., et al., 2000. Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice. Journal of Clinical Investigation 105:271–278.

AMP-Activated Protein Kinase

Moitra, J., Mason, M. M., et al., 1998. Life without white fat: A transgenic mouse. Genes and Development 12:3168–3181.

Hardie, D. G., 2007. AMP-activated/SNF 1 protein kinases: Conserved guardians of cellular energy. Nature Reviews Cell Molecular Biology 8:774–785.

Leptin and Hormonal Regulation of Eating Behaviour

ATP Coupling and the Thermodynamics of Metabolism

Hardie, D. G., Hawley, S. A., et al, 2007. AMP-activated protein kinase: Development of the energy sensor concept. Journal of Physiology 574:7–15. McGee, S. L., and Hargreaves, M., 2008. AMPK and transcriptional regulation. Frontiers in Bioscience 13:3022–3033. Metabolic Relationships between Organ Systems Harris, R., and Crabb, D. W., 1997. Metabolic interrelationships. In Textbook of Biochemistry with Clinical Correlations, 4th ed., Devlin, T. M., Ed. New York: Wiley-Liss. Sugden, M. C., Holness, M. J., et al, 1989. Fuel selection and carbon flux during the starved-to-fed transition. Biochemical Journal 263: 313–323.

Barinaga, M., 1995. “Obese” protein slims mice. Science 269:475–476, and references therein. Buettner, C., 2007. Does FASing out new fat in the hypothalamus make you slim? Cell Metabolism 6:249–251. Clement, K., Vaisse, C., et al., 1998. A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 392:398–401. Coll, A. P., Farooqi, S., et al., 2007. The hormonal control of food intake. Cell 129:251–262. Saper, C. B., Chou, T. C., et al., 2002. The need to feed: Homeostatic and hedonic control of eating. Neuron 36:199–211. Schwartz, M. W., and Morton, G. J., 2002. Obesity: Keeping hunger at bay. Nature 418:595–597.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

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Vaisse, C., Clement, K., et al., 2000. Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity. Journal of Clinical Investigation 106:253–262. Zhou, Y-T., Shimabukuro, M., et al., 1997. Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation. Proceedings of the National Academy of Sciences U.S.A. 94:6386–6390. Caloric Restriction and Longevity Dasgupta, B., and Milbrandt, J., 2007. Resveratrol stimulates AMP kinase activity in neurons. Proceedings of the National Academy of Science U.S.A. 104:7217–7222. Denu, J. M., 2005. The Sir2 family of protein deacetylases. Current Opinion in Chemical Biology 9:431–440.

Guarente, L., 2005. Caloric restriction and SIR2 genes—Towards a mechanism. Mechanisms of Aging and Development 126:923–928. Guarente, L., and Picard, F., 2005. Caloric restriction—The SIR2 connection. Cell 120:473–482. Michan, S., and Sinclair, D., 2007. Sirtuins in mammals: Insights into their biological function. Biochemical Journal 404:1–13. Milne, J. C., Lambert, P. D., et al., 2007. Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 450:712–716. Moynihan, K. A., and Imai, S.-I., 2006. Sirt1 as a key regulator orchestrating the response to caloric restriction. Drug Discovery Today: Disease Mechanisms 3:11–17.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Abbreviated Answers to Problems

For detailed answers to the end-of-chapter problems as well as additional problems to solve, see Student Solutions Manual, Study Guide and Problems Book by David Jemiolo and Steven Theg that accompanies this textbook.

CHAPTER 1 1. Because bacteria (compared with humans) have simple nutritional requirements, their cells obviously contain enzyme systems that allow them to convert rudimentary precursors (even inorganic substances such as NH4+, NO3-, N2, and CO2) into complex biomolecules—proteins, nucleic acids, polysaccharides, and complex lipids. On the other hand, animals have an assortment of different cell types designed for specific physiological functions; these cells possess a correspondingly greater repertoire of complex biomolecules to accomplish their intricate physiology. 2. Consult Figures 1.21 to 1.23 to confirm your answer. 3. a. Laid end to end, 250 E. coli cells would span the head of a pin. b. The volume of an E. coli cell is about 10-15 L. c. The surface area of an E. coli cell is about 6.3 * 10-12 m2. Its surface-to-volume ratio is 6.3 * 106 m-1. d. 600,000 molecules. e. 1.7 nM. f. Because we can calculate the volume of one ribosome to be 4.2 * 10-24 m3 (or 4.2 * 10-21 L), 15,000 ribosomes would occupy 6.3 * 10-17 L, or 6.3% of the total cell volume. g. Because the E. coli chromosome contains 4600 kilobase pairs (4.6 * 106 bp) of DNA, its total length would be 1.6 mm—approximately 800 times the length of an E. coli cell. This DNA would encode 4300 different proteins, each 360 amino acids long. 10-16 L

4. a. The volume of a single mitochondrion is about 4.2 * (about 40% the volume calculated for an E. coli cell in problem 3). b. A mitochondrion would contain on average fewer than eight molecules of oxaloacetate. 5. a. Laid end to end, 25 liver cells would span the head of a pin. b. The volume of a liver cell is about 8 * 10-12 L (8000 times the volume of an E. coli cell). c. The surface area of a liver cell is 2.4 * 10-9 m2; its surfaceto-volume ratio is 3 * 105 m-1, or about 0.05 (1/20) that of an E. coli cell. Cells with lower surface-to-volume ratios are limited in their exchange of materials with the environment. d. The number of base pairs in the DNA of a liver cell is 6 * 109 bp, which would amount to a total DNA length of 2 m (or 6 feet of DNA!) contained within a cell that is only

20 mm on a side. Maximal information content of liver-cell DNA = 3 * 109 bp, which, expressed in proteins 400 amino acids in length, could encode 2.5 * 106 proteins. 6. The amino acid side chains of proteins provide a range of shapes, polarity, and chemical features that allow a protein to be tailored to fit almost any possible molecular surface in a complementary way. 7. Biopolymers may be informational molecules because they are constructed of different monomeric units (“letters”) joined head to tail in a particular order (“words, sentences”). Polysaccharides are often linear polymers composed of only one (or two repeating) monosaccharide unit(s) and thus display little information content. Polysaccharides with a variety of monosaccharide units may convey information through specific recognition by other biomolecules. Also, most monosaccharide units are typically capable of forming branched polysaccharide structures that are potentially very rich in information content (as in cell surface molecules that act as the unique labels displayed by different cell types in multicellular organisms). 8. Molecular recognition is based on structural complementarity. If complementary interactions involved covalent bonds (strong forces), stable structures would be formed that would be less responsive to the continually changing dynamic interactions that characterize living processes. 9. Two carbon atoms interacting through van der Waals forces are 0.34 nm apart; two carbon atoms joined in a covalent bond are 0.154 nm apart. 10. Slight changes in temperature, pH, ionic concentrations, and so forth may be sufficient to disrupt weak forces (H bonds, ionic bonds, van der Waals interactions, hydrophobic interactions). 11. Living systems are maintained by a continuous flow of matter and energy through them. Despite the ongoing transformations of matter and energy by these highly organized, dynamic systems, no overt changes seem to occur in them: They are in a steady state. 12. The fraction of the M. genitalium genes encoding proteins = 0.925. Genes not encoding proteins encode RNA molecules. (0.925) (580,074 base pairs) = 536,820 base pairs devoted to protein-coding genes. Since 3 base pairs specify an amino acid in a protein, 369 amino acids are found in the average M. genitalium protein. If each amino acid contributes on average 120 D to the mass of a protein, the mass of an average M. genitalium protein is 44,280 D. 13. (0.925) (206) = 191 proteins. Assuming its genes are the same size as M. genitalium, the minimal genome would be 228,480 base pairs.

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

A-2

Abbreviated Answers to Problems

14. Given 1109 nucleotides (or base pairs) per gene, the minimal virus, with a 3500-nucleotide genome, would have only 3 genes; the maximal virus, with a 280,000-bp genome, would have 252 genes.

18. a.

10

15. Fate of proteins synthesized by the rough ER: a. Membrane proteins would enter the ER membrane, and, as part of the membrane, be passed to the Golgi, from which vesicles depart and fuse with the plasma membrane. b. A secreted protein would enter the ER lumen and be transferred as a luminal protein to the Golgi, from which vesicles depart. When the vesicle fuses with the plasma membrane, the protein would be deposited outside the cell.

pH

8

3. a.

pKa = 2.3

2

0

b. 9.85;

c. 5.7;

d. 12.5;

e. 4.4;

= 2.51 *

10-5 M;

b. Ka = 3.13 *

0.5

1.0 equivalents OH–

f. 6.97.

2. a. 1.26 mM; b. 0.25 mM; c. 4 * 10-12 M; d. 2 * 10-4 M; e. 3.16 * 10-10 M; f. 1.26 * 10-7 M (0.126 mM). [H+]

pKa = 8.3

6

4

CHAPTER 2 1. a. 3.3;

12

10-8 M;

b.

5. Combine 187 mL of 0.1 M acetic acid with 813 mL of 0.1 M sodium acetate. 6. [HPO4-2]>[H2PO4 -] = 0.398. 7. Combine 555.7 mL of 0.1 M Na3PO4 with 444.3 mL of 0.1 M H3PO4. Final concentrations of ions will be [H2PO4-] = 0.0333 M; [HPO42-] = 0.0667 M; [Na+] = 0.1667 M; [H+] = 3.16 * 10-8 M. 8. Add 432 mL of 0.1 N MCI to 1 L 0.05 M BICINE. [BICINE]total = 0.05 M/1.432 L = 0.0349 M [protonated form] = 0.0302 M.

NCH2COO– CH2CH2OH

c. The relevant pKa for this calculation is 8.3. Combine 400 mL of 0.1 M bicine at its pHI (pH 5.3) with 155 mL of 0.1 M NaOH and 345 mL of water. d. The relevant pKa for this calculation is 2.3. The concentration of fully protonated form of bicine at pH 7.5 is 2.18 * 10-7 M. 19. a. 0.063 M 8

b.

6

pH

9. a. Fraction of H3PO4: @pH0 = 0.993; @pH 2 = 0.58; @pH 4 = 0.01; negligible @pH 6. b. Fraction of H2PO4-: @pH0 = 0.007; @pH 2 = 0.41; @pH 4 = 0.986; @pH 6 = 0.94; @pH 8 = 0.14; negligible @pH 10. c. Fraction of HPO42-: negligible @pH 0, 2, and 4; @pH 6 = 0.06; @pH 8 = 0.86; @pH 10 ! 1.0; @pH 12 = 0.72. d. Fraction of PO43-: negligible at any pH 6 10; @pH 12 = 0.28. 10. At pH 5.2, [H3A] = 4.33 * 10-5 M; [H2A-] = 0.0051 M; [HA2-] = 0.014 M; [A3 - ] = 0.0009 M.

pKa = 4.7

4

2

0

0.2

11. a. pH = 7.02; [H2PO4-] = 0.0200 M; [HPO42 - ] = 0.0133 M. b. pH = 7.38; [H2PO4-] = 0.0133 M; [HPO42 - ] = 0.0200 M.

20. 1.53 nmol/mL • sec

12. [H2CO3] = 2.2 mM; [CO2(d)] = 0.75 mM. When [HCO3-] = 15 mM and [CO2(d)] = 3 mM, pH = 6.8.

22. c.

13. Titration of the fully protonated form of anserine will require the addition of three equivalents of OH-. The pKa values lie at 2.64 (COOH); 7.04 (imidazole-N+H); and 9.49 (NH3+). Its isoelectric point lies midway between pK2 and pK3, so pHI = 8.265. To prepare 1 L of 0.04 M anserine buffer, add 164 mL of 0.1 M HCl to 400 mL of 0.1 M anserine at its isoelectric point, and make up to 1 L final volume. 14. Add 410 mL of 0.1 M NaOH to 250 mL of 0.1 M HEPES in its fully protonated form and make up to 1 L final volume. 15. 166.7 g/mole. 16. Add 193 mL of water and 307 mL of 0.1 M HCl to 500 mL of 0.1 M triethanolamine. 17. Combine 200 mL of 0.1 M Tris-H+ with 732 mL water and 68 mL of 0.1 M NaOH.

2.0

CH2CH2OH

pKa = 7.5.

4. a. pH = 2.38; b. pH = 4.23.

1.5

0.4 0.6 equivalents OH–

0.8

1.0

21. A drop in blood pH would occur.

CHAPTER 3 1. Keq = 613 M; !G " = -15.9 kJ/mol. 2. !G " = 1.69 kJ/mol at 20"C; !G " = - 5.80 kJ/mol at 30°C. !S " = 0.75 kJ/mol # K. 3. !G = -24.8 kJ/mol. 4. State functions are quantities that depend on the state of the system and not on the path or process taken to reach that state. Volume, pressure, and temperature are state functions. Heat and all forms of work, such as mechanical work and electrical work, are not state functions. 5. !G "= = !G " - 39.5 n (in kJ/mol), where n is the number of H+ produced in any process. So !G " = !G "= + 39.5 n = -30.5 kJ/mol + 39.5(1) kJ/mol. !G " = 9.0 kJ/mol at 1 M [H+]. NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Abbreviated Answers to Problems

6. a. Keq(AC) = (0.02 * 1000) = 20. b.

= 10.1 kJ/mol. !G"(BC) = -17.8 kJ/mol. !G"(AC) = - 7.7 kJ/mol. Keq = 20. !G"(AB)

7. See The Student Solutions Manual, Study Guide and Problems Book for resonance structures. 8. Keq = [Cr][Pi]/[CrP][H2O]. Keq = 3.89 * 107.

9. CrP in the amount of 135.3 moles would be required per day to provide 5860 kJ energy. This corresponds to 17,730 g of CrP per day. With a body content of 20 g CrP, each molecule would recycle 886 times per day. Similarly, 637 moles of glycerol-3-P, or 108,300 g of glycerol-3-P, would be required. Each molecule would recycle 5410 times/day. 10. !G = -46.1 kJ/mol. 11. The hexokinase reaction is a sum of the reactions for hydrolysis of ATP and phosphorylation of glucose: ATP + H2O ∆ ADP + Pi Glucose + Pi ∆ G-6-P + H2O Glucose + ATP ∆ G-6-P + ADP The free energy change for the hexokinase reaction can thus be obtained by summing the free energy changes for the first two reactions listed here.

pH-dependent, because the enolase reaction (see Figure 3.11) neither consumes nor produces protons. !G " = !G "= = 1.8 kJ/mol. 18. The magnitude of !G "= for ATP hydrolysis is sufficiently great that it provides ample energy to drive the conversion of A to B, even though the reaction is unfavorable. The result is that the equilibrium ratio of B to A is more than 108 greater when the reaction is coupled to ATP hydrolysis. If A S B were coupled instead to 1,3-bisphosphoglycerate hydrolysis, the ratio of B to A would be even greater, since the !G "= for hydrolysis of 1,3-bisphosphoglycerate is substantially greater than that of ATP. Using the concentrations for 1,3-BPG and 3-PG in Table 17.2, and repeating the calculations on page 72 yields a ratio of [Beq]/[Aeq] = 4.14 * 109, an even greater ratio than that calculated on page 72. 19. This exercise is left to the student. Use Figure 3.8 as a guide. 20. Without pyrophosphate cleavage, the acyl-CoA synthetase reaction is only slightly favorable, with a !G "= of 0.8 kJ/mol. With pyrophosphate cleavage included, the net !G "= for the reaction is #33.6 kJ/mol, a far more favorable value. CHAPTER 4 1. Structures for glycine, aspartate, leucine, isoleucine, methionine, and threonine are presented in Figure 4.3. 2. Asparagine $ Asn $ N. Arginine $ Arg $ R. Cysteine $ Cys $ C. Lysine $ Lys $ K.

!G "= for hexokinase = -30.5 kJ/mol + 13.9 kJ/mol = -16.6 kj/mol 12. Comparing the acetyl group of acetoacetyl-CoA and the methyl group of acetyl-CoA, it is reasonable to suggest that the acetyl group is more electron-withdrawing in nature. For this reason, it tends to destabilize the thiol ester of acetoacetylCoA, and the free energy of hydrolysis of acetoacetyl-CoA should be somewhat larger than that of acetyl-CoA. In fact, !G "= = - 43.9 kJ/mol, compared with #31.5 kJ/mol for acetyl-CoA.

A-3

Proline $ Pro $ P. Tyrosine $ Tyr $ Y. Tryptophan $ Trp $ W. 3.

13. Carbamoyl phosphate should have a somewhat larger free energy of hydrolysis than acetyl phosphate, at least in part because of greater opportunities for resonance stabilization in the products. In fact, the free energy of hydrolysis of carbamoyl phosphate is #51.5 kJ/mol, compared with #43.3 kJ/mol for acetyl phosphate. 14. The denaturation of chymotrypsinogen is spontaneous at 58°C, because the !G " at this temperature is negative (at approximately #2.8 kJ/mol). The native and denatured forms are in equilibrium at approximately 56.6°C. 15. The positive values for !Cp for the protein denaturations described in Table 3.1 reflect an increase in motion of the peptide chain in the denatured state. This increased motion provides new ways to store heat energy. 16. Keq = 2.04 * 10-2 at 298K; Keq = 9 at 320 K. !H "= = 219 kJ/mol !G "= = 9.64 kJ/mol !S "= = 702 J/mol # K

17. The value of !G " is determined at standard state, which includes 1M [H+]. Using Equation 3.23, a value for !G " of #3.36 kJ/mol can be calculated. This value applies at pH 2, 7, and 12 because, of course, !G " is pH-independent. However, Equation 3.23 can also be used to determine that !G "= is #14.77 kJ/mol at pH 2 and #71.82 kJ/mol at pH 12. Finally, !G "= for enolase is not NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

A-4

Abbreviated Answers to Problems

4. The proximity of the a-carboxyl group lowers the pKa of the a-amino group. 5. Equivalence point

6. Denoting the four histidine species as His2+, His+, His0, and His-, the concentrations are: pH 2: [His2+] = 0.097 M , [His+] = 0.153 M , [His0] = 1.53 * 10-5 M , [His-] = 9.6 * 10-13 M .

13 12

pH 6.4: [His2+] = 1.78 * 10-4 M , [His+] = 0.071 M , [His0] = 0.179 M , [His-] = 2.8 * 10-4 M .

11

%-NH3&

10

pK a $ 9.8

pH 9.3: [His2+] = 1.75 * 10-12 M , [His+] - 5.5 * 10-5 M , [His0] = 0.111 M , [His-] = 0.139 M .

9

pH

8

7. pH = pKa + log (2/1) = 4.3 + 0.3 = 4.6.

7

The g-carboxyl group of glutamic acid is 2/3 dissociated at pH = 4.6.

6 5 4

8. pH = pKa + log (1/4) = 10.5 + (-0.6) = 9.9.

R group pK a $ 3.9

3

9. a. The pH of a 0.3 M leucine hydrochloride solution is approximately 1.46. b. The pH of a 0.3 M sodium leucinate solution is approximately 11.5. c. The pH of a 0.3 M solution of isoelectric leucine is approximately 6.05.

%-COOH pK a $ 2.1

2 1 0

0

1

2 Equivalents

3

10. pI = (2 + 8.4)/2 = 5.2 11. pI = (2 + 4 + 9 + 12.5)/4 = approximately 6.9 12. The sequence of reactions shown would demonstrate that L(–)-serine is related stereochemically to L(–)-glyceraldehyde: COOH H2N

H

COOH H 2N

CH2OH L-(#)-Serine

CH3 L-(#)-Alanine

CHO HO

H

H

HO

H CH2OH acid

L-Glyceric

Br

CH3 2-Bromopropanoic acid

COOH

CH2OH L-(#)-Glyceraldehyde

H

COOH

COOH HO

H

CH3 2-Hydroxypropanoic acid

Straight arrows indicate reactions that occur with retention of configuration. Looped arrows indicate inversion of configuration. (From Kopple, K. D., 1966. Peptides and Amino Acids. New York: Benjamin Co.) 13. Cystine (disulfide-linked cysteine) has two chiral carbons, the two a-carbons of the cysteine moieties. Each chiral center can exist in two forms, so there are four stereoisomers of cystine.

14.

However, it is impossible to distinguish the difference between L-cysteine/D-cysteine and D-cysteine/L-cysteine conjugates. So three distinct isomers are formed:

15. There are eight Tyr residues in the protein. 16. A water is removed from each amino acid when it is incorporated into a protein, so the “molecular weight” of a residue is lower by 18 units. Also, most proteins have relatively more small side chains (Gly, Ala) and fewer Trp side chains than a statistical average would predict. 17. Aspartame is composed of aspartic acid and phenylalanine, with a carboxymethyl cap. These amino acids are linked by a peptide NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Abbreviated Answers to Problems

(amide) bond in aspartame. Heating can cleave amide linkages. For this reason, drinks such as coffee and hot chocolate must be consumed relatively quickly after preparation. Aspartame kept hot for several hours is quite bitter tasting (based on the experience of one of the authors). 18. Phenylketonuria is an autosomal recessive genetic disease caused by a deficiency or absence of the enzyme phenylalanine hydroxylase (PAH), an essential enzyme that converts phenylalanine to tyrosine. Without sufficient PAH activity, phenylalanine accumulates and is converted to phenylpyruvate (which can be detected in the urine). Without treatment, phenylketonurics eventually experience progressive mental retardation and seizures. Phenylketonuria can be controlled by eliminating phenylalanine from the diet, and phenylketonurics should be advised not to use aspartame. 19. The process for distinguishing R- and S-configurations of chiral molecules is described in the text on page 91. Enzymes discriminate between isomers of chiral molecules thanks to the asymmetric arrangement of amino acid residues in the enzyme active site. 20. Appropriate ranges for buffering: Alanine—1.4–3.4, 8.7–10.7 Histidine—0.8–2.8, 5.0–7.0, 8.2–10.2 Aspartic acid—1.1–4.9, 8.8–10.8 Lysine—1.2–3.2, 8–11.5

LK

1. The central rod domain of keratin is composed of distorted a-helices, with 3.6 residues per turn, but a pitch of 0.51 nm, compared with 0.54 nm for a true a-helix. (0.51 nm/turn)(312 residues)/(3.6 residues/turn) = 44.2 nm $ 442 Å.

2. The collagen helix has 3.3 residues per turn and 0.29 nm per residue, or 0.96 nm/turn. Then:

3. Seven fragments

F

CHAPTER 6

The distance between residues is 0.347 nm for antiparallel b-sheets and 0.325 nm for parallel b-sheets. So 312 residues of antiparallel b-sheet amount to 1083 Å and 312 residues of parallel b-sheet amount to 1014 Å.

2. M-S-F-Y

AVLY

15. A graph of n versus [L] reveals that at n = 0.5, [L] = KD = 2.4 mM. 16. IRS-1 has 1242 amino acids. Its average molecular mass is 131,590.97. The amino acid sequence of the tryptic peptide of IRS-1 of mass of 1741.9629 is LNSEAAAVVLQLMNIR. The sequence of the tryptic fragment containing the SHPTP-2 site is LCGAAGGLENGLNYIDLDLVK.

(0.54 nm/turn)(312 residues)/(3.6 residues/turn) = 46.8 nm $ 468 Å.

1. Nitrate reductase is a dimer (2 Mo/240,000 Mr).

TSR

14. Unlike any amino acid side chain, the phosphate group (or more appropriately, the phosphoryl group) bears two equivalents of negative charge at physiological pH. Furthermore, replacing an H atom on an S, T, or Y side chain with a phosphoryl group introduces a very bulky substituent into the protein structure where none existed before.

For an a-helix, the length would be:

CHAPTER 5

M

HINDR

M

(4 in/year)(2.54 cm/in)(107 nm/cm)/(0.96 nm/turn) = 1.06 * 108 turns/year. (1.06 *

CnBR

Trypsin Chymotrypsin Trypsin

Trypsin

4. Phe-Asp-Tyr-Met-Leu-Met-Lys. 5. Tyr-Asn-Trp-Met-(Glu-Leu)-Lys. Parentheses indicate that the relative positions of Glu and Leu cannot be assigned from the information provided. 6. Ser-Glu-Tyr-Arg-Lys-Lys-Phe-Met-Asn-Pro. 7. Ala-Arg-Met-Tyr-Asn-Ala-Val-Tyr or Asn-Ala-Val-Tyr-AlaArg-Met-Tyr sequences both fit the results. (That is, in one-letter code, either ARMYNAVY or NAVYARMY.) 8. Gly-Arg-Lys-Trp-Met-Tyr-Arg-Phe. 9. Actually, there are four possible sequences: NIGIRVIA, GINIRVIA, VIRNIGIA, and of course, VIRGINIA. 10. Gly-Trp-Arg-Met-Tyr-Lys-Gly-Pro. 11. Leu-Met-Cys-Val-Tyr-Arg-Cys-Gly-Pro. S

A-5

S

12. Alanine, attached to a solid-phase matrix via its a-carboxyl group, is reacted with diisopropylcarbodiimide-activated lysine. Both the a-amino and e-amino groups of the lysine must be blocked with 9-fluorenyl-methoxycarbonyl (Fmoc) groups. To add leucine to Lys-Ala to form a linear tripeptide, precautions must be taken to prevent the incoming Leu a-carboxyl group from reacting inappropriately with the Lys e-amino group instead of the Lys a-amino group. 13. The mass of the myoglobin chain is calculated to be 16,947 { 1 daltons.

108 turns/year)(1 year/365 days)(1 day/24 hours) (1 hour/60 minutes) = 201 turns/minute.

3. Asp: The ionizable carboxyl can participate in ionic and hydrogen bonds. Hydrophobic and van der Waals interactions are negligible. Leu: The leucine side chain does not participate in hydrogen bonds or ionic bonds, but it will participate in hydrophobic and van der Waals interactions. Tyr: The phenolic hydroxyl of tyrosine, with a relatively high pKa, will participate in ionic bonds only at high pH but can both donate and accept hydrogen bonds. Uncharged tyrosine is capable of hydrophobic interactions. The relatively large size of the tyrosine side chain will permit substantial van der Waals interactions. His: The imidazole side chain of histidine can act as both an acceptor and donor of hydrogen bonds and, when protonated, can participate in ionic bonds. Van der Waals interactions are expected, but hydrophobic interactions are less likely in most cases. 4. As an imino acid, proline has a secondary nitrogen with only one hydrogen. In a peptide bond, this nitrogen possesses no hydrogens and thus cannot function as a hydrogen-bond donor in a-helices. On the other hand, proline stabilizes the cis-configuration of a peptide bond and is thus well suited to b-turns, which require the cis-configuration. 5. For a right-handed crossover, moving in the N-terminal to C-terminal direction, the crossover moves in a clockwise direction when viewed from the C-terminal side toward the N-terminal side. The reverse is true for a left-handed crossover;

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

A-6

Abbreviated Answers to Problems

that is, movement from N-terminus to C-terminus is accompanied by counterclockwise rotation. 6. The Ramachandran plot reveals allowable values of f and c for a-helix and b-sheet formation. The plots consider steric hindrance and will be somewhat specific for individual amino acids. For example, peptide bonds containing glycine can adopt a much wider range of f and c angles than can peptide bonds containing tryptophan. 7. The protein appears to be a tetramer of four 60-kD subunits. Each of the 60-kD subunits in turn is a heterodimer of two peptides, one of 34 kD and one of 26 kD, joined by at least one disulfide bond. 8. Hydrophobic interactions frequently play a major role in subunit–subunit interactions. The surfaces that participate in subunit–subunit interactions in the B4 tetramer are likely to possess larger numbers of hydrophobic residues than the corresponding surfaces of protein A.

CHAPTER 7 1. See structures in The Student Solutions Manual, Study Guide and Problems Book. 2. See structures and titration curve in The Student Solutions Manual, Study Guide and Problems Book. 3. Glycated hemoglobin can be separated from ordinary hemoglobin on the basis of charge difference (by ion-exchange chromatography, high-performance liquid chromatography [HPLC] electrophoresis, and isoelectric focusing) or on the basis of structural difference (by affinity chromatography). 4. The systematic name for trehalose is a-D-glucopyranosyl(1 S 1)-a-D-glucopyranoside. Trehalose is not a reducing sugar. Both anomeric carbons are occupied in the disaccharide linkage. 5. See structure in The Student Solutions Manual, Study Guide and Problems Book.

9. The length is given by (53 residues) * (0.15 nm run/residue) = 7.95 nm. The number of turns in the helix is given by (53 residues)/(3.6 residues/turn) = 14.7 turns. There are 49 hydrogen bonds in this helix.

6. A sample that is 0.69 g a-D-glucose/mL and 0.31 g b-D-glucose/ mL will produce a specific rotation of 83°.

10. Glycines are essential components of tight turns (b-turns) and thus are often essential for maintenance of protein structure.

8. A 0.2 g sample of amylopectin corresponds to 0.2 g/162 g/ mole or 1.23 * 10-3 mole glucose residues; 50 mmole is 0.04 of the total sample or 4% of the residues. Methylation of such a sample should yield 1,2,3,6-tetramethylglucose for the glucose residues on the reducing ends of the sample. The amylopectin sample contains 1.2 * 1018 reducing ends.

11. Denatured ribonuclease (in the presence of urea and betamercaptoethanol) possessed no enzymatic activity. Subsequent removal of the beta-mercaptoethanol (but not urea) lead to some of the ribonuclease activity being restored. This was attributed to the formation of random (albeit not correct) disulfide bridges upon renaturation. However, when both the betamercaptoethanol and urea were removed simultaneously, the protein could refold into its native conformation and form the correct disulfide bridges. Enzymatic activity was also restored. Thus, information needed for protein folding resides within the primary sequence of ribonuclease which dictates which noncovalent interactions are necessary for the correct structure to form first, followed by correct disulfide bridge formation. 12. Asp, Glu, Ser, Thr, His, and perhaps also Asn, Gln, Cys, Arg, Lys. 13. The ability of poly-Glu to form a-helices requires that the glutamate carboxyls be protonated. Deprotonation produces a polyanionic peptide that is not amenable to helix formation. 14. A coiled-coil formed from a-helices with 3.5 residues per turn would form a symmetrical seven-residue-repeating structure that would place the first and fourth residues of the seven-residue repeat at the same positions about the helix axis in every sevenresidue repeat. This would allow the two helices of a coiled-coil structure to lie side by side with no twist about the coiled-coil axis. Such a structure would probably not be as stable as the twisted structure of coiled coils formed from a-helices with 3.6 residues per turn. 15. a. The third sequence would place hydrophobic residues on both sides of a b-strand and could thus be found in a parallel b-sheet. b. The second sequence would place hydrophobic residues on just one side of a b-strand and could thus be found in an anti-parallel b-sheet. c. The sixth sequence consists of GPX repeats (where X is any amino acid) and could thus be part of a tropocollagen molecule. d. The first sequence consists of seven-residue repeats, with first and fourth residues hydrophobic, and could thus be part of a coiled coil structure.

7. See structures in The Student Solutions Manual, Study Guide and Problems Book.

9. There are several target sites for trypsin and chymotrypsin in the extracellular sequence of glycophorin, and it would be reasonable to expect that access to these sites by trypsin and chymotrypsin would be restricted by the presence of oligosaccharides in the extracellular domain of glycophorin. 10. Energy yield upon combustion (whether by metabolic pathways or other reactions) depends on the oxidation level. Carbohydrate and protein are at approximately the same oxidation level, and both of these are significantly less than that of fat. 11. This mechanism could involve either an SN1 or SN2 mechanism. In the former, protonation of the bridging oxygen would result in dissociation to produce a carbo-cation intermediate, which could be attacked by the phosphate nucleophile. The observation of retention of configuration at the anomeric carbon favors this mechanism. An SN2 mechanism would presumably involve water attack at the anomeric carbon, with dissociation of the oxygen of the carbohydrate chain. This would be followed by SN2 attack by phosphate. Two SN2 attacks would result in retention of configuration at the anomeric carbon atom. 12. Laetrile contains a cyanide group. Breakdown of laetrile and release of this cyanide function in the body would be highly toxic. 13. Chondroitin and glucosamine are amino sugar components of cartilage and connective tissue. Dietary supplement with these substances could help replenish the cartilage matrix proteoglycan, relieving pain and restoring the proper function of connective tissue structures. 14. Two of the sugar units in stachyose are glucose and fructose, and the bond joining them is easily cleaved by stomach enzymes. However, the other two sugar units in stachyose are galactoses in b(1 S 6)-linkages, which are not broken down by human enzymes. The result is that stachyose loses a fructose in the NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

A-7

Abbreviated Answers to Problems

stomach but the resulting trisaccharide passes into the intestines, where bacterial enzymes degrade it, producing intestinal gas in the process. Beano contains an enzyme that hydrolyzes b(1 S 6)galactose linkages. Taking several Beano tablets before a meal of beans or legumes facilitates complete breakdown of stachyose and other related oligosaccharides in the stomach, avoiding the production of intestinal gas. The active enzyme in Beano is referred to as a b(1 S 6)-galactosidase. 15. b-D-Fructofuranosyl-O-a-D-galactopyranosyl-(1 S 6)-O-aD-galactopyranosyl-(1 S 6)-a-D-glucopyranoside 16. Starch phosphorylase cleaves glucose units, one at a time, from starch chains until a (1 S 6)-branch is encountered. Thus, limit dextrins are amylopectin fragments with a glucose in (1 S 6)linkage at each nonreducing end. The mechanism of the reaction that cleaves these glucose units is essentially the same as that for starch phosphorylase, except that it occurs at a glucose unit that is (1 S 6)-linked. 17. In the beer-making process, the mash fermented by yeast contains starch, which is partially broken down by the amylase from the malt to produce limit dextrins (see problem 16). These limit dextrins add significant caloric content to regular beers. Joseph Owades used the enzyme amyloglucosidase, which hydrolyzes both (1 S 4) and (1 S 6) linkages in starch, thus breaking down the limit dextrins to simple glucose, which is fermented normally. (This raises the alcohol content of the beer above what would normally be produced, and water is typically added to adjust the alcohol content.) 18. As described in problem 14, Beano is a b(1 S 6)-galactosidase. This enzyme can also hydrolyze (1 S 6)-glucose linkages, although somewhat more slowly than for its natural substrate. Thus, Beano can slowly convert the limit dextrins produced in the beer-brewing process into simple glucose units, much like the enzyme used by Joseph Owades (see problem 17). Amateur brewers have learned to use Beano to make their own light beer, often referred to as Beano beer. 19. Assuming each glucose unit contributes 0.55 nm to the growing cellulose polymer (and the plant length), a growth rate of 1 foot per day corresponds to a rate for cellulose synthase of 6,400 glucose units per second. 20. Basic amino acid side chains (Arg, His, Lys) in antithrombin III present positively charged side chains for ionic interactions with sulfate functions on heparin; H-bond donating amino acid side chains could form H bonds with O atoms in :OH groups on the heparin carbohydrate residues; H-bond accepting amino acid side chains could form H bonds with H atoms in :OH groups of heparin. 21. Because these glycosaminoglycans are rich in hydroxyl groups, amine groups, and anionic functions (carboxylates, sulfates), they interact strongly with water. The heavily hydrated proteoglycans formed from these glycosaminoglycans are reversibly dehydrated in response to the pressure imposed on a joint during normal body movements. This dehydration has a cushioning effect on the joint; when the pressure is relieved, the glycosaminoglycans are spontaneously rehydrated due to their affinity for water. CHAPTER 8 1. Because the question specifically asks for triacylglycerols that contain stearic acid and arachidonic acid, we can discount the triacylglycerols that contain only stearic or only arachidonic acid. In this case, there are six possibilities:

S

S

A

S

A

S

A

S

S

S

A

A

A

S

A

A

A

S

2. See The Student Solutions Manual, Study Guide and Problems Book for a discussion. 3. a. Phosphatidylethanolamine and phosphatidylserine have a net positive charge at low pH. b. Phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and diphosphatidylglycerol normally carry a net negative charge. c. Phosphatidylethanolamine and phosphatidylcholine carry a net zero charge at neutral pH. 4. Diets high in cholesterol contribute to heart disease and stroke. On the other hand, plant sterols bind to cholesterol receptors in the intestines but are not taken up by the cells containing these receptors. There is substantial evidence that a diet that includes plant sterols can reduce serum cholesterol levels significantly. 5. Former Interior Secretary James Watt was well known during the Reagan administration for his comments on several occasions that trees caused and produced air pollution. As noted in this chapter, it is of course true that trees emit isoprenes that are the cause of the blue–gray haze that is common in still air in mid- to late-summer in the eastern United States. However, these isoprene compounds are not significantly toxic to living things, and it would be misleading to call them pollutants. 6. Louis L’Amour clearly knew his biochemistry. His protagonist knew that fat carries a higher energy content than protein or carbohydrate. If meals are going to be scarce (as for a person living in the wild and on the run), it is wise to consume fat rather than protein or carbohydrate. The same reasoning applies for migratory birds in the weeks preceding their long flights. 7. Phospholipase A2 from snake bites operates without regulation or control, progressively breaking down cell membranes. Phospholipase A2 action to produce cell signals is under precise control and is carefully regulated to produce just the right amounts of required cell signals. 8. The lethal dose for 50% of animals is referred to as the LD50. The LD50 for dogs is approximately 3 mg/kg of body weight. Thus, for a 40-lb dog (18.2 kg), consumption of approximately 55 mg of warfarin would be lethal for 50% of animals. 9. See the Student Solution Guide and www.cengage.com/login for the solutions to this problem. 10. Humans require approximately 2000 kcal per day. Seal blubber is predominantly triglycerides, which yield approximately 9 kcal per gram. This means that a typical human would need to consume 222 grams of seal blubber per day (about half a pound) in order to obtain all of his or her calories from this energy source. 11. Results for this problem will depend on the particular cookies chosen by the student. 12. The only structural differences between cholesterol and stigmasterol are the double bond of stigmasterol at C22 9C23, and the ethyl group at C24. 13. Androgens mediate the development of sexual characteristics and sexual function in animals. Glucocorticoids participate in the control of carbohydrate, protein, and lipid metabolism. Mineralocorticoids regulate salt (Na+, K+, and Cl-) balances in tissues. 14. The answers to this question will depend on the household products chosen and the isoprene substances identified.

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A-8

Abbreviated Answers to Problems

15. Hydroxide ions (in lye) catalyze the breakdown of triglycerides to produce fatty acid salts such as sodium stearate and sodium palmitate and leaving glycerol as a byproduct. Micelle formation by these “soaps” leads to emulsification of fats and other nonpolar substances. 16. Amphipathic phosphatidylcholine from egg yolk exerts a detergentlike action on mixtures of vegetable oil and water. Micelles made primarily of egg phosphatidylcholine emulsify the vegetable oil, forming a stable suspension that persists indefinitely. (Thus, mayonnaise does not need to be mixed or shaken before use, whereas oil and vinegar mixtures do.) The micellar particles of mayonnaise are large enough to scatter visible light, so mayonnaise appears milky white, whereas the separated layers of oil and water in containers of oil and vinegar appear clear. 17. Stanol esters function in cholesterol reduction by binding to cholesterol receptors in the intestines. However, each serving of stanol esters consumed only blocks a fraction of all intestinal receptors. Regular consumption of stanol esters eventually blocks all or most available receptors. Binding of stanol esters is tight and long lasting, but not indefinite, so stanol ester consumption must be continued to maintain the beneficial results. The graph on page 247 shows that reductions of serum cholesterol levels can approach 15%; given that the typical diet accounts for about 15% of total serum cholesterol, it may be concluded that stanol esters are highly effective in preventing uptake of dietary cholesterol.

in denial of its use, and many have been stripped of medals and awards for athletic accomplishments aided by THG. CHAPTER 9 1. The equations needed for this problem are found on page 261. See The Student Solutions Manual, Study Guide and Problems Book for details. 2. Carbons C-1 and C-6 of glucose become the methyl carbons of acetyl-CoA that is the substrate for fatty acid synthesis. Carbons C-2 and C-5 of glucose become the carboxyl carbon of acetylCoA for fatty acid synthesis. Only citrate that is immediately exported to the cytosol provides glucose carbons for fatty acid synthesis. Citrate that enters the TCA cycle does not immediately provide carbon for fatty acid synthesis. 3. A suitable model, based on the evidence presented in this chapter, would be that the fundamental regulatory mechanism in ACC is a polymerization-dependent conformation change in the protein. All other effectors—palmitoyl-CoA, citrate, and phosphorylation-dephosphorylation—may function primarily by shifting the inactive protomer-active polymer equilibrium. Polymerization may bring domains of the protomer (that is, bicarbonate-, acetyl-CoA-, and biotin-binding domains) closer together or may bring these domains on separate protomers close to each other. See The Student Solutions Manual, Study Guide and Problems Book for further details.

18. Serum cholesterol is partly derived from diet and partly from synthesis in the liver. Stanol esters prevent uptake of dietary cholesterol but do not affect synthesis in the liver. Cholesterollowering drugs, on the other hand, block cholesterol synthesis but have no effect on uptake from diet. Thus, the effects of these two classes of cholesterol-lowering agents are additive.

4. The pantothenic acid group may function, at least to some extent, as a flexible “arm” to carry acyl groups between the malonyl transferase and ketoacyl-ACP synthase active sites. The pantothenic acid moiety is approximately 1.9 nm in length, setting an absolute upper-limit distance between these active sites of 3.8 nm. However, on the basis of modeling considerations, it seems likely that the distance between these sites is smaller than this upper-limit value.

19. Research has shown that consumption of substantial quantities of plant fats can lead to significant reduction of serum cholesterol. The content of so-called phytosterols varies depending on the source. However, a sterol- or stanol-fortified spread like Benecol probably provides the highest concentration of dietary agents for cholesterol lowering.

5. Two electrons pass through the chain from NADH to FAD to the two cytochromes of the cytochrome b5 reductase and then to the desaturase. Together with two electrons from the fatty acyl substrate, these electrons reduce an O2 to two molecules of water. The hydrogen for the waters that are formed in this way comes from the substrate (2H) and from two protons from solution.

20. Tetrahydrogestrinone (THG) is synthesized by the catalytic hydrogenation of gestrinone (which has an acetylene group in place of the ethyl group on the D-ring of tetrahydrogestrinone). Patrick Arnold, known as the “father of prohormones,” is credited with the synthesis of THG from gestrinone and the promotion of THG as an anabolic steroid in preparations such as The Clear and The Cream. These substances were marketed aggressively to top athletes in several sports by Arnold and Victor Conte of the Bay Area Laboratory Co-Operative. THG (at that time) was undetectable by existing laboratory analyses, and its use was not specifically prohibited in athletic competitions by the World Anti-Doping Code. Use of THG has since been prohibited by nearly all sports regulatory agencies, and effective methods for its detection have been developed. Numerous athletes have by now admitted to use of THG or have been convicted for perjury

6. Ethanolamine + glycerol + 2 fatty acyl-CoA + 2 ATP + CTP + H2O S phosphatidylethanolamine + 2 ADP + 2 CoA + CMP + PPi + Pi 7. The conversion of acetyl-CoA to lanosterol can be written as: 18 Acetyl-CoA + 13 NADPH + 13 H+ + 18 ATP + 0.5 O2 S lanosterol + 18 CoA + 13 NADP+ + 18 ADP + 6 Pi + 6 PPi + CO2 The conversion of lanosterol to cholesterol is complicated; however, in terms of carbon counting, three carbons are lost in the conversion to cholesterol. This process might be viewed as 1.5 acetate groups for the purpose of completing the balanced equation. 8. The numbers 1–4 in the cholesterol structure indicate the carbon positions of mevalonate as shown (note that the numbering shown here is not based on the systematic numbering of mevalonate):

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Abbreviated Answers to Problems

11. It “costs” 1 ATP to form a malonyl-CoA. Each cycle of the fatty acyl synthase consumes 2 NADPH molecules, each worth 3.5 ATP. Thus, each of the seven cycles required to form a palmitic acid consumes 8 ATPs. A total of 56 ATPs are consumed to synthesize one molecule of palmitic acid.

9. The O-linked saccharide domain of the LDL receptor probably functions to extend the receptor domain away from the cell surface and above the glycocalyx coat so that the receptor can recognize circulating lipoproteins. 10. As shown in Figures 9.19 and 9.22, the syntheses (in eukaryotes) of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol are dependent upon CTP. A CTP deficiency would be likely to affect all these synthetic pathways.

12. The mechanism of the 3-ketosphinganine synthase reaction: is shown in the following figure.

H #OOC

C

CH2OH

CH2OH

NH2 H

C

H H

O C

O

N O# OH

2#O

3PO

Schiff base formation

&

N

&

N

H

H

CH2OH

CH2OH H

C#

H H

H

N

& CO2

N OH

OH

3PO

C

C

C 2#O

C

C OH

2#O PO 3

&

N

N

H

H C

S

CoA

O H2O

H C O

C

CH2OH

NH3&

2 S-3-Ketosphinganine

13. FAD is required for the eukaryotic reaction that converts stearic acid to oleic acid because the oxidation of a single bond to a double bond in stearic acid involves NADH and thus requires a two-electron transfer, whereas the reoxidation of FADH2 to FAD is accomplished by electron transfer to cytochrome b5.

A-9

Cytochromes are capable of one-electron transfers only, so FADH2/FAD is required because it can participate both in one-electron and two-electron transfers. 14. See Chapter 22, problem 13, for a mechanism for the HMG-CoA synthase reaction.

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A-10

Abbreviated Answers to Problems

15. A mechanism for the b-ketoacyl ACP synthase reaction:

16. This exercise is left to the student. 17. This exercise is left to the student. 18. This exercise is left to the student. CHAPTER 10 1. Glycerophospholipids with an unsaturated chain at the C-1 position and a saturated chain at the C-2 position are rare to nonexistent. Glycerophospholipids with two unsaturated chains, or with a saturated chain at C-1 and an unsaturated chain at C-2, are commonly found in biomembranes.

7. 8.

2. The phospholipid/protein molar ratio in purple patches of H. halobium is 10.8. 3. See The Student Solutions Manual, Study Guide and Problems Book for plots of sucrose solution density versus percent by weight and by volume. The plot in terms of percent by weight exhibits a greater curve, because less water is required to form a solution that is, for example, 10% by weight (10 g sucrose/100 g total) than to form a solution that is 10% by volume (10 g sucrose/100 mL solution).

9.

4. r = (4Dt)1/2. According to this equation, a phospholipid with D = 1 * 10-8 cm2 will move approximately 200 nm in 10 milliseconds.

10.

5. Fibronectin: For t = 10 msec, r = 1.67 * 10-7 cm = 1.67 nm. Rhodopsin: r = 110 nm. All else being equal, the value of D is roughly proportional to (Mr)-1/3. Molecular weights of rhodopsin and fibronectin are 40,000 and 460,000, respectively. The ratio of diffusion coefficients is thus expected to be (40,000)-1/3/(460,000)-1/3 = 2.3. On the other hand, the values given for rhodopsin and fibronectin give an actual ratio of 4286.The explanation is that fibronectin is anchored in the membrane via interactions with cytoskeletal proteins, and its diffusion is severely restricted compared with that of rhodopsin. 6. a. Divalent cations increase Tm. b. Cholesterol broadens the phase transition without significantly changing Tm. c. Distearoylphosphatidylserine should increase Tm, due to increased chain length and also to the favorable interactions between the more negative PS head group and the more positive PC head groups.

11.

d. Dioleoylphosphatidylcholine, with unsaturated fatty acid chains, will decrease Tm. e. Integral proteins will broaden the transition and could raise or lower Tm, depending on the nature of the protein. !G = RT In([C2]/[C1]). !G = + 4.0 kJ/mol. !G = RT In([Cout]/[Cin]) + Zf!c !G = + 4.08 kJ/mol - 2.89 kJ/mol. !G = +1.19 kJ/mol. The unfavorable concentration gradient thus overcomes the favorable electrical potential and the outward movement of Na+ is not thermodynamically favored. One could solve this problem by going to the trouble of plotting the data in y vs. [S], 1/y vs. 1/[S], or [S]/y vs. [S] plots, but it is simpler to examine the value of [S]/y at each value of [S]. The Hanes–Woolf plot makes clear that [S]/y should be constant for all [S] for the case of passive diffusion. In the present case, [S]/y is a constant value of 0.0588 (L/min)-1. It is thus easy to recognize that this problem describes a system that permits passive diffusion of histidine. This is a two-part problem. First calculate the energy available from ATP hydrolysis under the stated conditions; then use the answer to calculate the maximal internal fructose concentration against which fructose can be transported by coupling to ATP hydrolysis. Using a value of #30.5 kJ/mol for the !G "= of ATP and the indicated concentrations of ATP, ADP, and Pi, one finds that the !G for ATP hydrolysis under these conditions (and at 298 K) is #52.0 kJ/mol. Putting the value of &52.0 kJ/mol into Equation 9.1 and solving for C2 yields a value for the maximum possible internal fructose concentration of 1300 M! Thus, ATP hydrolysis could (theoretically) drive fructose transport against internal fructose concentrations up to this value. (In fact, this value is vastly in excess of the limit of fructose solubility.) Each of the transport systems described can be inhibited (with varying degrees of specificity). Inhibition of the rhamnose transport system by one or more of these agents would be consistent with involvement of one of these transport systems with rhamnose transport. Thus, nonhydrolyzable ATP analogs should inhibit ATP-dependent transport systems, ouabain should specifically block Na+ (and K+) transport, uncouplers should NEL

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Abbreviated Answers to Problems

inhibit proton gradient–dependent systems, and fluoride should inhibit the PTS system (via inhibition of enolase). 12. N-myristoyl lipid anchors are found linked only to N-terminal Gly residues. Only the peptide in (e) of this problem contains an N-terminal Gly residue. 13. Only the peptide in (a) possesses a CAAX sequence (where C $ Cys, A $ aliphatic (Ala, Val, Leu, Ile), and X $ any amino acid). 14. The hydropathy plot of a soluble protein should show no substantial stretches of hydrophobic residues, except for the signal sequence. 15. Prolines are destabilizing to a-helices, and a short helix with a proline would not be likely to be stable. Where a proline occurs in an a-helix, the helix is bent or kinked. Helices with a proline kink tend to be longer than average, presumably because longer helices are more stable and more able to tolerate the loss of H bonds in the kink region. 16. The structural consequences of a proline-induced kink in an a-helix are described in detail in the references provided in this problem. 17. Porin proteins typically consist of 18-stranded b-barrels. With 9 to 11 residues per strand, about 180–200 residues would be required to form the barrel, with a roughly equivalent number required to form the loops between strands. (The maltoporin chains from E. coli consists of about 420 residues.) The transmembrane domain of Wza consists of eight a-helical segments with about 25 residues per helix, about 200 residues in all. Thus, the number of residues needed to create the transmembrane pore in these two proteins is about the same, even though the number of residues per membrane spanning segment is less for a b-strand (9–11) than for an a-helix (21–25).

A-11

moieties in the protein. For example, a conformation change that made the environment around an Asp carboxyl more polar would reduce the pKa of that group, promoting dissociation (i.e., proton release). An appropriate sequence of such changes could accomplish transmembrane proton transport. 23. Point (b)—that proteins can be anchored to the membrane via covalent links to lipid molecules—was not part of the Singer– Nicolson fluid mosaic model. CHAPTER 11 1. Polypeptide hormones constitute a larger and structurally more diverse group of hormones than either the steroid or amino acid–derived hormones, and it thus might be concluded that the specificity of polypeptide hormone-receptor interactions, at least in certain cases, should be extremely high. The steroid hormones may act either by binding to receptors in the plasma membrane or by entering the cell and acting directly with proteins controlling gene expression, whereas polypeptides and amino acid–derived hormones act exclusively at the membrane surface. Amino acid–derived hormones can be rapidly interconverted in enzyme-catalyzed reactions that provide rapid responses to changing environmental stresses and conditions. See The Student Solutions Manual, Study Guide and Problems Book for additional information.

18. From Figure 10.29, the area within a typical “fenced” area is about 0.3 mm * 0.3 mm, or 9 * 106 Å2. Dividing by 60 Å2, it appears that there are about 150,000 phospholipids in a monolayer-fenced area in the membrane (assuming a membrane of pure phospholipid, with no cholesterol, etc.).

2. The cyclic nucleotides are highly specific in their action, because cyclic nucleotides play no metabolic roles in animals. Ca2+ ion has an advantage over many second messengers because it can be very rapidly “produced” by simple diffusion processes, with no enzymatic activity required. IP3 and DAG, both released by the metabolism of phosphatidylinositol, form a novel pair of effectors that can act either separately or synergistically to produce a variety of physiological effects. DAG and phosphatidic acid share the unique property that they can be prepared from several different lipid precursors. Nitric oxide, a gaseous second messenger, requires no transport or translocation mechanisms and can diffuse rapidly to its target sites.

19. The lysine side chain N is about 5.5 Å from the a-carbon (about 1.1 Å per bond). If a Lys side chain was reoriented toward the membrane center, the maximum change in position relative to the membrane center would be 10–11 Å (assuming the position of the a-carbon did not change). Figure 10.15 indicates that the energy of a Lys side chain would change by 4kT over the 15-Å distance from the membrane surface to the membrane center. A movement of 10 Å would thus correspond to 2/3 of 4kT, or 8kT/3—almost twice the average translation kinetic energy of a molecule in the gas phase (3kT/2).

3. Nitric oxide functions primarily by binding to the heme prosthetic group of soluble guanylyl cyclase, activating the enzyme. An agent that could bind in place of NO—but that does not activate guanylyl cyclase—could reverse the physiological effects of nitric oxide. Interestingly, carbon monoxide, which has long been known to bind effectively to heme groups, appears to function in this way. Solomon Snyder and his colleagues at Johns Hopkins University have shown that administration of CO to cells that have been stimulated with nitric oxide causes attenuation of the NO-induced effects.

20. In a hydrophobic environment, the aspartate carboxyl group will be more stable in the protonated (uncharged) form than in the deprotonated (charged) form.

4. Herbimycin, whose structure is shown in the figure below, reverses the transformation of cells by Rous sarcoma virus, presumably as a direct result of its inactivation of the viral tyrosine kinase. The manifestations of transformation (on rat kidney cells, for example) include rounded cell morphology, increased glucose uptake and glycolytic activity, and the ability to grow without being anchored to a physical support (termed anchorage-independent growth). Herbimycin reverses all these phenotypic changes. On the basis of these observations, one might predict that herbimycin might also inactivate tyrosine kinases that bear homology to the viral pp60v–src tyrosine kinase. This inactivation has in fact been observed, and herbimycin is

21. In a hydrophobic environment, the side chains of lysine and arginine would be more stable in their deprotonated (uncharged) forms than in the protonated (charged) forms. As a result, the pKa values of these residues would be lowered significantly in a hydrophobic environment. 22. Based on the discussion in problems 20 and 21 in this chapter, it would be reasonable to imagine that light-induced conformation changes could alter the pKa values of the proton-transferring

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A-12

Abbreviated Answers to Problems

used as a diagnostic tool for implicating tyrosine kinases in cellsignaling pathways.

12. The formation and breakdown of cAMP: O O

#

O

P

O

P

O O

O

O#

P

O

CH2

Adenine

O

O

#

H

H OH

5. The identification of phosphorylated tyrosine residues on cellular proteins is difficult. Quantities of phosphorylated proteins are generally extremely small, and to distinguish tyrosine phosphorylation from serine/threonine phosphorylation is tedious and laborious. On the other hand, monoclonal antibodies that recognize phosphotyrosine groups on protein provide a sensitive means of detecting and characterizing proteins with phosphorylated tyrosines, using, for example, Western blot methodology. 6. Hormones act at extremely low concentrations, but many of the metabolic consequences of hormonal activation (release of cyclic nucleotides, Ca2+ ions, DAG, etc., and the subsequent alterations of metabolic pathways) occur at and involve higher concentrations of the affected molecular species. As we have seen in this chapter, most of the known hormone receptors mediate hormonal signals by activating enzymes (adenylyl cyclase, phospholipases, protein kinases, and phosphatases). One activated enzyme can produce many thousands of product molecules before it is inactivated by cellular regulation pathways. 7. Vesicles with an outside diameter of 40 nm have an inside diameter of approximately 36 nm and an inside radius of 18 nm. The data correspond to a volume of 2.44 * 10-20 L. Then 10,000 molecules/6.02 * 1023 molecules/mole = 1.66 * 10-20 mole. The concentration of acetylcholine in the vesicle is thus 1.66/2.44 M or 0.68 M. 8. The evidence outlined in this problem points to a role for cAMP in fusion of synaptic vesicles with the presynaptic membrane and the release of neurotransmitters. GTPgS may activate an inhibitory G protein, releasing Gai(GTPgS), which inhibits adenylyl cyclase and prevents the formation of requisite cAMP. 9. In both cases, the fraction of receptor that is bound with hormone is approximately 50%. 10. Statin drugs inhibit the synthesis of new cholesterol in human subjects, but they have no effect on dietary intake of cholesterol. Dietary cholesterol accounts for approximately 2/3 of all body cholesterol. Moreover, synthesis of steroid hormones is not generally dependent on ambient levels of cholesterol in the body. Thus the taking of statin drugs would not be expected to influence the levels and functions of steroid hormones in the body. 11. b-Strands indeed provide more genetically economical means of traversing a membrane. However, the only significant classes of proteins that employ b-strands for membrane traversal are the porins and toxins such as hemolysin. These b-strand structures provide no easy way to communicate conformational changes across the membrane. On the other hand, bundles of a-helices are more able to convey and transmit conformational changes across a bilayer membrane.

O O

#

OH

O

P

O

O#

P

O

&

O#

O# O

H

O

H

P BH

O

Adenine H

O

O# B

CH2

OH

cAMP

H O HO

P

O

CH2

O

Adenine

O

#

H

H OH

OH

13. The point at which no ion flow occurs is the point of equilibrium that balances the chemical and electrical forces across a membrane. The Nernst equation is obtained by setting DG to zero in Equation 10.2. Rearrangement yields RT ln (C2/C1) = -Zf!c Using the data in Figure 11.49, one can use this equation to yield an equilibrium potential for K+ of #77 mV and an equilibrium potential for Na+ of &53.4 mV. 14. Using the Goldman equation, one can calculate !c = - 60 mV, in agreement with values measured experimentally in neurons. 15. The resting potential in neurons is #60 mV. Local depolarization of the membrane causes the potential to rise approximately 20 mV to about #40 mV. This change causes the Na+ channels to open and Na+ ions begin to flow into the cell. This causes the potential to continue increasing to a value of about &30 mV. Because this value is close to the equilibrium potential for Na+, the Na+ channels begin to close and K+ channels open. K+ rushes out of the neuron, returning the membrane potential to a very negative value. There is a slight overshoot of the potential, and the K+ channels close and the resting membrane potential is restored. 16. Pathway steps (in Figure 11.4) that involve amplification include the tyrosine kinase of the receptor protein, Raf, MAPKK, and MAPK. 17. If Ras were mutated so as to have no GTPase activity, it would activate Raf indefinitely, and the pathway would run as if hormones were continually activating the receptor tyrosine kinase. 18. There are several possible answers to this problem. One is that the original assays of kinase activities that reflected signaling effects were carried out on whole-cell preparations, in which the G-protein-dependent and G-protein-independent activities

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Abbreviated Answers to Problems

would appear as components of the overall activation. The availability of G-protein-uncoupled receptor mutants and ligands that could stimulate arrestin recruitment without G-protein activation made it possible to distinguish the G-protein-dependent and G-protein-independent effects. (See the Further Reading references in this chapter by Luttrell for additional discussion of these issues.)

1. y/Vmax = 0.8.

2. y = 91 mmol/mL # sec. 3. Ks = 1.43 * 10-5 M; Km = 3 * 10-4 M. Because k2 is 20 times greater than k-1, the system behaves like a steady-state system. 4. If the data are graphed as double-reciprocal Lineweaver–Burk plots: a. Vmax = 51 mmol/mL # sec and Km = 3.2 mM. b. Inhibitor (2) shows competitive inhibition with a KI = 2.13 mM. Inhibitor (3) shows noncompetitive inhibition with a KI = 4 mM. 5.

In (a), Vmax doubles, but Km is constant. In (b), Vmax halves, but Km is constant. In (c), Vmax is constant, but the apparent Km increases. In (d), Vmax decreases, but Km is constant. In (e), Vmax decreases, Km decreases, but the ratio Km/Vmax is constant. 6.

CHAPTER 12

(a)

v

A-13

a. The slope is given by KmB/Vmax (KSA/[A] + 1). b. y-intercept = ((KmA/[A]) + 1)1/Vmax . c. The horizontal and vertical coordinates of the point of intersection are 1/[B] = -KmA/KSAKmB and 1/y = 1/Vmax (1 - (KmA/KSA)).

7. Top left: (1) Competitive inhibition (I competes with S for binding to E). (2) I binds to and forms a complex with S. Top right: (1) Pure noncompetitive inhibition. (2) Random, single-displacement bisubstrate reaction, where A doesn’t affect B binding, and vice versa. (Other possibilities include [3] Irreversible inhibition of E by I; [4] 1/y vs. 1/[S] plot at two different concentrations of enzyme, E.)

2E

Bottom left: (1) Mixed noncompetitive inhibition. (2) Ordered single-displacement bisubstrate mechanism.

E

Bottom right: (1) Uncompetitive inhibition. (2) Doubledisplacement (ping-pong) bisubstrate mechanism. 8. Clancy must drink 694 mL of wine, or about one 750-mL bottle. 9. a. KS = 5mM; b. Km = 30 mM; c. kcat = 5 * 103 sec-1; d. kcat/Km = 1.67 * 108 M-1sec-1; e. Yes, because kcat/Km approaches the limiting value of 109 M-1sec-1; f. V max = 10-5 mol/mL # sec; g. [S] = 90 mM; h. Vmax would equal 2 * 10-5 mol/mL # sec, but Km = 30 mM, as before.

[S] (b) E v

1/2 E

[S]

11. a. Vmax = 1.6 mmol mL-1 sec-1; b. y = 1.45 mmol mL-1 sec-1;

(c) O &I

v

[S] (d)

O

v

10. a. Vmax = 132 mmol mL-1 sec-1; b. kcat = 44,000 sec-1; c. kcat/Km = 24.4 * 108 M-1 sec-1; d. Yes! kcat/Km actually exceeds the theoretical limit of 109 M-1 sec-1 in this problem; e. The rate at which E encounters S; the ultimate limit is the rate of diffusion of S.

&I

c. kcat/Km = 1.6 * 108 M-1 sec-1; d. Yes. 12. a. Vmax = 6 mmol mL-1 sec-1; b. kcat = 1.2 * 106 sec-1; c. kcat/Km = 1 * 108 M-1 sec-1; d. Yes! kcat/Km approaches the theoretical limit of 109 M-1 sec-1; e. The rate at which E encounters S; the ultimate limit is the rate of diffusion of S. 13. a. Vmax = 64 mmol mL-1 sec-1; b. kcat = 1.28 * 104 sec-1; c. kcat/Km = 1.4 * 108 M-1 sec-1; d. Yes! kcat/Km approaches the theoretical limit of 109 M-1 sec-1. 14. a. Vmax = 120 mmol mL-1 sec-1; b. y = 104.7 mmol mL-1 sec-1; c. kcat/Km = 3.64 * 108 M-1 sec-1; d. Yes! kcat/Km approaches the theoretical limit of 109 M-1 sec-1. 15. a. Starting from Vmaxf = k2[ET] and Vmaxr = k-1[ET] for the maximal rates of the forward and reverse reactions respectively, and the Michaelis constants KmS = (k-1 + k2)/k1 and KmP = (k-1 + k2)/k-2 for S and P, respectively,

[S] (e) O

y = (Vmaxf[S]/KmS) - Vmaxr[P]/Kmp)/(1 + [S]/KmS + [P]/KmP) b. At equilibrium, y = 0 and Keq = [P]/[S] = Vmaxf KmP/Vmaxr KmS.

v

&I

[S]

16. a. S is the preferred substrate; its Km is smaller than the Km for T, so a lower [S] will give y = Vmax/2, compared with [T]. b. kcat/Km defines catalytic efficiency. kcat/Km for S = 2 * 107 M-1 sec-1; kcat/Km for T = 4 * 107 M-1 sec-1, so the enzyme is a more efficient catalysis with T as substrate.

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A-14

Abbreviated Answers to Problems

17. a. Because the enzyme shows maximal activity at or below 40°C, it seems more like a mammalian enzyme than a plant enzyme, which would be expected to have a broader temperature optimum because plants experience a broader range of temperatures. b. An enzyme from a thermophilic bacterium growing at 80°C would show an activity versus temperature profile similar to this one but shifted much farther to the right.

Because pepstatin more closely fits the profile of a pepsin substrate, we would surmise that it is a better inhibitor of pepsin than of HIV-1 protease. In fact, pepstatin is a potent inhibitor of pepsin (KI 6 1 nm) but only a moderately good inhibitor of HIV-1 protease, with a KI of about 1 mM. 4. The enzyme-catalyzed rate is given by: y = ke[ES] = ke=[EX[]

CHAPTER 13 1. a. Nucleophilic attack by an imidazole nitrogen of His57 on the 9CH2 9 carbon of the chloromethyl group of TPCK covalently inactivates chymotrypsin. (See The Student Solutions Manual, Study Guide and Problems Book for structures.) b. TPCK is specific for chymotrypsin because the phenyl ring of the phenylalanine residue interacts effectively with the binding pocket of the chymotrypsin active site. This positions the chloromethyl group to react with His57. c. Replacement of the phenylalanine residue of TPCK with arginine or lysine produces reagents that are specific for trypsin. 2. a. The structures proposed by Craik et al., 1987 (Science 237:905–907) are shown here. (If you look up this reference, note that the letters A and B of the figure legend for Figure 3 of this article actually refer to parts B and A, respectively. Reverse either the letters in the figure or the letters in the figure legend and it will make sense.)

Ke[ =

[EX[] [ES]

!Ge[ = -RT ln Ke[ Ke[ = e-!Ge /RT [

[EX[] = Ke[[ES] = e-!Ge /RT[ES] [

So

ke[ES] = ke=e-!Ge /RT[ES]

or

ke = ke=e-!Ge /RT

[

[

Similarly, for the uncatalyzed reaction: ku = ku=e-!Gu /RT [

Assuming that ku= " ke= Then

ke ku ke ku

e-!Ge /RT [

=

e-!Gu /RT

= e(!Gu

[

[ - !G [)/RT e

5. This problem is solved best by using the equation derived in problem 4. ke [ [ = e(!Gu - !Ge )/RT ku Using this equation, we can show that the difference in activation energies for the uncatalyzed and catalyzed hydrolysis reactions (!Gu - !Gc) is 92 kJ/mol. 6. Trypsin catalyzes the conversion of chymotrypsinogen to p-chymotrypsin, and chymotrypsin itself catalyzes the conversion of p-chymotrypsin to a-chymotrypsin. B

N

56 N

H

57

D102N mutant

H O His57

H Asn102

7. The mechanism suggested by Lipscomb is a general base pathway in which Glu270 promotes the attack of water on the carbonyl carbon of the substrate:

N

N

N G

H O Ser214

H

H

Ser195 O

H

b. Asn102 of the mutant enzyme can serve only as a hydrogenbond donor to His57. It is unable to act as a hydrogen-bond acceptor, as aspartate does in native trypsin. As a result, His57 is unable to act as a general base in transferring a proton from Ser195. This presumably accounts for the diminished activity of the mutant trypsin. 3. a. The usual explanation for the inhibitory properties of pepstatin is that the central amino acid, statine, mimics the tetrahedral amide hydrate transition state of a good pepsin substrate with its unique hydroxyl group. b. Pepsin and other aspartic proteases prefer to cleave peptide chains between a pair of hydrophobic residues, whereas HIV-1 protease preferentially cleaves a Tyr-Pro amide bond. NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Abbreviated Answers to Problems

8. Using the equation derived in problem 4, it is possible to calculate the ratio ke/ku as 1.86 * 1012.

3. Monod, Wyman, Changeux allosteric system: Lineweaver–Burk plot

9. If the concentration of free enzyme is equal to the concentration of enzyme–ligand complex, the concentration of ligand would be 1 * 10-27 M. This corresponds to 1.67 * 103 liters per molecule.

I

1/v

O A

10. !G = - 154 kJ/mol. This value is intermediate between noncovalent forces (H bonds are typically 10–30 kJ/mol) and covalent bonds (300–400 kJ/mol). 11. Assuming that the rate of gluconeogenesis would be equal to the slowest step in the pathway, with k = 2 * 10-20/sec, and assuming a cellular concentration of fructose-1,6-bisphosphatase of 0.031 (see Table 17.2), the rate of sugar synthesis would be 2 * 10-20/sec * 0.031 mM, or 6.2 * 10-22 mM/sec. Assuming a total human cell volume of 40 L, this corresponds to 2.48 * 10-23 moles glucose/sec. Assuming 30 ATP per glucose (under cellular conditions) and 50 kJ/mole of ATP hydrolyzed, we find that the rate of energy production is (2.48 * 10-23) * (30 ATP/glucose) * (30.5 kJ/mole), or 2.269 * 10-20 kJ/sec. Converting to kilocalories and years, we find that the time to synthesize the needed 480 kilocalories with an uncatalyzed reaction would be 2.8 * 1015 years, or roughly 200,000 times the lifetime of the universe so far.

1/[S] Hanes–Woolf plot [S]/v

I O

12. The correct answer is c. The stomach is a very acidic environment, whereas the small intestine is slightly alkaline. The lower part of the figure shows that enzyme X has optimal activity near pH 2, whereas enzyme Y works best at a pH near 8. 13. The correct answer is a. The two enzymes have nonoverlapping pH ranges, so it is highly unlikely that they could operate in the same place at the same time. 14. The correct answer is b. Only enzyme A has a temperature range that encompasses human body temperature (37°C).

A

[S]

4. Using the curves for negative cooperativity as shown in Figure 14.9 as a guide: M-M

15. The correct answer is d. The activities of the two enzymes overlap between 40° and 50°C. 16. The correct answer is c. We have no information on the pH behavior of enzymes A and B, nor on the behavior of X and Y as a function of temperature. The only answer that is appropriate to the data shown is c.

Negative cooperativity v

17. The only possible answer is b, because a “leveling off ” implies that all the enzyme is saturated with S.

[S]

18. The correct answer is c. In order to bring the substrate into the transition state, an enzyme must enjoy environmental conditions that favor catalysis. There is no activity apparent for enzyme Y below pH 5.5. CHAPTER 14 1. a. As [P] rises, the rate of P formation shows an apparent decline, as enzyme-catalyzed conversion of P S S becomes more likely. b. Availability of substrates and cofactors. c. Changes in [enzyme] due to enzyme synthesis and degradation. d. Covalent modification. e. Allosteric regulation. f. Specialized controls, such as zymogen activation, isozyme variability, and modulator protein influences. 2. Proteolytic enzymes have the potential to degrade the proteins of the cell in which they are synthesized. Synthesis of these enzymes as zymogens is a way of delaying expression of their activity to the appropriate time and place.

Lineweaver–Burk

1 — v

Michaelis– Menten

Negative cooperativity

1/ [S] Negative cooperativity Michaelis– Menten

Hanes–Woolf

[S] — v

[S]

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A-15

A-16

Abbreviated Answers to Problems

5. For n = 2.8, Ylungs = 0.98 and Ycapillaries = 0.77.

11. Pro: Lactate is a metabolic indicator of the need for oxygen; it binds to Mb at a distinct site (the allosteric site?) and it lowers Mb’s affinity for O2.

For n = 1.0, Ylungs = 0.79 and Ycapillaries = 0.61. Thus, with an n of 2.8 and a P50 of 26 torr, hemoglobin becomes almost fully saturated with O2 in the lungs and drops to 77% saturation in resting tissue, a change of 21%. If n of 1.0 and a P50 of 26 torr, hemoglobin would become only 79% saturated with O2 in the lungs and would drop to 61% in resting tissue, a change of 18%. The difference in hemoglobin O2 saturation conditions between the values for n = 2.8 and 1.0 (21% - 18%, or 3% saturation) seems small, but note that the potential for O2 delivery (98% saturation versus 79% saturation) is large and becomes crucial when pO2 in actively metabolizing tissue falls below 40 torr.

12.

Con: Mb is a monomeric protein. Traditionally, allosteric phenomena have been considered the realm of oligomeric proteins. How then is Mb “allosteric”? Since a ligand-induced conformational change in a monomeric proteins can affect binding of “substrate” (i.e., O2), the definition of allostery may need to be broadened. S!

6. An excess of a negatively cooperative allosteric inhibitor could never completely shut down the enzyme. Because the enzyme leads to several essential products, inhibition by one product might starve the cell for the others.

S!

7. a. -R(R/K)X(S*/T*)b. -KRKQIAVRGL8. Ligand binding is the basis of allosteric regulation, and allosteric effectors are common metabolites whose concentrations reflect prevailing cellular conditions. Through reversible binding of such ligands, enzymatic activity can be adjusted to the momentary needs of the cell. On the other hand, allosteric regulation is inevitably determined by the amounts of allosteric effectors at any moment, which can be disadvantageous. Covalent modification, like allosteric regulation, is also rapid and reversible, because the converter enzymes act catalytically. Furthermore, covalent modification allows cells to escape allosteric regulation by covalently locking the modified enzyme in an active (or inactive) state, regardless of effector concentrations. One disadvantage is that covalent modification systems are often elaborate cascades that require many participants.

The wedge-shaped protein monomers (red) assemble into trimers, but the alternative conformation for the monomer (square, green) forms tetramers. The substrate or allosteric regulator (yellow) binds only to the square conformation, and its binding prevents the square from adopting the wedge conformation. Thus, if S or the allosteric regulator is present, equilibrium favors a greater population of square tetramers among the morpheein ensemble at the expense of round trimers. 13.

1.0

ATP or CTP

0.8 Glutamine

9. Sickle-cell anemia is the consequence of Hb S polymerization through hydrophobic contacts between the side chain of Valbb6 and a pocket in the EF corner of b-subunits. Potential drugs might target this interaction directly or indirectly. For example, a drug might compete with Valb6 side chain for binding in the EF corner. Alternatively, a useful drug might alter the conformation of Hb S such that the EF corner was no longer accessible or accommodating to the Valb6 side chain. Another possibility might be to create drugs that deter the polymerization process in other ways through alterations in the surface properties between Hb S molecules. 10. Nitric oxide is covalently attached to Cys93b. Thus, the interaction is not reversible binding, as in allosteric regulation. On the other hand, the reaction of NO• with this cysteine residue is apparently spontaneous, and no converter enzyme is needed to add or remove it. Thus, the regulation of covalent modification that is afforded by converter enzyme involvement is obviated. The Hb:NO• interaction illustrates that nature does not always neatly fit the definitions that we create.

0.6 v

Vmax 0.4

0.2

0

3

6 [S]

9

K 0.5

14. Negative cooperativity in NAD+ binding to glyceraldehyde-3-P dehydrogenase:

Where affinity for NADH is

'

NAD&

'

NAD& NAD&

Induced conformation change

NAD& NAD&

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Abbreviated Answers to Problems

15. Hyperventilation results in decreased blood pCO2, which in turn leads to an increase in pH. Thus, the affinity of Hb for O2 increases (less O2 will be released to tissues). Hypoventilation has exactly the opposite effects on pCO2 and pH; thus, hypoventilation leads to a diminished affinity of Hb for O2 (more O2 will be released to the tissues). CHAPTER 15 1. The pronghorn antelope is truly a remarkable animal, with numerous specially evolved anatomical and molecular features. These include a large windpipe (to draw in more oxygen and exhale more carbon dioxide), lungs that are three times the size of those of comparable animals (such as goats), and lung alveoli with five times the surface area so that oxygen can diffuse more rapidly into the capillaries. The blood contains larger numbers of red blood cells and thus more hemoglobin. The skeletal and heart muscles are likewise adapted for speed and endurance. The heart is three times the size of that of comparable animals and pumps a proportionally larger volume of blood per contraction. Significantly, the muscles contain much larger numbers of energy-producing mitochondria, and the muscle fibers themselves are shorter and thus designed for faster contractions. All these characteristics enable the pronghorn antelope to run at a speed nearly twice the top speed of a thoroughbred racehorse and to sustain such speed for up to 1 hour. 2. Refer to Figure 15.9. The step in which the myosin head conformation change occurs, is the step that should be blocked by b,g-methylene-ATP, because hydrolysis of ATP should occur in this step and b,g-methylene-ATP is nonhydrolyzable. 3. Phosphocreatine is synthesized from creatine (via creatine kinase) primarily in muscle mitochondria (where ATP is readily generated) and then transported to the sarcoplasm, where it can act as an ATP buffer. The creatine kinase reaction in the sarcoplasm yields the product creatine, which is transported back into the mitochondria to complete the cycle. Like many mitochondrial proteins, the expression of mitochondrial creatine kinase is directed by mitochondrial DNA, whereas sarcoplasmic creatine kinase is derived from information encoded in nuclear DNA. 4. Note in step 3 of Figure 15.9 that it is ATP that stimulates dissociation of myosin heads from the actin filaments—the dissociation of the cross-bridge complex. When ATP levels decline (as happens rapidly after death), large numbers of myosin heads are unable to dissociate from actin and the muscle becomes stiff and unable to relax.

A-17

7800 tubulin monomers using the value of 25.6 Å calculated in problem 6. 8. Movement at 2 to 5 m/sec would correspond to a time of 5.6 to 14 hours to traverse this distance. 9. 14 nm is 140 Å, which would be approximately 93 residues of a coiled coil. 10. Using Equation 10.1, one can calculate a !G of 17,100 J/mol for this calcium gradient. 11. Using Equation 3.13, one can calculate a cellular !G of - 48,600 J/mol for ATP hydrolysis. 12. 17,100 J are required to transport 1 mole of calcium ions. Two moles of calcium would require 34,200 J, and three would cost 51,300 J. Thus, the gradient of problem 10 would provide enough energy to drive the transport of two calcium ions per ATP hydrolyzed. 13. Energy (or work) = force * distance. If 1 ATP is hydrolyzed per step and the cellular value of !G "= for ATP hydrolysis is #50 kJ/mol, then the calculation of force exerted by a motor is force = 50 kJ/mol/(step size). The SI unit of force is the newton, and 1 newton # meter = 1 J. For the kinesin-1 motor, 50 kJ/mol/(8 * 10-9 m) = 6.25 * 1012 newtons. For the myosin-V motor, 50 kJ/mol/(36 * 10-9 m) = 1.39 * 1012 newtons. For the dynein motor, 50 kJ/mol/(28 * 10-9 m) = 1.78 * 1012 newtons. 14. Perhaps the simplest way to view this problem is to consider the potential energy change for lifting a 10-kg weight 0.4 m. E = mgh = (10 kg) # (9.8 m/sec2) # (0.4 m) = 39.2 J. Now, if 1 ATP is expended per myosin step along an actin filament, 39.2 J/(50 kJ/mol) = 7.8 * 10-4 mol.

Then, (7.8 * 10-4 mol) # (6.02 * 1023) = 4.72 * 1020 molecules of ATP expended, one per step. So there must be 4.72 * 1020 myosin steps along actin. However, the stepping process is almost certainly not 100% efficient. As shown in the box on page 514, a step size of 11 nm against a force of 4 pN would correspond to an energy expended per myosin per step of 4.4 * 10-20 J. With this assumed energy expended per step, we can calculate 39 J/(4.4 * 10-20 J) = 8.86 * 1020 steps total.

If we take the step size value for skeletal myosin from the box on page 514 as 11 nm, one myosin head would have to take 0.4 m/11 * 10-9 m or 3.64 * 107 steps to raise the weight a distance of 0.4 m. Taking (8.86 * 1020 steps total)/(3.64 * 107 steps per myosin) = 2.43 * 1013. myosin heads involved per 11-nm step.

5. The skeletal muscles of the average adult male have a crosssectional area of approximately 35,000 cm2. The gluteus maximus muscles represent approximately 300 cm2 of this total. Assuming 4 kg of maximal tension per square centimeter of cross-sectional area, one calculates a total tension for the gluteus maximus of 1200 kg (as stated in the problem). The same calculation shows that the total tension that could be developed by all the muscles in the body is 140,000 kg (or 154 tons)!

15. All these are smooth muscle except for the diaphragm. (See http://chanteur.net/contribu/index.htm#http://chanteur.net/ contribu/cJMdiaph.htm for an explanation of the common misconception that the diaphragm is smooth muscle.)

6. Taking 55,000 g/mol divided by 6.02 × 1023/mol and by 1.3 g/mL, one obtains a volume of 7.03 × 10−20 mL. Assume a sphere and use the volume of a sphere (V = (4/3)!r3) to obtain a radius of 25.6 Å. The diameter of the tubulin dimer (see Figure 15.12) is 8 nm, or 80 Å, and two times the radius we calculated here is approximately 51 Å, a reasonable value by comparison.

CHAPTER 16

7. A liver cell is 20,000 nm long, which would correspond to 5000 tubulin monomers if using the value of Figure 15.12, and about

16. 1, a; 2, e; 3, d; 4, c; 5, b. 17. This exercise is left to the student and will presumably be different for every student.

1. 6.5 * 1012 (6.5 trillion) people. 2. Consult Table 16.2. 3. O2, H2O, and CO2. 4. See Section 16.2.

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A-18

Abbreviated Answers to Problems

5. Consult Figure 16.8. 6. Consult Corresponding Pathways of Catabolism and Anabolism Differ in Important Ways, p. 520. See also Figure 16.8. 7. See The ATP Cycle, p. 521; NAD+ Collects Electrons Released in Catabolism, p. 522; and NADPH Provides the Reducing Power for Anabolic Processes, p. 523. 8. In terms of quickness of response, the order is allosteric regulation > covalent modification > enzyme synthesis and degradation. See The Student Solutions Manual, Study Guide and Problems Book for further discussions. 9. See Metabolic Pathways Are Compartmentalized within Cells, p. 527, and discussions in The Student Solutions Manual, Study Guide and Problems Book. 10. Many answers are possible here. Some examples: Large numbers of metabolites in tissues and fluids; great diversity of structure of biomolecules; need for analytical methods that can detect and distinguish many different metabolites, often at very low concentrations; need to understand relationships between certain metabolites; time and cost required to analyze and quantitate many metabolites in a tissue or fluid. 11. Mass spectrometry provides great sensitivity for detection of metabolites. NMR offers a variety of methods for resolving and discriminating metabolites in complex mixtures. Other comparisons and contrasts are discussed in the references at the end of the chapter. 12. See Figure 12.23; The Coenzymes of the Pyruvate Dehydrogenase Complex, page 597, and the Activation of Vitamin B12, page 751. 13. The genome is the entire hereditary information in an organism, as encoded in its DNA or (for some viruses) RNA. The transcriptome is the set of all messenger RNA molecules (transcripts) produced in one cell or a population of cells under a defined set of conditions. The proteome is the entire complement of proteins produced by a genome, cell, tissue, or organism under a defined set of conditions. The metabolome is the complete set of low molecular weight metabolites present in an organism or excreted by it under a given set of physiological conditions. 14. A mechanism for liver alcohol dehydrogenase:

16. Most discussions of ocean sequestration of CO2 are devoted to capture of CO2 by phytoplankton or injection of CO2 deep in the ocean. However, the box on page 539 offers another possibility—carbon sequestration in the shells of corals, mollusks, and crustaceans. This approach may be feasible. However, there are indications that these classes of sea organisms are already suffering from global warming and from pollution of ocean water. 17.

32P

and 35S both decay via beta particle emission. A beta particle is merely an electron emitted by a neutron in the nucleus. Thus, beta decay does not affect the atomic mass, but it does convert a neutron to a proton. Thus, beta emission changes 32P to 32S and converts 35S to 35Cl: 32P S b - + 32S, t 1/2 = 14.3 days. 35S

S b- +

35Cl, t

1/2

= 87.1 days.

The decay equation is a first-order decay equation: A/A0 = e -0.693/ty2 Thus, after 100 days of decay, the fraction of 32P remaining would be 0.786% and the fraction of 35S remaining would be 45%. CHAPTER 17 1. a. Phosphoglucoisomerase, fructose bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde-3-P dehydrogenase, phosphoglycerate mutase, and enolase. b. Hexokinase/glucokinase, phosphofructokinase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase. c. Hexokinase and phosphofructokinase. d. Phosphoglycerate kinase and pyruvate kinase. e. According to Equation 3.13, reactions in which the number of reactant molecules differs from the number of product molecules exhibit a strong dependence on concentration. That is, such reactions are extremely sensitive to changes in concentration. Using this criterion, we can predict from Table 18.1 that the free energy changes of the fructose bisphosphate aldolase and glyceraldehyde-3-P dehydrogenase reactions will be strongly influenced by changes in concentration. f. Reactions that occur with !G near zero operate at or near equilibrium. See Table 17.1 and Figure 17.22. 2. The carboxyl carbon of pyruvate derives from carbons 3 and 4 of glucose. The keto carbon of pyruvate derives from carbons 2 and 5 of glucose. The methyl carbon of pyruvate is obtained from carbons 1 and 6 of glucose. 3. Increased [ATP], [citrate], or [glucose-6-phosphate] inhibits glycolysis. Increased [AMP], [fructose-1,6-bisphosphate], or [fructose-2,6-bisphosphate] stimulates glycolysis.

15. TCA cycle: in mitochondria; converts acetate units to CO2 plus NADH and FADH2. Glycolysis: in cytosol; converts glucose to pyruvate. Oxidative phosphorylation: in mitochondria; uses electrons (from NADH and FADH2) to produce ATP. Fatty acid synthesis: in cytosol; uses acetate units to synthesize fatty acids.

4. See The Student Solutions Manual, Study Guide and Problems Book for discussion. 5. The mechanisms for fructose bisphosphate aldolase and glyceraldehyde-3-P dehydrogenase are shown in Figures 17.12 and 17.13, respectively. 6. The relevant reactions of galactose metabolism are shown in Figure 17.24. See The Student Solutions Manual, Study Guide and Problems Book for mechanisms.

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Abbreviated Answers to Problems

7. Iodoacetic acid would be expected to alkylate the reactive activesite cysteine that is vital to the glyceraldehyde-3-P dehydrogenase reaction. This alkylation would irreversibly inactivate the enzyme.

18.

CH2 C

OH

+

O

CH2

8. Ignoring the possibility that 32P might be incorporated into ATP, the only reaction of glycolysis that utilizes Pi is glyceraldehyde-3-P dehydrogenase, which converts glyceraldehyde-3-P to 1,3-bisphosphoglycerate. 32Pi would label the phosphate at carbon 1 of 1,3-bisphosphoglycerate. The label will be lost in the next reaction, and no other glycolytic intermediates will be directly labeled. (Once the label is incorporated into ATP, it will also show up in glucose-6-P, fructose-6-P, and fructose-1, 6-bisphosphate.)

A-19

H2N

Lys

PO32–

O

Formation of Schiff base intermediate

OH

CH2 C

H

9. The sucrose phosphorylase reaction leads to glucose-6-P without the need for the hexokinase reaction. Direct production offers the obvious advantage of saving a molecule of ATP.

H

B

–

Lys

H

H

CH2

H

10. All of the kinases involved in glycolysis, as well as enolase, are activated by Mg2+ ion. A Mg2+ deficiency could lead to reduced activity for some or all of these enzymes. However, other systemic effects of a Mg2+ deficiency might cause even more serious problems.

+ N

O

PO32–

Borohydride reduction of Schiff base intermediate

11. a. 7.5. b. 0.0266 mM.

CH2 H

12. +1.08 kJ/mol.

C

13. a. -13.8 kJ/mol. b. Keq = 194,850.

OH N

Lys

H CH2

c. [Pyr]/[PEP] = 24,356.

PO32–

O

Stable (trapped) E–S derivative

14. a. -13.8 kJ/mol. b. Keq = 211. c. [FBP]/[F-6-P] = 528.

Degradation of enzyme (acid hydrolysis)

15. a. -19.1 kJ/mol. b. Keq = 1651. c. [ATP]/[ADP] = 13.8. 16. !G "= = -33.9 kJ/mol.

CH2

17. An 8% increase in [ATP] changes the [AMP] concentration to 20 mM (from 5 mM).

H

C CH2

OH N H

Lys OH

N 6-dihydroxypropyl-L-lysine

19.

UDPGlc

Glc-1-P

Gal-1-P

UDPGal

k1

k–1

k3

k4

k6

k–6

E

E • UDPGlc

E-UDPGal

E

k2 k–2

E-UMP • Glc-1-P

k–3

E-UMP

20. Pyruvate kinase deficiency primarily affects red blood cells, which lack mitochondria and can produce ATP only via glycolysis. Absence of pyruvate kinase reduces the production of ATP by glycolysis in red cells, which in turn reduces the activity of

k–4

E-UMP • Gal-1-P

k5 k–5

the Na,K-ATPase (see Chapter 10). This results in alterations of electrolyte (Na+ and K+) concentrations, which leads to cellular distortion, rigidity, and dehydration. Compromised red blood cells are destroyed by the spleen and liver.

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A-20

Abbreviated Answers to Problems

CHAPTER 18 1. Glutamate enters the TCA cycle via a transamination to form a-ketoglutarate. The g-carbon of glutamate entering the cycle is equivalent to the methyl carbon of an entering acetate. Thus, no radioactivity from such a label would be lost in the first or second cycle, but in each subsequent cycle, 50% of the total label would be lost (see Figure 18.15). 2. a. 4 ATP, 10 NADH; b. 3 ATP, 5 NADH; c. 2 ATP, 4 NADH 3. a. 4 CO2 b. 2 CO2 c. 2 CO2 4. NAD+ activates pyruvate dehydrogenase and isocitrate dehydrogenase and thus would increase TCA cycle activity. If NAD+ increases at the expense of NADH, the resulting decrease in NADH would likewise activate the cycle by stimulating citrate synthase and a-ketoglutarate dehydrogenase. ATP inhibits pyruvate dehydrogenase, citrate synthase, and isocitrate dehydrogenase; reducing the ATP concentration would thus activate the cycle. Isocitrate is not a regulator of the cycle, but increasing its concentration would mimic an increase in acetate flux through the cycle and increase overall cycle activity. 5. For most enzymes that are regulated by phosphorylation, the covalent binding of a phosphate group at a distant site induces a conformation change at the active site that either activates or inhibits the enzyme activity. On the other hand, X-ray crystallographic studies reveal that the phosphorylated and unphosphorylated forms of isocitrate dehydrogenase share identical structures with only small (and probably insignificant) conformation changes at Ser113, the locus of phosphorylation. What phosphorylation does do is block isocitrate binding (with no effect on the binding affinity of NADP+). As shown in Figure 2 of the paper cited in the problem (Barford, D., 1991. Molecular mechanisms for the control of enzymic activity by protein phosphorylation. Biochimica et Biophysica Acta 1133:55–62), the g-carboxyl group of bound isocitrate forms a hydrogen bond with the hydroxyl group of Ser113. Phosphorylation apparently prevents isocitrate binding by a combination of a loss of the crucial H bond between substrate and enzyme and by repulsive electrostatic and steric effects. 6. A mechanism for the first step of the a-ketoglutarate dehydrogenase reaction:

7. Aconitase is inhibited by fluorocitrate, the product of citrate synthase action on fluoroacetate. In a tissue where inhibition has occurred, all TCA cycle metabolites should be reduced in concentration. Fluorocitrate would replace citrate, and the concentrations of isocitrate and all subsequent metabolites would be reduced because of aconitase inhibition. 8. FADH2 is colorless, but FAD is yellow, with a maximal absorbance at 450 nm. Succinate dehydrogenase could be conveniently assayed by measuring the decrease in absorbance in a solution of the flavoenzyme and succinate. 9. The central (C-3) carbon of citrate is reduced, and an adjacent carbon is oxidized in the aconitase reaction. The carbon bearing the hydroxyl group is obviously oxidized by isocitrate dehydrogenase. In the a-ketoglutarate dehydrogenase reaction, the departing carbon atom and the carbon adjacent to it are both oxidized. Both of the i CH2i carbons of succinate are oxidized in the succinate dehydrogenase reaction. Of these four molecules, all but citrate undergo a net oxidation in the TCA cycle. 10. Several TCA metabolite analogs are known, including malonate, an analog of succinate, and 3-nitro-2-S-hydroxypropionate, the anion of which is a transition-state analog for the fumarase reaction.

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Abbreviated Answers to Problems

A-21

11. A mechanism for pyruvate decarboxylase: R R'

B

S

E

H

+ N

S

R

CH3

–

+ N

R'

R"

R

O ....H B

C

E

R'

COO–

R"

CH3

S

C

+ N R"

OH

C O–

O

Pyruvate

CO2 CH3

S

R

C

R'

OH

–

+ N

CH3

S

R

C N

R'

R"

OH

R"

Resonance-stabilized carbanion on substrate H+

CH3

S

R

+ N

R'

C

O

R

H

H

R'

R"

2 incorporation into TCA via the pyruvate carboxylase reaction would label the :CH2 :COOH carboxyl carbon in oxaloacetate. When this entered the TCA cycle, the labeled carbon would survive only to the a-ketoglutarate dehydrogenase reaction, where it would be eliminated as 14CO2. 14C

incorporated in a reversed TCA cycle would label the two carboxyl carbons of oxaloacetate in the first pass O E

HC

B

E

COO–

H2C

+ B H

COO–

H

OH

O SCoA

C

O HC

O C

CH3

15. The labeling pattern would be the same as if methyl-labeled acetyl-CoA was fed to the conventional TCA cycle (see Figure 18.15).

Glyoxalate H

+

through the cycle. One of these would be eliminated in its second pass through the cycle. The other would persist for more than two cycles and would be eliminated slowly as methyl carbons in acetyl-CoA in the reversed citrate synthase reaction.

14CO

16.

+ N

Acetaldehyde

12. [isocitrate]/[citrate] $ 0.1. When [isocitrate] = 0.03 mM, [citrate] = 0.3 mM.

14.

–

R"

Hydroxyethyl-TPP

13.

O

S

–

H2C

C

HC SCoA H2C

Acetyl-CoA

COO– O C

SCoA H2O

CoASH OH HC

COO–

H2C

COO–

Malate NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

A-22

Abbreviated Answers to Problems

17.

Cytosol

Mitochondrion Pyruvate

Pyruvate CO2

NADPH

+

H+

CO2

NADP+ Malate

Malate NAD+

CO2

NAD+

Malate dehydrogenase

Malate dehydrogenase NADH

+

H+

NADH

Oxaloacetate Acetyl-CoA

+

+

H+

Oxaloacetate

H2O

ADP

Citrate synthase

+

Acetyl-CoA

Pi

ATP–Citrate lyase

CoA

CoA

ATP Citrate

Citrate Mitochondrial membrane

18. For the malate dehydrogenase reaction, Malate +

NAD+

∆ oxaloacetate + NADH +

4-hydroxy-trans-aconitate. This product remains tightly bound at the aconitase active site, inactivating the enzyme. Studies by Trauble et al. have shown that the inhibitory product can be displaced by a 106-fold excess of isocitrate.

H+

with the value of !G "= being +30 kJ/mol. Then !G "= = -RT ln Keq = - (8.314 J/mol # K)(298) ln a

-30,000 J/mol = ln (x/4.4 * 10-3) 2478 J/mol

[1]x [20][2.2 *

10-4]

b

-12.1 = ln (x/4.4 * 10-3) x = -RT ln Keq x = [oxaloacetate] = 0.024 mM Using the dimensions given in the problem, one can calculate a mitochondrial volume of 1.57 * 10-15 L. (0.024 * 10-6 M) # (1.57 * 10-15L) # (6.02 * 1023 molecules/mole) $ 14.4, or about 14 molecules of OAA in a mitochondrion (“pOAA” $ 7.62). 19. This exercise is left to the student. Review of the calculation of oxidation numbers should be a prerequisite for answering this problem. 20. This exercise is left to the student. 21. 2R, 3R-fluorocitrate is converted by aconitase to 2-fluoro-cisaconitate. This intermediate then rotates 180 degrees in the active site. Addition of hydroxide at the C4 position is followed by double bond migration from C3-C4 to C2-C3, to form

CHAPTER 19 1. The cytochrome couple is the acceptor, and the donor is the (bound) FAD/FADH2 couple, because the cytochrome couple has a higher (more positive) reduction potential. !! = = 0.254 V - 0.02 V* o

* This is a typical value for enzyme-bound [FAD]. !G = - nf!!o= = - 43.4 kJ/mol. 2. !!o= = - 0.03 V; !G = + 5790 J/mol. 3. This situation is analogous to that described in Section 3.3. The net result of the reduction of NAD+ is that a proton is consumed. The effect on the calculation of free energy change is similar to that described in Equation 3.26. Adding an appropriate term to Equation 19.12 yields: ! = !o= - RT ln [H+] + (RT/nf ln ([ox]/[red]) 4. Cyanide acts primarily via binding to cytochrome a3, and the amount of cytochrome a3 in the body is much lower than the amount of hemoglobin. Nitrite anion is an effective antidote for cyanide poisoning because of its unique ability to oxidize ferrohemoglobin to ferrihemoglobin, a form of hemoglobin that competes very effectively with cytochrome a3 for cyanide. The amount of ferrohemoglobin needed to neutralize an otherwise NEL

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Abbreviated Answers to Problems

lethal dose of cyanide is small compared with the total amount of hemoglobin in the body. Even though a small amount of hemoglobin is sacrificed by sequestering the cyanide in this manner, a “lethal dose” of cyanide can be neutralized in this manner without adversely affecting oxygen transport. 5. You should advise the wealthy investor that she should decline this request for financial backing. Uncouplers can indeed produce dramatic weight loss, but they can also cause death. Dinitrophenol was actually marketed for weight loss at one time, but sales were discontinued when the potentially fatal nature of such “therapy” was fully appreciated. Weight loss is best achieved by simply making sure that the number of calories of food eaten is less than the number of calories metabolized. A person who metabolizes about 2000 kJ (about 500 kcal) more than he or she consumes every day will lose about a pound in about 8 days.) 6. The calculation in Section 19.4 assumes the same conditions as in this problem (1 pH unit gradient across the membrane and an electrical potential difference of 0.18 V). The transport of 1 mole of H+ across such a membrane generates 23.3 kJ of energy. For three moles of H+, the energy yield is 69.9 kJ. Then, from Equation 3.13, we have: 69,900 J/mol = 30,500 J/mol + RT ln ([ATP]/[ADP][Pi]) 39,400 J/mol = RT ln ([ATP]/[ADP][Pi]) [ATP]/[ADP][Pi] = 4.36 * 106 M-1 at 37 "C. In the absence of the proton gradient, this same ratio is 7.25 * 10-6 M-1 at equilibrium! 7. The succinate/fumarate redox couple has the highest (that is, most positive) reduction potential of any of the couples in glycolysis and the TCA cycle. Thus, oxidation of succinate by NAD+ would be very unfavorable in the thermodynamic sense. Oxidation of succinate by NAD+: !!o= ! - 0.35 V; !G "= ! 67,500 J/mol. On the other hand, oxidation of succinate is quite feasible using enzyme-bound FAD. Oxidation of succinate by [FAD]: !!o= ! 0, !G "= ! 0, depending on the exact reduction potential for bound FAD. 8. a. -73.3 kJ/mol. b. Keq = 7.1 *

1012.

c. [ATP]/[ADP] = 19.7. 9. a. -151.5 kJ/mol. b. Keq = 3.54 * 1026. c. [ATP]/[ADP] = 8.8. 10. a. -219 kJ/mol. b. Keq = 2.63 * 1038. c. [ATP]/[ADP] = 54.4. 11. a. -217 kJ/mol. b. Keq = 1.08 * 1038. c. [ATP]/[ADP] = -3479. 12. a. -26 kJ/mol. b. Keq = 3.68 * 104. c.

[NAD+]/[NADH]

= 1.48.

13. a. Keq = 4.8 * 103. b. +48 kJ/mol. c. [ATP]/[ADP] = 2.3.

A-23

14. This answer should be based on the information on pages 633–635 and should be in the student’s own words. 15. This answer should be based on the information on pages 633–635 and should be in the student’s own words. 16. Several mechanisms are possible. See, for example, the mechanisms in the following references: Pryor, W. A., and Porter, N. A., 1990. Suggested mechanisms for the production of 4-hydroxy-2-nonenal from the autoxidation of polyunsaturated fatty acids. Free Radical Biology and Medicine 8:541–543; and Niki, E., Yoshida, Y., Saito, Y., and Noguchi, N., 2005. Lipid peroxidation: Mechanisms, inhibition, and biological effects. Biochemical and Biophysical Research Communications 338:668–676. 17. A calculation similar to that in problem 18 in Chapter 18 yields about 15 H+ in a typical mitochondrion. 18. In the succinate dehydrogenase reaction, a two-electron transfer reaction (the conversion of succinate to fumarate) must be coupled to iron–sulfur centers, which can only transfer one electron at a time. FAD can carry on both one-electron and two-electron transfers and is thus ideally suited for this reaction. CHAPTER 20 1. Efficiency = !G "=/(light energy input) = nf!!o=/(Nhc/l) = 0.564. 2. !G "= = - nf!!o=, so !G "=/ - n f = !!o=. n = 4 for 2 H2O S 4e- + 4 H+ + O2. !!o= = (!o=(primary oxidant) !o= (12 O2/H2O)) = + 0.0648 V. Thus, !o=(primary oxidant) = 0.881 V. 3. !G for ATP synthesis = +50,336 J/mol. !!o= = - 0.34 V. 4. Reduced plastoquinone = PQH2; oxidized plastoquinone $ PQ; Reduced plastocyanin = PC(Cu+); plastocyanin = PC(Cu2+): PQH2 + 2 H+stroma + 2 PC(Cu2+) S PQ + 2 PC(Cu+) + 4 H+ thylakoid lumen 5. Noncyclic photophosphorylation: 2.57 ATP would be synthesized from 8 quanta, which equates to 3.11 hy/ATP. Cyclic photophosphorylation theoretically yields 2 H+/hy, so if 14 H+ yield 3 ATP, 7 hy yield 3 ATP, which equates to 2.33 hy/ATP. 6. In mitochondria, H+ translocation leads to a decline in intermembrane space pH and hence cytosolic pH, because the outer mitochondrial membrane is permeable to protons. Thus, the eukaryotic cytosol pH is at risk from mitochondrial proton translocation. So, mitochondria rely on a greater membrane potential (!c) and a smaller !pH to achieve the same protonmotive force. Because proton translocation in eukaryotic photosynthesis deposits H+ into the thylakoid lumen, the cytosol does not experience any pH change and !pH is not a problem. Moreover, the light-induced efflux of Mg2+ from the lumen diminishes !c across the thylakoid membrane, so a greater contribution of !pH to the proton-motive force is warranted. 7. Replacement of Tyr by Phe would greatly diminish the possibility of e- transfer between water and P680+, limiting the ability of P680+ to regain an e- and return to the P680 ground state. 8. Assuming 12 c-subunits means 12 H+ are needed to drive one turn of the c-subunit rotor and the synthesis of 3 ATP by the CF1 part of the ATP synthase. If the R. viridis cytochrome bc1 complex drives the translocation of 2 H+/e-, then 2 hy gives 4 H+, and 6 hy gives 12 H+ and thus 3 ATP (thus, 2 hy yield 1 ATP).

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A-24

Abbreviated Answers to Problems

9. !G " = - nf!!o= = - 2(96,485 J/volt # mol) (!o=(1,3-BPG/Gal3P) - !o=(NADP+/NADPH)) = -(192,970 J/volt # mol)(-0.29 V - (-0.32 V)) = -192,970 (0.03) J/mol = -5,789 J/mol. 10. Use the first eight reactions in Table 20.1 to show that: CO2 + Ru-1,5-BP + 3 H2O + 2 ATP + 2 NADPH + 2 H+ S

glucose + 2 ADP + 4 Pi + 2 NADP+

11. Radioactivity will be found in C-1 of 3-phosphoglycerate; C-3 and C-4 of glucose; C-1 and C-2 of erythrose-4-P; C-3, C-4, and C-5 of sedoheptulose-1,7-bisP; and C-1, C-2, and C-3 of ribose-5-P. 12. Light induces three effects in chloroplasts: (1) pH increase in the stroma, (2) generation of reducing power (as ferredoxin), and (3) Mg2+ efflux from the thylakoid lumen. Key enzymes in the Calvin–Benson CO2 fixation pathway are activated by one or more of these effects. In addition, rubisco activase is activated indirectly by light, and, in turn, activates rubisco. 13. The following series of reactions accomplishes the conversion of 2-phosphoglycolate to 3-phosphoglycerate: 1. 2 phosphoglycolate + 2 H2O S 2 glycolate + 2 Pi 2. 2 glycolate + 2 O2 S 2 glyoxylate + 2 H2O2 3. 2 glyoxylate + 2 serine S 2 hydroxypyruvate + 2 glycine 4. 2 glycine S serine + CO2 + NH3

5. hydroxypyruvate + glutamate S serine + a-ketoglutarate (an aminotransferase reaction) 6. a-ketoglutarate + NH3 + NADH+ + H+ S glutamate + H2O + NAD+ (the glutamate dehydrogenase reaction) 7. hydroxypyruvate + NADH+ + H+ S glycerate + NAD+ 8. glycerate + ATP S 3-phosphoglycerate + ADP Net: 2 phosphoglycolate + 2 NADH+ + 2 H+ + ATP + 2 O2 + H2O S 3-phosphoglycerate + CO2 + 2 Pi + ADP + 2 H2O2

+ 2 NAD+

14. Considering the reactions involving water separately: 1. ATP synthesis: 18 ADP + 18 Pi S 18 ATP + 18 H2O

2. NADP+ reduction and the photolysis of water: 12 H2O + 12 NADP+ S 12 NADPH + 12 H+ + 6 O2 3. Overall reaction for hexose synthesis: 6 CO2 + 12 NADPH + 12 H+ + 18 ATP + 12 H2O S

glucose + 12 NADP+ + 6 O2 + 18 ADP + 18 Pi Net: 6 CO2 + 6 H2O S glucose + 6 O2

Of the 12 waters consumed in O2 production in reaction 2 and the 12 waters consumed in the reactions of the Calvin–Benson cycle (reaction 3), 18 are restored by H2O release in phosphoric anhydride bond formation in reaction 1. 15. a. If the number of c-subunits increases, the H+/ATP ratio will increase. b. If the number of c-subunits increases, !p can be smaller. (More protons moving down a shallower !p will yield the same overall !G for ATP synthesis.) 16. a. 870-nm light: !G = -192 kJ/mol b. 700-nm light: !G = - 259 kJ/mol c. 680-nm light: !G = -269 kJ/mol 17. !G = +35.1 kJ 18. !G = -950 kJ

CHAPTER 21 1. The reactions that contribute to the equation on page 700 are: 2 Pyruvate + 2 H+ + 2 H2O S glucose + O2 2 NADH + 2 H+ + O2 S 2 NAD+ + 2 H2O 4 ATP + 4 H2O S 4 ADP + 4 Pi 2 GTP + 2 H2O S 2 GDP + 2 Pi Summing these four reactions produces the equation on page 700. 2. This problem essentially involves consideration of the three unique steps of gluconeogenesis. The conversion of PEP to pyruvate was shown in Chapter 17 to have a !G "= of #31.7 kJ/mol. For the conversion of pyruvate to PEP, we need only add the conversion of a GTP to GDP + Pi (equivalent to ATP to ADP + Pi) to the reverse reaction. Thus: Pyruvate S PEP: !G "= = +31.7 kJ/mol - 30.5 kJ/mol = 1.2 kJ/mol Then, using Equation 3.13: !G = 1.2 kJ/mol + RT ln ([PEP][ADP]2[Pi]/[Py][ATP]2) !G (in erythrocytes) = -30.1 kJ/mol In the case of the fructose-1,6-bisphosphatase reaction, !G "= = - 16.3 kJ/mol (see Equation 18.6). !G = -16.7 kJ/mol + RT ln ([F-6-P][Pi]/[F-1,6-BP]) !G (in erythrocytes) = -36.5 kJ/mol For the glucose-6-phosphatase reaction, !G "= = #13.9 kJ/mol (see Table 3.3). !G = -13.9 kJ/mol + RT ln ([Glu][Pi]/ [G-6-P]) = -21.1 kJ/mol. From these !G values and those in Table 17.1, !G for gluconeogenesis = -87.7 kJ/mol. 3. Inhibition by 25 mM fructose-2,6-bisphosphate is approximately 94% at 25 mM fructose-1,6-bisphosphate and approximately 44% at 100 mM fructose-1,6-bisphosphate. 4. The hydrolysis of UDP–glucose to UDP and glucose is characterized by a !G "= of #31.9 kJ/mol. This is more than sufficient to overcome the energetic cost of synthesizing a new glycosidic bond in a glycogen molecule. The net !G "= for the glycogen synthase reaction is #13.3 kJ/mol. 5. According to Table 9.1, a 70-kg person possesses 1920 kJ of muscle glycogen. Without knowing how much of this is in fast-twitch muscle, we can simply use the fast-twitch data from A Deeper Look (page 718) to calculate a rate of energy consumption. The plot on page 718 shows that glycogen supplies are exhausted after 60 minutes of heavy exercise. Ignoring the curvature of the plot, 1920 kJ of energy consumed in 60 minutes corresponds to an energy consumption rate of 533 J/sec. 6. Although other inhibitory processes might also occur, enzymes with mechanisms involving formation of Schiff base intermediates with active-site lysine residues are likely to be inhibited by sodium borohydride (see Chapter 17, problem 18). The transaldolase reaction of the pentose phosphate pathway involves this type of active-site intermediate and would be expected to be inhibited by sodium borohydride. 7. Glycogen molecules do not have any free reducing ends, regardless of the size of the molecule. If branching occurs every 8 residues and each arm of the branch has 8 residues (or 16 per branch point), a glycogen molecule with 8000 residues would have about 500 ends. If branching occurs every 12 residues, a glycogen molecule with 8000 residues would have about 334 ends.

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Abbreviated Answers to Problems

f. Increasing AMP would have the same effect as decreasing ATP—stimulation of glycolysis and inhibition of gluconeogenesis. g. Fructose-6-phosphate is not a regulatory molecule and decreases in its concentration would not markedly affect either glycolysis or gluconeogenesis (ignoring any effects due to decreased [G-6-P] as a consequence of decreased [F-6-P]).

8. a. Increased fructose-1,6-bisphosphate would activate pyruvate kinase, stimulating glycolysis. b. Increased blood glucose would decrease gluconeogenesis and increase glycogen synthesis. c. Increased blood insulin inhibits gluconeogenesis and stimulates glycogen synthesis. d. Increased blood glucagon inhibits glycogen synthesis and stimulates glycogen breakdown. e. Because ATP inhibits both phosphofructokinase and pyruvate kinase, and because its level reflects the energy status of the cell, a decrease in tissue ATP would have the effect of stimulating glycolysis.

9. At 298 K, assuming roughly equal concentrations of glycogen molecules of different lengths, the glucose-1-P concentration would be about 0.286 mM.

10. All four of these reactions begin with the formation of N-carboxybiotin: O

ADP ATP & HCO3#

#O

C

O O

Pi O#

P

O #O

C

O

N

O#

NH

O HN

S

NH

S

R

R

The carboxylation of b-methylcrotonyl-CoA occurs as follows: B H H2C

O C

CH

C

H 3C

C

#

CH

C

H3C #O

C

SCoA

CH3

O

H2C

SCoA

#OOC

CH2

C

O CH

C

SCoA

O O N

NH

S

A-25

R

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A-26

Abbreviated Answers to Problems

The carboxylations of geranyl-CoA and urea are shown below: O SCoA H H

B

O

O SCoA

#

SCoA COO#

#O

C

O O N

NH

S

R O

H N

C

NH2

H #O

C

O

O O

#OOC

NH

N

S

N H

C

NH2

R

The mechanism of the carboxyltransferase reaction is a combination of the pyruvate carboxylase mechanism (shown in Figure 21.3) and a reverse propionyl-CoA carboxylase reaction.

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Abbreviated Answers to Problems

11. a. The mechanism of the acetolactate synthase reaction. B

S

R R(

+ N

E

S

R

H

CH3

–

+ N

R(

R((

O.... H B

C

E

R(

COO–

R((

S

R

Pyruvate

+ N R(( O

CH3 C

OH

C O–

CO2 CH3

S

R

+ N

R(

C

OH

–

R(

R((

CH3

S

R

C N

OH

R(( CH3

COO–

C O

Condensation

CH3

S

R R(

+ N

C

O

C

COO–

R(( CH3

H

OH

OH

S

R R(

+ N

–

R((

+

CH3

C

C

O

CH3

COO–

α-Acetolactate

Valine, Leucine

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A-27

A-28

Abbreviated Answers to Problems

b. The phosphoketolase reaction. S

R

CH2

–

+ N R(

C

R((

HO

OH

CH2

S

R

C

+ N

O R(

CH

OH

H

O

R((

OH

CH HCOH

HCOH

HCOH

HCOH

CH2OPO32–

CH2OPO32– Fructose-6-P Erythrose-4-P

CH2

S

R

C

+ N R(

...

..

H+

OH

OH

+ N

H

R(

E

B

R((

CH2OH

S

R

C

OH

–

CH2OH

S

R

C N

R(

+ R(( H

OH

R((

H2O

..... H2C

S

R

O

–

R(

E

B

+

CH3

C

+ N

H

O

+ N

CH3

S

R

R((

S

R

E

C

+ N R(

+

.... H B

C

O

PO32–

Acetyl phosphate

R((

12. Metformin both stimulates glucose uptake by peripheral tissues and enhances the binding of insulin to its receptors. Glipizide complements the actions of metformin by stimulating increased insulin secretion by the pancreas. 13. Epalrestat and tolrestat do not resemble the transition state for aldose reductase, and evidence from studies by Franklin

R(

O

–

PO32–

O R(( Acetyl-TPP

CH3

S

R

+ N R(

C

O–

O

PO32–

R((

Prendergast and others (Ehrig, T., Bohren, K., Prendergast, F., and Gabbay, K., 1994. Mechanism of aldose reductase inhibition: Binding of NADP+/NADPH and alrestatin-like inhibitors. Biochemistry 33:7157–7165) show that these inhibitors probably bind to the enzyme:NADP+ complex at a site other than the active site.

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Abbreviated Answers to Problems

14. Glucose-6-P

Fructose-6-P ATP Fructose-1,6-bisP

DHAP Glyceraldehyde-3-P TK Xylulose-5-P

Erythrose-4-P TA Sedoheptulose-7-P Ru-5-P Epimerase

Glyceraldehyde-3-P TK

Xylulose-5-P

Ribulose-5-P

Ribose-5-P

Ru-5-P Isomerase Ribose-5-P

15. Carbons 1, 2, and 5 of ribose-5-P will be labeled. 16. 6 NADP+ 6 NADPH Glucose-6-P

6 NADP+

6 NADPH

6-Phosphogluconate

Ribulose-5-P

6 CO2

Xylulose-5-P

Ribose-5-P TK

Glyceraldehyde-3-P

Sedoheptulose-7-P

Xylulose-5-P

TA Fructose-6-P

Erythrose-4-P TK Fructose-6-P

Gluconeogenesis 4 Glucose-6-P

Glyceraldehyde-3-P Gluconeogenesis 1 Glucose-6-P

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A-29

A-30

Abbreviated Answers to Problems

17. 3 NADP+ Glucose-6-P

3 NADP+ 3 NADPH

3 NADPH

6-Phosphogluconate

Ribulose-5-P

3 CO2

Xylulose-5-P

Xylulose-5-P

Ribose-5-P TK

Glyceraldehyde-3-P

Sedoheptulose-7-P TA

Fructose-6-P

Erythrose-4-P TK

2 ATP PFK

Fructose-6-P

Glyceraldehyde-3-P

2 ADP

Fructose-1,6-bisP

DHAP

Glyceraldehyde-3-P

5 NAD+

Glycolysis

5 NADH Pyruvate

+ 10 ATP

18. Carbons 2 and 4 end up in carbons 1 and 3 of pyruvate. 19. The reference by Hurley et al. in the Further Reading for this chapter suggests a mechanism based on structural data on glycogenin. Nucleophilic attack by Asp162 on the C-1 of glucose (in UDP-glucose) forms a covalent enzyme-substrate intermediate. Subsequent nucleophilic attack by the hydroxyl oxygen of Tyr194 of glycogenin produces the tyrosyl glucose that forms the foundation for synthesis of glycogen particles.

20. During the first few seconds of exercise, existing stores of ATP are consumed and creatine phosphate then provides additional ATP via the creatine kinase reaction. For the next 90 seconds or so, anaerobic metabolism (conversion of glucose to lactate via glycolysis) provides energy. At this point, aerobic metabolism begins in earnest, delivering significant energy resources to sustain long-term exercise. 21. Creatine phosphate provides ATP to muscle via the creatine kinase reaction:

During periods of energy abundance, muscles store ATP equivalents in the form of creatine phosphate. When the muscles demand energy, creatine kinase runs in the reverse direction, converting creatine phosphate to ATP.

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Abbreviated Answers to Problems

CHAPTER 22 1. Assuming that all fatty acid chains in the triacylglycerol are palmitic acid, the fatty acid content of the triacylglycerol is 95% of the total weight. On the basis of this assumption, one can calculate that 30 lb of triacylglycerol will yield 118.7 L of water. 2. 11-cis-Heptadecenoic acid is metabolized by means of seven cycles of b-oxidation, leaving a propionyl-CoA as the final product. However, the fifth cycle bypasses the acyl-CoA dehydrogenase reaction, because a cis-double bond is already present at the proper position. Thus, b-oxidation produces 7 NADH ( = 17.5 ATP), 6 FADH2 ( = 9 ATP), and 7 acetyl-CoA ( = 70 ATP), for a total of 96.5 ATP. Propionyl-CoA is converted to succinyl-CoA (with expenditure of 1 ATP), which can be converted to oxaloacetate in the TCA cycle (with production of 1 GTP, 1 FADH2, and 1 NADH). Oxaloacetate can be converted to pyruvate (with no net ATP formed or consumed), and pyruvate can be metabolized in the TCA cycle (producing 1 GTP, 1 FADH2, and 4 NADH). The net for these conversions of propionate is 16.5 ATP. Together with the results of b-oxidation, the total ATP yield for the oxidation of one molecule of 11-cis-heptadecenoic acid is 113 ATP. 3. Instead of invoking hydroxylation and b-oxidation, the best strategy for oxidation of phytanic acid is a-hydroxylation, which places a hydroxyl group at C-2. This facilitates oxidative a-decarboxylation, and the resulting acid can react with CoA to form a CoA ester. This product then undergoes six cycles of b-oxidation. In addition to CO2, the products of this pathway are three molecules of acetyl-CoA, three molecules of propionyl-CoA, and one molecule of 2-methylpropionyl-CoA. 4. Although acetate units cannot be used for net carbohydrate synthesis, oxaloacetate can enter the gluconeogenesis pathway in the PEP carboxykinase reaction. (For this purpose, it must be converted to malate for transport to the cytosol.) Acetate labeled at the carboxyl carbon will first label (equally) the C-3 and C-4 positions of newly formed glucose. Acetate labeled at the methyl carbon will label (equally) the C-1, C-2, C-5, and C-6 positions of newly formed glucose. 5. This exercise is left to the student, in consultation with the references suggested in the problem. 6. Fat is capable of storing more energy (37 kJ/g) than carbohydrate (16 kJ/g). Ten pounds of fat contains 10 * 454 * 37 = 167,980 kJ of energy. This same amount of energy would require 167,980/16 = 10,499 g, or 23 lb, of stored carbohydrate.

A-31

7. The enzyme methylmalonyl-CoA mutase, which catalyzes the third step in the conversion of propionyl-CoA to succinyl-CoA, is B12-dependent. If a deficiency in this vitamin occurs, and if large amounts of odd-carbon fatty acids were ingested in the diet, L-methymalonyl-CoA could accumulate. 8. a. Myristic acid: CH3(CH2)12COOH + 92 Pi + 92 ADP + 20 O2 S 92 ATP + 14 CO2 + 106 H2O b. Stearic acid: CH3(CH2)16COOH + 120 Pi + 120 ADP + 26 O2 S 120 ATP + 18 CO2 + 138 H2O c. a-Linolenic acid: C17H29COOH + 113.5 Pi + 113.5 ADP + 24.5 O2 S 113.5 ATP + 18 CO2 + 128.5 H2O d. Arachidonic acid: C19H31COOH + 125 Pi + 125 ADP + 27 O2 S 125 ATP + 20 CO2 + 141 H2O

+ CoA

+ CoA

+ CoA

+ CoA

9. During the hydration step, the elements of water are added across the double bond. Also, the proton transferred to the acetyl-CoA carbanion in the thiolase reaction is derived from the solvent, so each acetyl-CoA released by the enzyme would probably contain two tritiums. Seven tritiated acetyl-CoAs would thus derive from each molecule of palmitoyl-CoA metabolized, each with two tritiums at C-2. 10. A carnitine deficiency would presumably result in defective or limited transport of fatty acids into the mitochondrial matrix and reduced rates of fatty acid oxidation. 11. 1.3 grams * 37 kJ/gram = 48,000 J. This at first seems like a remarkably small amount of energy to sustain the hummingbird during a 500-mile flight at 50 mph. However, keep in mind that the hummingbird weighs only 3 to 4 grams, and see problem 12 following. 12. 48,000 J in 10 hours is 4800 J/hr. If the hummingbird consumes 250 mL per hour during migration, this means that it is consuming 4800/250 or 19.2 J/mL of oxygen consumed. If a human consumes 12.7 kcal/min while running, this is equivalent to 12,700 * 4.184 J/cal = 53.1 kJ/min; 48,000 J/53,100 J/min = 0.9 minute. So a human could run for only less than a minute on the energy consumed by the hummingbird (only 3 to 4 g) on a 500-mile flight. A typical person would have to run about 40 miles to lose 1 lb of fat. 13. A mechanism for the HMG-CoA synthase reaction:

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A-32

Abbreviated Answers to Problems

14.

H

_

H

COO

C

C

H

H

C

O

H

COO

C

C

H

C

O

SCoA CH2

O

A

CH2

OH OH

SCoA O

A

CH3

OH OH

N N Co3+ N N

N N Co2+ N N

C O

A

H

CH2 O

SCoA

CH2

OH OH

N N Co3+ N N

O

A

H

_ COO

C

C

C

O

SCoA

H

H

CH2

OH OH

N N Co2+ N N

A

N N Co2+ N N

_

H2C

O

OH OH

COO

CH2

_

H

O

A

OH OH

N N Co2+ N N

15. The changes in oxidation state of cobalt in the course of any B12-dependent reaction arise from homolytic cleavages of the Co3+ i C bonds involved. Moreover, in the Co3+ i C bonds shown in a typical B12-dependent reaction, the two electrons of the bond are not ascribed to the Co3+ atom in the calculation of the Co oxidation state. Thus, when the Co i C bond is cleaved homolytically, one electron reverts to the Co, changing its oxidation state from 3+ to 2+.

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Abbreviated Answers to Problems

16. See the following mechanisms. !-Methyleneglutarate mutase

H #OOC

H

C

#OOC

CH2

H

C

B12

C

CH2

COO#

COO#

C

CH2

CH2 H #OOC

H #OOC

C

CH2

C

COO# H

CH2

C

CH3

C

COO#

CH2

B12

Diol dehydrase

H H3C

CH

C

OH

H

H H3C

OH

CH

C

OH

OH

B12 H H3C

CH

C

O

B

H

OH

H B12

H H3C

B H

CH2

C O

&H2O

Glycerol dehydrase

H CH2

CH

C

OH

OH

OH

H H B12

CH2

CH

C

OH

OH

OH

H CH2

CH

OH B12

H B

C

OH

O

H

H CH2 OH

B

H

CH2

C O

&H2O

Ethanolamine ammonia-lyase

H CH2

C

NH3& OH

H CH2

H

C

NH3& OH

B12

H B12

H

CH2 B H

C

H O

NH3&

B

H CH3

C O

&NH4&

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A-33

A-34

Abbreviated Answers to Problems

17. H

CH2 H2C

C

CH

CH2

COO–

C

NH3+ Hypoglycin A

CoASH

H2C

C

CH

H3C

O

CH2 CH2

C

H2C

SCoA

N

Methylenecyclopropylacetyl-CoA (MCPA-CoA)

CH2 H2C

C

CH

– CH

C

CH

CH

N

O

C

SCoA

NH O

O

H2C – C

SCoA

C

N

H+ O

O

CH

CH

C

SCoA

+

FAD

H2C Reactive intermediate

18. This exercise is left to the student and should be based on the reference provided in the problem. 19. It may be presumed that the oxidation of the acyl chain is accomplished via a two-electron transfer, whereas the steps involved in reoxidation of FADH2 by ETF are one-electron transfers. FAD/FADH2 can participate both in one-electron and two-electron transfers, whereas NAD+/NADH can participate only in two-electron transfers. 20. The sequence of reactions involving creation of a double bond, then hydration across it, followed by oxidation is what happens to succinate in the TCA cycle. (This same sequence of reactions is also employed in fatty acid synthesis and in both the catabolism and anabolism of amino acids.) Clearly, this sequence of three reactions must represent an optimal mechanistic strategy for the chemistry achieved. CHAPTER 23 1. The oxidation number of N in nitrate is &5; in nitrite, &3; in NO, &2; in N2O, &1; and in N2, 0. 2. a. Assume that nitrate assimilation requires 4 NADPH equivalents per NO3- reduced to NH4+. Four NADPH have a metabolic value of 12 ATP. b. Nitrogen fixation requires 8 e- (see Equation 23.3) and 16 ATPs (see Figure 23.6) per N2 reduced. If 4 NADH provide the requisite 8 e-, each NADH having a metabolic value of 3 ATPs, then 28 ATP equivalents are consumed per N2 reduced in biological nitrogen fixation (or 14 ATP equivalents per NH4+ formed). 3. a. [ATP] increase will favor adenylylation; the value of n will be greater than 6 (n 7 6). b. An increase in PIIA/PIID will favor adenylylation; the value of n will be greater than 6 (n 7 6). c. An increase in the [aKG]/[Gln] ratio will favor deadenylylation; n 6 6. d. [Pi] decrease will favor adenylylation; n 7 6.

4. Two ATPs are consumed in the carbamoyl-P synthetase-I reaction, and 2 phosphoric anhydride bonds (equal to 2 ATP equivalents) are expended in the argininosuccinate synthetase reaction. Thus, 4 ATP equivalents are consumed in the urea cycle, as 1 urea and 1 fumarate are formed from 1 CO2, 1 NH3, and 1 aspartate. 5. Protein catabolism to generate carbon skeletons for energy production releases the amino groups of amino acids as excess nitrogen, which is excreted in the urine, principally as urea. 6. One ATP in reaction 1, one NADPH in reaction 2, one NADPH in reaction 11, and one succinyl-CoA in reaction 12 add up to 10 ATP equivalents, assuming each NADPH is worth 4 ATPs. (The succinyl-CoA synthetase reaction of the citric acid cycle [see p. 603] fixes the metabolic value of succinyl-CoA versus succinate at 1 GTP [ = 1 ATP].) 7. From Figure 23.36: 14C-labeled carbon atoms derived from 14C-2 of PEP are shaded yellow. 8. 1. 2 aspartate S transamination S 2 oxaloacetate. 2. 2 oxaloacetate + 2 GTP S PEPcarboxykinase S 2 PEP + 2 CO2 + 2 GDP. 3. 2 PEP + 2 H2O S enolase S 2 2-PG. 4. 2 2-PG S phosphoglyceromutase S 2 3-PG. 5. 2 3-PG + 2 ATP S 3-P glycerate kinase S 2 1,3-bisPG + 2 ADP. 6. 2 1,3-bisPG + 2 NADH + 2 H+ S G-3-P dehydrogenase S 2 G-3-P + 2 NAD+ + 2 Pi. 7. 1 G-3-P S triose-P isomerase S 1 DHAP. 8. G-3-P + DHAP S aldolase S fructose-1,6-bisP. 9. F-1,6-bisP + H2O S FBPase S F-6-P + Pi. 10. F-6-P S phosphoglucoisomerase S G-6-P. 11. G-6-P + H2O S glucose phosphatase S glucose + Pi. NEL

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A-35

Abbreviated Answers to Problems

Net: 2 aspartate + 2 GTP + 2 ATP + 2 NADH + 6 H+ + 4 H2O S glucose + 2 CO2 + 2 GDP + 2 ADP + 4 Pi + 2 NAD+ (Note that 4 of the 6 H+ are necessary to balance the charge on the 4 carboxylate groups of the 2 OAA and the loss of amino groups from Asp is ignored.) (As a consequence of reaction [1], 2 a-keto acids [for example, a-ketoglutarate] will receive amino groups to become 2 a-amino acids [for example, glutamate].) 9. Alanine: glutamate:pyruvate aminotransferase. Arginine: from glutamate via ornithine, so it’s glutamate dehydrogenase. Aspartate: glutamate:oxaloacetate aminotransferase. Asparagine: from aspartate, so it’s the glutamate:oxaloacetate aminotransferase. Cysteine: cysteine is formed from serine, so it’s glutamate via 3-phosphoserine aminotransferase. Glutamate: glutamate dehydrogenase. Glutamine: glutamine synthetase.

13. Aspartame is a N-a-L-aspartyl-L-phenylalanine-1-methyl ester that is broken down in the digestive tract and phenylalanine is released. Phenylalanine is the substance that phenylketonurics must avoid. 14. Glyphosate inhibits 3-enolpyruvylshikimate-5-P synthase, an essential enzyme in the biosynthesis of chorismate. Not only is chorismate the precursor for synthesis of the aromatic amino acids Phe, Tyr, and Trp, it is also the precursor for formation of lignin, a major structural component in plant cell walls, as well as other essential substances such as folate, coenzyme Q, plastoquinone, and vitamins E and K. 15. Glycine is formed from serine, which is formed from 3-phosphoglycerate. Carbons 3 and 4, 2 and 5, and 1 and 6 of glucose contribute carbons 1, 2, and 3 of 3-phosphoglycerate, respectively. The b-carbon of serine derives from the 3-C in 3-phosphoglycerate, the Ca-carbon atom in serine comes from 3-phosphoglycerate C-2, and the carboxyl-C in serine comes from C-1 of 3-PG. It is these latter two carbons of 3-PG that are found in glycine; that is, C-1 and C-2 of 3-PG; C-1 of 3-PG came from C-3 and C-4 in glucose, and C-2 of 3-PG came from C-2 and C-5 in glucose.

Glycine: glycine is formed from serine, so it’s glutamate via 3-phosphoserine aminotransferase.

16. Serine is an important precursor for glycine synthesis.

Histidine: from glutamate via l-histidinol phosphate aminotransferase.

18. The liver converts amino acids to glucose to provide a source of energy for other cells, such as nerve and red blood cells. Conversion of amino acids to glucose by gluconeogenesis requires energy; for example, six ATP equivalents are needed to form glucose from aspartic acid.

Isoleucine: glutamate:a-keto-b-methylvalerate aminotransferase. Leucine: glutamate:a-ketoisocaproate aminotransferase. Lysine: from glutamate via saccharopine formation by a glutamate-dependent NADPH dehydrogenase; in bacteria, lysine is synthesized from aspartate, so it’s glutamate:oxaloacetate aminotransferase. Methionine: from aspartate, so it’s glutamate:oxaloacetate aminotransferase. Phenylalanine: glutamate:phenylpyruvate aminotransferase ($ phenylalanine aminotransferase).

17. Consult www.pdb.org to view pdb id = 1LM1.

CHAPTER 24 1.

H &

O HO

P

OCH2 5(

#O

Proline: from glutamine, so it’s glutamate dehydrogenase.

H

N N

O

O H

N

NH2

N

H

H

H OH OH

Serine: glutamate via 3-phosphoserine aminotransferase. Threonine: from aspartate, so it’s glutamate:oxaloacetate aminotransferase.

2. See Figure 24.15.

Tryptophan: from serine via tryptophan synthase, so its a-amino group comes from serine, which gets its amino group from glutamate via 3-phosphoserine aminotransferase.

4. The number of A and T residues = 3.14 * 109 each; the number of G and C residues = 2.68 * 109 each.

Tyrosine: glutamate:4-hydroxyphenylpyruvate aminotransferase ($ tyrosine aminotransferase).

6. 5=-ATCGCAACTGTCACTA-3=.

Valine: glutamate:a-ketoisovalerate aminotransferase. 10. Pyridoxal (vitamin B6), because it is the precursor to pyridoxal-P, the key coenzyme in aminotransferase reactions, as well as other aspects of amino acid metabolism. 11. The conversion of homocysteine to methionine is folatedependent; dietary folate absorption is dependent on vitamin B12 for removal of methyl groups added to folate during digestion; finally, the a-amino group of homocysteine formed in the methione biosynthetic pathway comes from aspartate via the pyridoxal-P–dependent glutamate:oxaloacetate aminotransferase. 12. The BCKAD complex is structurally and functionally analogous to the pyruvate dehydrogenase complex and the a-ketoglutarate dehydrogenase complexes. See Figure 18.4; the reaction mechanism for the pyruvate dehydrogenase complex is essentially the same as that of the BCKAD complex.

3. fA = 0.29; fG = 0.22; fC = 0.25; fT = 0.28.

5. 5=-TAGTGACAGTTGCGAT-3=. 7. 5=-TACGGTCTAAGCTGA-3=. 8. There are two possibilities, a and b. (E $ EcoRI site; B $ BamHI site.) Note that b is the reverse of a. a. B 1

E B 3

0.5

5.5

b. B E 5.5

0.5

B 3

9. a. GGATCCCGGGTCGACTGCAG; b. GTCGACCCGGGATCCTGCAG.

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1

A-36

Abbreviated Answers to Problems

phosphodiester bond formation in polynucleotide synthesis.

SmaI products: a. GGATCCC and GGGTCGACTGCAG. b. GTCGACCC and GGGATCCTGCAG. 10. Synthesis of a polynucleotide 100 residues long requires formation of 99 phosphodiester bonds. !G "= for phosphodiester synthesis (assuming it is the same magnitude but opposite sign as that for phosphoric anhydride cleavage of an NTP to give NMP + PPi) is &32.3 kJ/mol. !G "=overall = (99)(+32.3) = 3198 kJ/mol.

15. Once in every 4.4 * 1012 nucleotides.

11. a. Hydrogen bonding and van der Waals interactions between amino acid side chains and DNA, and ionic interactions of amino acid side chains with the nucleic acid backbone phosphate groups. Double-helical DNA does not present hydrophobic regions for interaction with proteins because of base-pair stacking. b. Proteins can recognize specific base sequences if they can fit within the major or minor groove of DNA and “read” the H-bonding pattern presented by the edges of the bases in the groove. The dimensions of an a-helix are such that it fits snugly within the major groove of B-DNA; then, depending on the amino acid sequence of the protein, the side chains displayed on the circumference of the a-helix have the potential to form H bonds with H-bonding functions provided by the bases.

18. 3.59 * 1012 D.

16. A DNA sequence 16 nucleotides long. 17. The strategy for protein sequencing by Edman degradation, as described on page 113, could be adapted, replacing Edman’s reagent with snake venom phosphodiesterase acting on an immobilized nucleotide sequence.

12. a. The restriction endonuclease must be able to recognize a specific nucleotide sequence in the DNA that has twofold rotational symmetry. b. In order to read a base sequence within DNA, either the restriction endonuclease must interact with the bases by direct access, for example, by binding in the major groove, or the restriction endonuclease must be able to “read” the base sequence by indirect means, for example, if the base sequence imparts some local variation in the cylindrical surface of the DNA that the enzyme might recognize. c. The restriction endonuclease must be able to cleave both DNA strands, often in a staggered fashion. d. An obvious solution to the requirements listed in (a) and (c) would be a homodimeric subunit organization for restriction endonucleases. 13. a. The ribose group of nucleosides greatly increases the water solubility of the base. b. Ribose has fewer hydroxyl groups and thus less likelihood to undergo unwanted side reactions. c. The absence of the 2-OH in 2-deoxyribose leads to a polynucleotide sugar–phosphate backbone that is more stable because it is not susceptible to alkaline hydrolysis. 14. a. Phosphate groups bear a negative electrical charge at neutral pH. b. Cleavage of phosphoric anhydride bonds is strongly exergonic and can provide the thermodynamic driving force for diverse metabolic reactions. c. Cleavage of phosphoric anhydride bonds is strongly exergonic and can provide the thermodynamic driving force for

CHAPTER 25 1. T C T A C G G A C T G A

2. Original nucleotide: 5´-GATAGCGCAAAGATCAACCTT. 3. a. 10.5 base pairs per turn; b. !f = 34.3"; c (true repeat) = 6.72 nm. 4. 27.3 nm; 122 base pairs. 5. 4.35 mm. 6. L0 = 160. If W = -12, L = T + W = 160 + (-12) = 148. s = !L/L0 = - 12/160 = -0.075. 7. For 1 turn of B-DNA (10 base pairs): LB = 1.0 + WB. For Z-DNA, 10 base pairs can only form 10/12 turn (0.833 turn), and LZ = 0.833 + WZ. For the transition B-DNA to Z-DNA, strands are not broken, so LB = LZ; that is, 1.0 + WB = 0.833 = WZ, or WZ - WB = +0.167. (In going from B-DNA to Z-DNA, the change in W, the number of supercoils, is positive. This result means that, if B-DNA contains negative supercoils, their number will be reduced in Z-DNA. Thus, all else being equal, negative supercoils favor the B S Z transition.) 8. 6 * 109 bp/200 bp = 3 * 107 nucleosomes. The length of B-DNA 6 * 109 bp long = (0.34 nm)(6 * 109) = 2.04 * 109 nm (more than 2 meters!). The height of 3 * 107 nucleosomes = (6 nm)(3 * 107) = 18 * 107 nm (0.18 meter). 9. From Figure 25.33: Similarly shaded regions indicate complementary sequences joined via intrastrand hydrogen bonds:

anticodon

0

10

20

30

40 nucleotide no.

CCA end

50

60

70

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Abbreviated Answers to Problems

10. Increasing order of Tm: yeast 6 human 6 salmon 6 wheat 6 E. coli. 11. In 0.2 M Na+, Tm("C) = 69.3 + 0.41(% G + C): Rats (% G + C) = 40%, Tm = 69.3 + 0.41(40) = 85.7 "C Mice (% G + C) = 44%, Tm = 69.3 + 0.41(44) = 87.3 "C Because mouse DNA differs in GC content from rat DNA, they could be separated by isopycnic centrifugation in a CsCl gradient. 12. GC content = 0.714 (from Table 24.1 and equations used in problem 3, Chapter 24). r = 1.660 + 0.098(GC) = 1.730 g/mL. 13. See Figure 25.35 and compare your structure with the base pair formed between G18 and c55 in yeast phenylalanine tRNA, which has a single H bond between the 2-amino group of G (2-NH2gO “ ) and the O atom at position 4 in c. 14. Assuming the plasmid is in the B-DNA conformation, where each pair contributes 0.34 nm to the length of the molecule, the circumference of a perfect circle formed from pBR322 would be (0.34 nm/bp)(4363 bp) = 1483 nm = 1.48 mm. The E. coli K12 chromosome laid out as a perfect circle would have a circumference of (0.34 nm/bp) (4,639,000 bp) = 1,577,260 nm = 1.58 mm. The diameter of a B-DNA molecule is about 2.4 nm (see Table 25.1). For pBR322, the length/diameter ratio would be 1480/2.4 = 616.7. For the E. coli K12 chromosome, the length/ diameter ratio would be 1,577,260/2.4 = 657,192. 15. (a) Z-DNA; (b) cruciform; (c) triplex DNA; (d) tRNA; (e) type-II restriction endonuclease site. 16. Rely on the features in Figure 25.33 to draw a tRNA cloverleaf with the anticodon positioned at nucleotides 34–36: CUG. 17. (a) b-globin; (b) hexosaminidase A (a-polypeptide); (c) insulin receptor; (d) neutral amino acid transporter. CHAPTER 26 1. See Figure 26.2 (purines) and Figure 26.13 (pyrimidines). 2. Assume ribose-5-P is available. Purine synthesis: 2 ATP equivalents in the ribose-5-P pyrophosphokinase reaction, 1 in the GAR synthetase reaction, 1 in the FGAM synthetase reaction, 1 in the AIR carboxylase reaction, 1 in the CAIR synthetase reaction, and 1 in the SACAIR synthetase reaction yields IMP, the precursor common to ATP and GTP. Net: 7 ATP equivalents. a. ATP: 1 GTP (an ATP equivalent) is consumed in converting IMP to AMP; 2 more ATP equivalents are needed to convert AMP to ATP. Overall, ATP synthesis from ribose-5-P onward requires 10 ATP equivalents. b. GTP: 2 high-energy phosphoric anhydride bonds from ATP, but 1 NADH is produced in converting IMP to GMP; 2 more ATP equivalents are needed to convert GMP to GTP. Overall, GTP synthesis from ribose-5-P onward requires 8 ATP equivalents. Pyrimidine synthesis: Starting from HCO3- and Gln, 2 ATP equivalents are consumed by CPS-II, and an NADH

A-37

equivalent is produced in forming orotate. OMP synthesis from ribose-5-P plus orotate requires conversion of ribose5-P to PRPP at a cost of 2 ATP equivalents. Thus, the net ATP investment in UMP synthesis is just 1 ATP. c. UTP: Formation of UTP from UMP requires 2 ATP equivalents. Net ATP equivalents in UTP biosynthesis = 3. d. CTP: CTP biosynthesis from UTP by CTP synthetase consumes 1 ATP equivalent. Overall ATP investment in CTP synthesis = 4 ATP equivalents. 3. a. See Figure 26.6. b. See Figure 26.17. c. See Figure 26.17. 4. a. Azaserine inhibits glutamine-dependent enzymes, as in steps 2 and 5 of IMP synthesis (glutamine:PRPP amidotransferase and FGAM synthetase), as well as GMP synthetase (step 2, Figure 26.5), and CTP synthetase (Figure 26.16). b. Methotrexate, an analog of folic acid, antagonizes THFdependent processes, such as steps 4 and 10 (GAR transformylase and AICAR transformylase) in purine biosynthesis (Figure 26.3), and the thymidylate synthase reaction (Figure 26.26) of pyrimidine metabolism. c. Sulfonamides are analogs of p-aminobenzoic acid (PABA). Like methotrexate, sulfonamides antagonize THF formation. Thus, sulfonamides affect nucleotide biosynthesis at the same sites as methotrexate, but only in organisms such as prokaryotes that synthesize their THF from simple precursors such as PABA. d. Allopurinol is an inhibitor of xanthine oxidase (Figure 26.10). e. 5-Fluorouracil inhibits the thymidylate synthase reaction (Figure 26.26). 5. UDP, via conversion to dUDP (Figure 26.24), is ultimately a precursor to dTTP, which is essential to DNA synthesis. 6. See Figure 26.23. 7. Ribose, as ribose-5-P, is released during nucleotide catabolism (as in Figure 26.8). Ribose-5-P is catabolized via the pentose phosphate pathway and glycolysis to form pyruvate, which enters the citric acid cycle. From Figure Chapter 21, problem 17, note that 3 ribose-5-P (rearranged to give 1 ribose-5-P and 2 xylulose-5-P) give a net consumption of 2 ATPs and a net production of 8 ATPs and 5 NADH ( = 15 ATPs), when converted to 5 pyruvate. If each pyruvate is worth 15 ATP equivalents (as in a prokaryotic cell), the overall yield of ATP from 3 ribose-5-P is 75 ATP + 23 ATP - 2 ATP = 96 ATP. Net yield per ribose-5-P is thus 32 ATP equivalents. 8. Comparing Figures 26.3 and 23.40, note that AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) is a common intermediate in both pathways. It is a product of step 5 of histidine biosynthesis (Figure 23.40) and step 9 of purine biosynthesis (Figure 26.3). Thus, formation of AICAR as a byproduct of histidine biosynthesis from PRPP and ATP bypasses the first nine steps in purine synthesis. However, cells require greater quantities of purine than of histidine, and these nine reactions of purine synthesis are essential in satisfying cellular needs for purines.

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A-38

Abbreviated Answers to Problems

9. See the following figure. STEP 6

O

H O

N

C

ADP

O

O#

H

NH

HN

P

#O

O H &N

O C

H

O #O

O #O

P

ADP

O

HC

O#

C

N

H2N

N

2#O

HC H B

GDP3-

4. 2 Urea + 2 H2O S urease S 2 CO2 + 2 HN3

a

b

C

N

H 2N

N

C

N CH N

COO# H2N

N

H

P2i

COO# COO# HC

NH

O C

N

COO# H2N

N

CH2

O N

+ + fumarate + 11. 1. Uric acid + 2 H2O + O2 S urate oxidase S allantoin + H2O2 + CO2 2. Allantoin + H2O S allantoinase S allantoic acid + glyoxylic acid 3. Allantoic acid + H2O S allantoicase S 2 urea

c

H2N

H B O

NH

10. Aspartate + GTP4- + H2O S NH+4

NH2

C

C

CH2

O O

3P

COO#

v

N

C

COO#

STEP 9

12.

H

CH

HN BH

B STEP 8

N

H

O#

H

NH

HN

P

B O#

H2N

C

N

H2N

N

13. ATCase (subunit organization a6b6) consists of 6 functional units, each composed of 1 a-subunit and 1 b-subunit, best described as (ab)6. 14. From these starting materials to UTP in a eukaryotic cell, where mitochondrial oxidation of dihydroorotate via a coenzyme Q–linked flavoprotein would yield 1.5 ATPs, has a net cost of 2.5 ATPs. From UTP to dTTP would require an ATP at the CTP synthetase step; recovery of an ATP in the nucleoside diphosphate kinase reaction ADP + CTP S CDP + ATP; an NADPH ( =4 ATP equivalents) in converting CDP S dCDP, recovery of an ATP equivalent in the deoxycytidylate kinase reaction dCDP + ADP S dCMP + ATP, deamination of dCMP to dUMP by dCMP deaminase; conversion of dUMP to dTMP by thymidylate synthase and then 2 ATP equivalents to form dTTP. Net: 7.5 ATP equivalents versus 10 ATP for ATP synthesis via the purine biosynthetic pathway. 15. 1. UMP + ATP S UDP + ADP 2. UDP + NADPH S dUDP + NADP+ + OH3. dUDP + ATP S dUTP + ADP 4. dUTP + H2O S dUMP + PPi 5. PPi + H2O S 2 Pi

[Aspartate]

6. dUMP + N5,N10-methylene-THF S dTMP + DHF NEL

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Abbreviated Answers to Problems

Net: UMP + N5,N10-methylene-THF + 2 ATP + NADPH + 2 H2O S dTMP + DHF + 2 ADP + 2 Pi + OH- + NADP+ 16. Possibilities include glutamine phosphoribosyl pyrophosphate amidotransferase (see Figure 26.3), adenine phosphoribosyl transferase and hypoxanthine–guanine phosphoribosyl transferase (see Figure 26.7), ribose-5-phosphate pyrophosphokinase (see Figure 26.3), and enzymes of the pyrimidine salvage pathway. 17. Explore pdb id = 7DFR. Note the proximity of the 4-position of the nicotinamide ring (the hydride donor) to the 7-position of folate (the hydride acceptor). Note that polar groups on the substrates are solvent-exposed. 18. View pdb id = 1RAA to see that the CTP-binding sites on the R-subunits are dominated by b-strands. View pdb id = 1RAA to see the ATCase substrate-binding sites (evident from the location of the substrate analog N-2-(phosphonoacetyl)L-asparagine). The allosteric (CTP-binding) sites and the active sites are at opposite ends of the protein. Thus, CTP binding is communicated to the active site via ligand-induced conformational changes extending across the entire protein. R-state ATCase is represented by pdb id = 2IPO; T-state by pdb id = 1RAA. CHAPTER 27 1. Linear and circular DNA molecules consisting of one or more copies of just the genomic DNA fragment; linear and circular DNA molecules consisting of one or more copies of just the vector DNA; linear and circular DNA molecules containing one or more copies of both the genomic DNA fragment and plasmid DNA. 2. -GAATTCCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCGAATTC GGATCC GTCGAC GCATGC EcoRI BamHI SalI SphI CCCGGG TCTAGA CTGCAG SmaI XbaI PstI 3. a. AAGCTTGAGCTCGAGATCTAGATCGAT HindIII Xho I Xba I Sac I Bg III Cla I b. Vector:

Hind III: 5(-A. . . . . . . .-gap-. . . . . . . . .CGAT-3(: ClaI 3(-TTCGA. . . .-gap-. . . . . . . . . . .TA-5(

Fragment: Hind III: 5(-AGCTT(NNNN-etc-NNNN)AT-3( 3(-A(NNNN-etc-NNNN)TAGC-5( 4. N = 3480. 5. N = 10.4 million. 6. 5´-ATGCCGTAGTCGATCAT and 5´-ATGCTATCTGTCCTATG. 7. -Thr-Met-Ile-Thr-Asn-Ser-Pro-Asp-Pro-Phe-Ile-His-Arg-ArgAla-Gly-Ile-Pro-Lys-Arg-Arg-Pro… The junction between b-galactosidase and the insert amino acid sequence is between Pro and Asp, so the first amino acid encoded by the insert is Asp. (The polylinker itself codes for Asp just at the BamHI site, but in constructing the fusion, this Asp and all of this downstream section of polylinker DNA is displaced to a position after the end of the insert.) 8. 5´(G)AATTCNGGNATGCAYCCNGGNAARCTTNYGCNAGYTGGTTYGTNGGGAATTCN-

A-39

(Note: The underlined triplet AGY represents the middle Ser residue. Ser codons are either AGY or TCN [where Y $ pyrimidine and N $ any base]; AGY was selected here so that the mutagenesis of this codon to a Cys codon [TGY] would involve only an A S T change in the nucleotide sequence.) Because the middle Ser residue lies nearer to the 3´-end of this EcoRI fragment, the mutant primer for PCR amplification should encompass this end. That is, it should be the primer for the 3 S 5= strand of the EcoRI fragment: 5´-NNNGAATTCCCNcACRcAACCARcCANcGC-3´ where the mutated Ser S Cys triplet is underlined, NNN $ several extra bases at the 5´-end of the primer to place the EcoRI site internal, Nc $ the nucleotide complementary to the nucleotide at this position in the 5= S 3= strand, and Rc $ the pyrimidine complementary to the purine at this position in the 5= S 3= strand. 9. The number of sequence possibilities for a polymer is given by xy, where x is the number of different monomer types and y is the number of monomers in the oligomers. Thus, for RNA oligomers 15 nucleotides long: xy = 415 = 1,073,741,824. For pentapeptides, xy = 205 = 3,200,000. 10. See Figure 27.17. You would need a GAL4-deficient yeast strain expressing a fusion protein composed of the GAL4 DB domain fused with a cytoskeletal protein (such as actin or tubulin) to serve as the bait. These GAL4- cells would be transformed with a cDNA library of human epithelial proteins constructed so that the human proteins were expressed as GAL4 TA fusion proteins; these fused proteins are the target. Interaction of a target protein with the cytoskeletal protein fused to the GAL4 DB domain will lead to GAL4-driven expression of the lacZ gene, whose presence can be revealed by testing for b-galactosidase activity. 11. See Figure 27.9 for isolation of mRNA and Figure 27.10 for preparation of a cDNA library from mRNA. The mRNA would be isolated, and the cDNA libraries would be prepared from two batches of yeast cells, one grown aerobically and one grown anaerobically. 12. See Figure 27.11. Differently labeled single-stranded cDNA from separate libraries prepared from aerobically versus anaerobically grown yeast cells would be hybridized with a DNA microarray (gene chip) of yeast genes. 13. An antibody against hexokinase A could be used to screen the yeast cDNA library to identify a yeast colony expressing this protein (see discussion on page 912–913). Once a sample of the putative yeast hexokinase A protein was isolated, its identity could be confirmed by peptide mass fingerprinting using mass spectrometry (see page 121). 14. The experiment in problem 12 identified the cDNA clone for the protein, but this clone will not have the regulatory elements (promoter) necessary for the experiment at hand. Using the cDNA clone as a probe, the genomic clone for this nox gene could be identified in a yeast genomic library. Cloning and sequencing a genomic clone would identify putative promoter regions that could be fused to the coding region of green fluorescent protein (GFP) to create a reporter gene construct (see page 915) that would “light up” more and more as oxygen levels declined. 15. Go to the NCBI site map at http://www.ncbi.nlm.nih.gov/ Sitemap/index.html. In the alphabetical index, click on Genomes and Maps to go to the Entrez Genome site at http://www.ncbi .nlm.nih.gov/Sitemap/index.html#Genomes.

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A-40

Abbreviated Answers to Problems

Under Organism Collections, Entrez Genomes section, click on the links for bacteria, archea, and eukaryotes to see: Number of bacterial genomes completed at http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/eub_g.html Number of archeal genomes completed at http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/a_g.html Completed eukaryotic genomes accessible at http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/euk_g.html CHAPTER 28 1. Because DNA replication is semiconservative, the density of the DNA will be (1.724 + 1.710)/2 = 1.717 g/mL. If replication is dispersive, the density of the DNA will also be 1.717 g/mL. To distinguish between these possibilities, heat the dsDNA obtained after one generation in 15N to 100°C to separate the strands; then examine the strands by density gradient ultracentrifugation. If replication is indeed semiconservative, two bands of ssDNA will be observed, the “heavy” one containing 15N atoms and the “light” one containing 14N atoms. If replication is dispersive, only a single band of intermediate density would be observed. 2. The 5´-exonuclease activity of DNA polymerase I removes mispaired segments of DNA sequence that lie in the path of the advancing polymerase. Its biological role is to remove mispaired bases during DNA repair. The 3´-exonuclease activity acts as a proofreader to see whether the base just added by the polymerase activity is properly base-paired with the template. If not (that is, if it is an improper base with respect to the template), the 3´-exonuclease removes it and the polymerase activity can try once more to insert the proper base. An E. coli strain lacking DNA polymerase I 3´-exonuclease activity would show a high rate of spontaneous mutation. 3. Assume that the polymerization rate achieved by each half of the DNA polymerase III homodimer is 750 nucleotides per sec. The entire E. coli genome consists of 4.64 * 106 bp. At a rate of 750 bp/sec per DNA polymerase III homodimer (one at each replication fork), DNA replication would take almost 3100 sec (51.7 min; 0.86 hr). When E. coli is dividing at a rate of once every 20 min, E. coli cells must be replicating DNA at the rate of 4.64 * 106 bp per 20 min (2.32 * 105 bp/min or 3867 bp/sec). To achieve this rate of replication would require initiation of DNA replication once every 20 min at ori, and a minimum of 3933/1500 = 2.57 replication bubbles per E. coli chromosome, or 5.14 replication forks. 4. (1)

(2)

(3)

6. Okazaki fragments are 1000 to 2000 nucleotides in length. Because DNA replication in E. coli generates Okazaki fragments whose total length must be 4.64 * 106 nucleotides, a total of 2300 to 4700 Okazaki fragments must be synthesized. Consider in comparison a human cell carrying out DNA replication. The haploid human genome is 3 * 109 bp, but most cells are diploid (6 * 109bp). Collectively, the Okazaki fragments must total 6 * 109 nucleotides in length, distributed over 3 to 6 million separate fragments (assuming they are 1–2 kb in length). 7. DNA gyrases are ATP-dependent topoisomerases that introduce negative supercoils into DNA. DNA gyrases change the linking number, L, of double-helical DNA by breaking the sugar–phosphate backbone of its DNA strands and then religating them (see Figure 25.23). In contrast, helicases are ATP-dependent enzymes that disrupt the hydrogen bonds between base pairs that hold double-helical DNA together. Helicases move along dsDNA, leaving ssDNA in their wake. 8. A diploid human cell contains 6 * 109 bp of DNA. One origin of replication every 300 kbp gives 20,000 origins of replication in 6 * 109 bp. If DNA replication proceeds at a rate of 100 bp/ sec at each of 40,000 replication forks (2 per origin), 6 * 109 bp could be replicated in 1500 sec (25 min). To provide 2 molecules of DNA polymerase per replication fork, a cell would require 80,000 molecules of this enzyme. 9. For purposes of illustration, consider how the heteroduplex bacteriophage chromosome in Figure 28.17 might have arisen: X

Y

Z

X

y

Z

nicking

strand invasion, ligation, branch migration

resolution of Holliday junction

+ _ +

(4)

The E. coli chromosome replicating once every 20 min (3 replication bubbles, 6 replication forks)

5. If there are 40 molecules of DNA polymerase III (as DNA polymerase III homodimers) per E. coli cell and E. coli growing at its maximum rate has about 5 replication forks per chromosome, DNA polymerase III availability is sufficient to sustain growth at this rate.

+ _ _ +

_ +

10. The mismatch repair system of E. coli relies on DNA methylation to distinguish which DNA strand is “correct” and which is “mismatched”; the unmethylated strand (the newly synthesized one that has not had sufficient time to become methylated is, by definition, the mismatched one). Homologous recombination involves DNA duplexes at similar stages of methylation, neither of which would be interpreted as “mismatched,” and thus the mismatch repair system does not act. 11. B-DNA normally has a helical twist of about 10 bp/turn, so the rotation per residue (base pair), !f, is 36°. If the DNA is unwound to 18.6 bp/turn, !f becomes 19.4°. Thus, the change in !f is 16.6°. NEL

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Abbreviated Answers to Problems

12. Consider the two DNA duplexes, WC and wc, respectively, displayed as “ladders” in the following figure. The gap at the right denotes the initial cleavage, in this case of the W and w strands. Cleavage by resolvase at the arrows labeled 1 and ligation of like strands (W with w and C with c) will yield patch recombinants; cleavage at the arrows labeled 2 and ligation of like strands gives splice recombinants. 1 w c

C W 1

13. The DNA is: GCTA CGAT a. HNO2 causes deamination of C and A. Deamination of C yields U, which pairs the way T does, giving: GTTA and ACTA and (rarely) ATTA CAAT TGAT TAAT Deamination of A yields I, which pairs as G does, giving: GCTG and GCCA and (rarely) GCCG CGAC CGGT CGGC b. Bromouracil usually replaces T, and pairs the way C does: GCCA and GCTG and (rarely) GCCG CGGT

CGAC

CHAPTER 29 1. The first AUG codon is underlined: 5´-AGAUCCGUAUGGCGAUCUCGACGAAGACUCCUAG GGAAUCC… Reading from the AUG codon, the amino acid sequence of the protein is: (N-term)Met-Ala-Ile-Ser-Thr-Lys-Thr-Pro-Arg-Glu-Ser-…

2 w C c W

2

A-41

CGGC

Less often, bromouracil replaces C, and mimics T in its base-pairing: GTTA and ACAT and (rarely) ATTA CAAT TGTA TAAT c. 2-Aminopurine replaces A and normally base-pairs with T but may also pair with C: GCTG and CGGT and (rarely) GCCG CGAC GCCA CGGC 14. Transposons are mobile genetic elements that can move from place to place in the genome. Insertion of a transposon within or near a gene may disrupt the gene or inactivate its expression. 15. The PrPSC form of the prion protein accumulates in individuals with mutations in the gene for PrPc and is the cause of inheritable (genetic) forms of Creutzfeldt-Jacob disease. Furthermore, prion-related diseases may be acquired by ingestion of PrPSC, indicating the infectious properties of this form of the protein. Variant CJD (vCJD) is an example of an infectious version of a prion disease. 16. Hexameric helicases unwind DNA at a cost of 1 ATP/ nucleotide. The genome of E. coli K12 consists of 4,686,137 nucleotides, so 4,686,137 ATP equivalents would be needed to completely unwind the E. coli K12 chromosome. 17. Antibodies against specific proteins are commonly used protein identification tools. Armed with an antibody specific for DNA polymerase III, another antibody specific for DNA polymerase IV, and a procedure to separate replication forks (large macromolecular complexes) from soluble proteins (gel filtration, centrifugation?), addition of DNA polymerase IV should lead to association of IV with the replication fork and release of III. IV should now be found in the large macromolecular complex and III among the soluble proteins.

2. See Figure 29.3 for a summary of the events in transcription initiation by E. coli RNA polymerase. A gene must have a promoter region for proper recognition and transcription by RNA polymerase. The promoter region (where RNA polymerase binds) typically consists of 40 bp to the 5 =-side of the transcription start site. The promoter is characterized by two consensus sequence elements: a hexameric TTGACA consensus element in the #35 region and a Pribnow box (consensus sequence TATAAT) in the #10 region (see Figure 29.4). 3. The fact that the initiator site on RNA polymerase for nucleotide binding has a higher Km for NTP than the elongation site ensures that initiation of mRNA synthesis will not begin unless the available concentration of NTP is sufficient for complete synthesis of an mRNA. 4. First, transcription is compartmentalized within the nucleus of eukaryotes. Furthermore, in contrast to prokaryotes (which have only a single kind of RNA polymerase), eukaryotes possess three distinct RNA polymerases—I, II, and III—acting on three distinct sets of genes (see Section 29.3). The subunit organization of these eukaryotic RNA polymerases is more complex than that of their prokaryotic counterparts. The three sets of genes are recognized by their respective RNA polymerases because they possess distinctive categories of promoters. In turn, all three polymerases interact with their promoters via particular transcription factors that recognize promoter elements within their respective genes. Proteincoding eukaryotic genes, in analogy with prokaryotic genes, have two consensus sequence elements within their promoters, a TATA box (consensus sequence TATAAA) located in the #25 region, and Inr, an initiator element that encompasses the transcription start site. In addition, eukaryotic promoters often contain additional short, conserved sequence modules such as enhancers and response elements for appropriate regulation of transcription. Transcription termination differs as well as prokaryotes and eukaryotes. In prokaryotes, two transcription basic termination mechanisms occur: rho protein-dependent termination and DNA-encoded termination sites. Transcription in eukaryotes tends to be imprecise, occurring downstream from consensus AAUAAA sequences known as poly(A) addition sites. An important aspect of eukaryotic transcription is post-transcriptional processing of mRNA (see Section 29.5). 5. When DNA-binding proteins “read” specific base sequences in DNA, they do so by recognizing a specific matrix of H-bond donors and acceptors displayed within the major groove of B-DNA by the edges of the bases. When DNA-binding proteins recognize a specific DNA region by “indirect readout,” the protein is discerning local conformational variations in the cylindrical surface of the DNA double helix. These conformational variations are a consequence of unique sequence information contributed by the base pairs that make up this region of the DNA. 6. The metallothionine promoter is about 265 bp long. Because each bp of B-DNA contributes 0.34 nm to the length of DNA, this promoter is about 90 nm long, covering 26.5 turns of B-DNA (10 bp/turn). Each nucleosome spans about 146 bp, so about 2 nucleosomes would be bound to this promoter.

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A-42

Abbreviated Answers to Problems

DNA backbone and the histone tails. Collectively, these modifications weaken histone:DNA interactions.

7. If cells contain a variety of proteins that share a leucine zipper dimerization motif but differ in their DNA recognition helices, heterodimeric bZIP proteins can form that will contain two different DNA contact basic regions (consider this possibility in light of Figures 29.35 and 29.36). These heterodimers are no longer restricted to binding sites on DNA that are dyad-symmetric; the repertoire of genes with which they can interact will thus be dramatically increased.

10. Because the lac repressor is tetrameric and each subunit has an inducer-binding site, it seems likely that inducer binding is cooperative. Cooperative binding of inducer would be advantageous, because the slope of the lac operon expression versus [inducer] would be steeper over a narrower [inducer] range. 11. Capping of the 5 =-end and polyadenylation of the 3 =-end of mRNA protect the RNA from both 5 =- and 3 = -exonucleolytic degradation. Methylations at the 5 = -end enhance the interaction between cap-binding protein eIF4E (see Figure 30.29), thus increasing the likelihood that the mRNA will be translated. Note also in Figure 30.29 that polyadenylylation provides a protein-binding site for proper assembly of the 40S initiation complex essential to translation.

8. Exon 17 of the fast skeletal muscle troponin T gene is one of the mutually exclusive exons (see Figure 29.46). Deletion of this exon would cut by half the alternative splicing possibilities, so only 32 different mature mRNAs would be available from this gene. If exon 7, a combinatorial exon, were duplicated, it would likely occur as a tandem duplication. The different mature mRNAs could contain 0, 1, or 2 copies of exon 7. The 32 combinatorial possibilities shown in the center of Figure 29.46 represent those with 0 or 1 copy of exon 7. Those with 2 copies are 16 in number: 456778, 56778, 46778, 6778, 45778, 4778, 5778, 778, 4577, 577, 5677, 45677, 4677, 477, 677, and 77. Given the mutually exclusive exons 17 and 18, 96 different mature mRNAs would be possible.

12. In the A Deeper Look box on page 935, the argument is made that polymerases have a common enzymatic mechanism for nucleotide addition to a growing polynucleotide chain based on two metal ions coordinated to the incoming nucleotide. These metal ions interact with two aspartate residues that are highly conserved in both DNA and RNA polymerases. The discovery of a second Mg 2 + ion in the RNA polymerase II active site confirms to the universality of this model.

9. Modifications include acetylation and methylation of Lys residues, thus suppressing their positive charge: methylation of Arg residues, again suppressing the positive charge, and phosphorylation of Ser residues, thus introducing negative charges. The positively charged Lys and Arg residues interact electrostatically with phosphate groups on the DNA backbone, so these interactions would be diminished through acetylation and methylation. Phosphorylation of Ser residues would lead to electrostatic repulsion between the

13. 50 bp of DNA is (50) (0.34 nm/bp) = 17 nm. Because 147 bp of DNA make 1.7 turns around the nucleosome (see Chapter 25) $ 86.4bp/turn of DNA, 50 bp = 0.58 turns, or slightly more than 1 2 turn of DNA around the histone core octamer. Promoter and response modules are about 20 to 25 bp.

14. O #

O

N

P

O

O

CH2

R

N

O

O O

#

N

NH2

OH

P

O

O

CH2

N

N A

N

O

N O

N

N

O# O

O

P

G O

CH2

N

O

N

N NH2

O #

O

P

O

O

Y

CH2

N

O

NH O

O O

P

OH O

#O

O #

O

P

OH O

O

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

A-43

Abbreviated Answers to Problems

15. a-Helices composed of 7 to 8 residues would have a length of 1.05 to 1.2 nm. The overall diameter of B-DNA is about 2.4 nm, but the diameter at the bottom of major groove is significantly less (as a reference, the C1= - C1= distance between base-paired nucleotides in separate chains is 1.1 nm). A distance along the B-DNA helix axis of 1.1 nm would correspond to about 3 base pairs, so if such an a-helix were laid along the major groove, it could not make contacts with more than 3 base pairs (see also Figure 29.33).

reading frame (a stretch of coding sequence devoid of stop codons): CAATACGAAGCAATCCCGCGACTAGACCTTAAC… (alternate codons underlined) The amino acid sequence it encodes is: Gln-Tyr-Glu-Ala-Ile-Pro-Arg-Leu-Asp-Leu-Asn…. Two of the three reading frames of the complementary DNA sequence also contain stop codons. The third may be an open reading frame:

16. Phe644 acts as a wedge to separate the template and nontemplate strands from one another; Phe644 is located at the end of the Y-helix.

…GTTAAGGTCTAGTCGCGGGATTGCTTCGTATTG (alternate codons underlined)

17. a-Amanitin binds in the large cleft between the two largest RNA polymerase II subunits, in particular to an a-helix, the “bridge” helix, running across this cleft. Its position in the cleft slows inhibits translocation of DNA and RNA and the entry of the next template nucleotide into the active site.

An unambiguous conclusion about the partial amino acid sequence of this cDNA cannot be reached. 2. A random (AG) copolymer would contain varying amounts of the following codons: AAA AAG AGA GAA AGG GAG GGA GGG

18. The two protein chains in pdb id = 1GU4 are identical through positions 268 (D) to 333 (Q). Leucines are found at positions 297, 304, 306, 313, 320, 324, 327, and 330. Those at positions 306, 313, 320, and 327 are seven residues apart, as in a leucine zipper. Basic residues (K and R) precede these leucines, occurring at positions, 268, 275, 277, 280, 286, 287, 289, 291, 293, 295, and 302.

codons for Lys Lys Arg Glu Arg Glu Gly Gly, respectively. Therefore, the random (AG) copolymer would direct the synthesis of a polypeptide consisting of Lys, Arg, Glu, and Gly. The relative frequencies of the various codons are a function of the probability that a base will occur in a codon. For example, if the A/G ratio is 5/1, the ratio of AAA/AAG is (5 * 5 * 5)/(5 * 5 * 1) = 5/1. If the 3A codon is assigned a value of 100, then the 2A1G codon has a frequency of 20. From this analysis, the relative abundances of these amino acids in the polypeptide should be:

CHAPTER 30 1. The cDNA sequence as presented: CAATACGAAGCAATCCCGCGACTAGACCTTAAC… represents six potential reading frames, the three inherent in the sequence as written and the three implicit in the complementary DNA strand, which is written 5= S 3=, is:

Lys = 120; Arg = 24; Glu = 24; Gly = 4.8 (Normalized to Lys = 100: Arg = 20; Glu = 20; Gly = 4) 3. Review the information in Section 30.2, noting in particular the two levels of specificity exhibited by aminoacyl-tRNA synthetases (1: at the level at ATP ∆ PPi exchange and aminoacyl adenylate synthesis in the presence of amino acid and the absence of tRNA; and 2: at the level of loading the aminoacyl group on an acceptor tRNA).

…GTTAAGGTCTAGTCGCGGGATTGCTTCGTATTG Two of the three reading frames of the cDNA sequence given contain stop codons. The third reading frame is a so-called open

4. Base pairs are drawn such that the B-DNA major groove is at the top (see following figure). H N H O N N R

N

H

N H

N

N H G C

N O

H

R

H

N

N N

H

N N

O

H

O

N

O

O

R

N

N R

N

N R

R

C G

5. The wobble rules state that a first-base anticodon U can recognize either an A or a G in the codon third-base position; first-base anticodon G can recognize either U or C in the codon third-base position; and first-base anticodon I can recognize either U, C, or A in the codon third-base position. Thus, codons with third-base A or G, which are degenerate for a particular amino acid (Table 30.3 reveals that all codons with third-base purines are degenerate, except those for Met and Trp), could be served by single tRNA species with first-base anticodon U. More emphatically, codons with third-base pyrimidines (C or U)

N

H

H

N O H

N H G U

O O

N N

R

N

H N O N R

H H

N

N N

R

N H

U G

are always degenerate and could be served by single tRNA species with first-base anticodon G. Wobble involving first-base anticodon I further minimizes the number of tRNAs needed to translate the 61 sense codons. Wobble tends to accelerate the rate of translation because the noncanonical base pairs formed between bases in the third position of codons and bases occupying the first-base wobble position of anticodons are less stable. As a consequence, the codon:anticodon interaction is more transient.

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A-44

Abbreviated Answers to Problems

6. The stop codons are UAA, UAG, and UGA. Sense Sense codons that are a single-base change from UAA include CAA (Pro), AAA (Lys), GAA (Glu), UUA (Leu), UCA (Ser), UAU (Tyr), and UAC (Tyr). Sense codons that are a single-base change from UAG include CAG (Gln), AAG (Lys), GAG (Glu), UCG (Ser), UUG (Leu), UGG (Trp), UAU (Tyr), and UAC (Tyr). Sense codons that are a single-base change from UGA include CGA (Arg), AGA (Arg), GGA (Gly), UUA (Leu), UCA (Ser), UGU (Cys), UGC (Cys), and UGG (Trp). That is, 19 of the 61 sense codons are just a single base change from a nonsense codon. 7. The list of amino acids in problem 6 is a good place to start in considering the answer to this question. Amino acid codons in which the codon base (the wobble position) is but a single base change from a nonsense codon are the more likely among this list, because pairing is less stringent at this position. These include UAU (Tyr) and UAC (Tyr) for nonsense codons UAA and UAG, and UGU (Cys), UGC (Cys), and UGG (Trp) for nonsense codon UGA. 8. The more obvious answer to this question is that eukaryotic ribosomes are larger, more complex, and hence slower than prokaryotic ribosomes. In addition, initiation of translation requires a greater number of initiation factors in eukaryotes than in prokaryotes. It is also worth noting that eukaryotic cells, in contrast to prokaryotic cells, are typically under less selective pressure to multiply rapidly. 9. Each amino acid of a protein in an extended b-sheet–like conformation contributes about 0.35 nm to its length. 10 nm , 0.35 nm per residue = 28.6 amino acids. 10. Larger, more complex ribosomes offer greater advantages in terms of their potential to respond to the input of regulatory influences, to have greater accuracy in translation, and to enter into interactions with subcellular structures. Their larger size may slow the rate of translation, which in some instances may be a disadvantage. 11. The universal organization of ribosomes as two-subunit structures in all cells—archaea, eubacteria, and eukaryotes—suggests that such an organization is fundamental to ribosome function. Translocation along mRNA, aminoacyl-tRNA binding, peptidyl transfer, and deacylated-tRNA release are processes that require repetitious uncoupling of physical interactions between the large and small ribosomal subunits. 12. Prokaryotic cells rely on N-formyl-Met-tRNAifMet to initiate protein synthesis. The tRNAifMet molecule has a number of distinctive features, not found in noninitiator tRNAs, that earmark it for its role in translation initiation (see Figure 30.16). Furthermore, N-formyl-Met-tRNAifMet interacts only with initiator codons (AUG or, less commonly, GUG). A second tRNAMet, designated tRNAmMet, serves to deliver methionyl residues as directed by internal AUG codons. Both tRNAifMet and tRNAmMet are loaded with methionine by the same methionyl-tRNA synthetase, and AUG is the Met codon, both in initiation and elongation. AUG initiation codons are distinctive in that they are situated about 10 nucleotides downstream from the Shine–Dalgarno sequence at the 5=-end of mRNAs; this sequence determines the translation start site (see Figure 30.18). Eukaryotic cells also have two tRNAMet species, one of which is a unique tRNAiMet that functions only in translation initiation. Eukaryotic mRNAs lack a counterpart to the prokaryotic Shine–Dalgarno sequence; apparently the eukaryotic small

ribosomal subunit binds to the 5=-end of a eukaryotic mRNA and scans along it until it encounters an AUG codon. This first AUG codon defines the eukaryotic translation start site. 13. The Shine–Dalgarno sequence is a purine-rich sequence element near the 5=-end of prokaryotic mRNAs (see Figure 30.18). It base-pairs with a complementary pyrimidine-rich region near the 3=-end of 16S rRNA, the rRNA component of the prokaryotic 30S ribosomal subunit. Base pairing between the Shine– Dalgarno sequence and 16S rRNA brings the translation start site of the mRNA into the P site on the prokaryotic ribosome. Because the nucleotide sequence of the Shine–Dalgarno element varies somewhat from mRNA to mRNA, whereas the pyrimidine-rich Shine–Dalgarno-binding sequence of 16S rRNA is invariant, different mRNAs vary in their affinity for binding to 30S ribosomal subunits. Those that bind with highest affinity are more likely to be translated. 14. The most apt account is b. Polypeptide chains typically contain hundreds of amino acid residues. Such chains, attached as a peptidyl group to a tRNA in the P site, would show significant inertia to movement, compared with an aminoacyl-tRNA in the A site. 15. Elongation factors EF-Tu and EF-Ts interact in a manner analogous to the GTP-binding G proteins of signal transduction pathways (see pages 367–370). EF-Tu binds GTP and in the GTP-bound form delivers an aminoacyl-tRNA to the ribosome, whereupon the GTP is hydrolyzed to yield EF-Tu:GDP and Pi. EF-Ts mediates an exchange of the bound GDP on EF-Tu:GDP with free GTP, regenerating EF-Tu:GTP for another cycle of aminoacyl-tRNA delivery. The a-subunit of the heterotrimeric GTP-binding G proteins also binds GTP. Ga has an intrinsic GTPase activity and the GTP is eventually hydrolyzed to form Ga:GDP and Pi. Guanine nucleotide exchange factors facilitate the exchange of bound GDP on Ga for GTP, in analogy with EF-Ts for EF-Tu. The amino acid sequences of EF-Tu and G proteins reveal that they share a common ancestry. 16. Four: two in the aminoacyl-tRNA synthetase reaction; one by EF-Tu; one by EF-G. 17. a. The proteins are shown as ribbons. b. The rRNA is shown as a strand (line). c. The tRNAs (shown with the sugar–phosphate backbone as a strand and the bases as ring structures) are oriented such that their acceptor stems extend away from the 30S subunit, in the direction from which a 50S subunit will add. d. The tRNA acceptor stems will lie within the peptidyl transferase center of the 50S subunit. 18. a. 64,281. b. If you use the cursor to rotate the structure, many bases around the periphery are easy to see (otherwise, the complexity of the structure makes it hard to see them). c. Many such regions are easy to find along the structure’s periphery. d. RNA. CHAPTER 31 1. mRNA half-life is defined as the length of time for which an mRNA is stable inside the cell. 2. P-bodies are described as cytoplasmic foci consisting of RNA-protein complexes or, in other words, dynamic aggregates of mRNPs (messenger ribonuclearproteins) and P-body components. 3. Once the polyA tail is shortened beyond the critical length, PABP can no longer bind to the mRNA polyA tail. NEL

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Abbreviated Answers to Problems

4. Up until the critical length. So long as the polyA tail is equal to or greater than approximately 25 nucleotides, adenosine molecules can be added back to lengthen the polyA tail. 5. polyA tail, PABP, eIF4G, deadenylases, critical. 6. No. Once an mRNA has its polyA tail removed, the mRNA is exposed to 3’ to 5’ degradation by the exosome.. 7. The AU-rich element enhances mRNA turnover by rendering the transcript less stable, potentially recruiting the exosome and other trans-acting mRNA degradation factors. 8. The half-life of the stable message will decrease (greater mRNA turnover). This effect is due to the presence of the ARE, which will render the stable message more labile. 9. Transcript C, which contains the most AREs, is likely to have the shortest half-life, while Transcript A, which does not contain any AREs, is likely to have the longest half-life. 10. Since both KSRP and TTP have been described as destabilizing RBPs, their function is to down-regulate the expression of labile genes, such as proto-oncogenes and cytokine genes. 11. A premature termination codon. 12. EJCs are deposited at exon-exon junctions during splicing, when the linking intron is removed. 13. 3’ to 5’, via the exosome. 14. Perfect complementarity is only required at the level of the seed element within a miRNA. 15. Pri-miRNA. Drosha converts pri-miRNA to pre-miRNA via cleavage. 16. Both. Perfect complementarity allows for cleavage of the target mRNA via Ago protein slicer activity and subsequent mRNA degradation. 17. Argonaute is an endoribonuclease, as it cleaves the target mRNA at an internal site. CHAPTER 32 1. Human rhodanese has 296 amino acid residues. Its synthesis would require the involvement of 4 ATP equivalents per residue or 1184 ATP equivalents. Folding of rhodanese by the Hsp60 a14 complex consumes another 130 ATP equivalents. The total number of ATP equivalents expended in the synthesis and folding of rhodanese is approximately 1314. 2. Because a single proteolytic nick in a protein can doom it to total degradation, nicked proteins are clearly not tolerated by cells and are quickly degraded. Cells are virtually devoid of partially degraded protein fragments, which would be the obvious intermediates in protein degradation. The absence of such intermediates and the rapid disappearance of nicked proteins from cells indicate that protein degradation is a rigorously selective, efficient cellular process. 3. Hsp70: Hsp70 binds to exposed hydrophobic regions of unfolded proteins. The Hsp70 domain involved in this binding is 18 kD in size and therefore would have a diameter of roughly 3 nm (see problem 5 for the math). Assuming the hydrophobic binding site of Hsp70 stretches across its diameter, it would interact with about 3 nm/0.35 nm = 8 or 9 amino acid residues. Multiple Hsp70 monomers could interact with longer hydrophobic stretches. So, for Hsp70 to interact with a polypeptide chain does not necessarily depend on the protein’s size, but it does depend on its hydrophobicity (and absence of charged groups). Hsp70 and Hsp60 chaperonins: Proteins that interact with both of these classes of chaperones not only must fit the description

A-45

for Hsp70 targets but must also be small enough to access the Hsp60 chaperonin chamber, which has a diameter of about 5 nm. This restriction means that Hsp60 cannot interact with proteins more than roughly 50 kD in mass (see problem 5). 4. Many eukaryotic proteins have a multidomain or modular organization, where each module is composed of a contiguous sequence of amino acid residues that folds independently into a discrete domain of structure (see Figure 6.26 for examples of this type of sequence and structure organization). Such proteins would be ideal for co-translational folding: As each newly synthesized contiguous sequence emerges from the ribosome tunnel, it begins folding into its characteristic domain structure. The final, fully folded state of the complete protein would be achieved when the various domains assumed their proper spatial relationships to one another through hinge motions occurring at intradomain regions of the protein. 5. The maximal diameter for a spherical protein would be 5 nm (radius = 2.5 nm). The volume of this protein is given by 4 V = pr3 = 4/3(3.14)(2.5 * 10-9 m)3 = 65.4 * 10-21 mL. 3 If its density is 1.25 g/mL, its mass would be (1.25 g)(65.4 * 10-21 mL) = 81.8 * 10-21 g/molecule, so its molecular weight = (6.023 * 1023)(81.8 * 10-21 g) = 492.5 * 102 g/mol = 49,250 g/mol, or about 50 kD. 6. There are 649 different phosphorylated forms for a protein having 7 separate phosphorylation sites. 7. GrpE catalyzes nucleotide exchange on DnaK, replacing ADP with ATP, which converts DnaK back to a conformational form having low affinity for its polypeptide substrate. This change leads to release of bound polypeptide, giving it the opportunity to fold, which is an important step in DnaK function. EF-Ts catalyzes nucleotide exchange on EF-Tu, converting it to the conformational form competent in aminoacyl-tRNA binding. Binding of the aminoacyl-tRNA:EF-Tu complex to the ribosome A site triggers the GTPase activity of EF-Tu as codon:anticodon recognition takes place and the aminoacyl-tRNA:EF-Tu complex conformationally adjusts to the A site. DnaJ delivers an unfolded polypeptide chain to DnaK, and its interaction with DnaK also triggers the ATPase activity of DnaK, whereupon the DnaK:ADP complex forms a stable complex with the unfolded polypeptide. In both instances, hydrolysis of bound nucleoside triphosphate is triggered and leads to conformational changes that stabilize the respective complexes. 8. Protein targeting information resides in more generalized features of the leader sequences such as charge distribution, relative polarity, and secondary structure, rather than amino acid sequence per se. Proteins destined for secretion have N-terminal amino acid sequence with one or more basic amino acids followed by a run of 6 to 12 hydrophobic amino acids. The sequence MRSLLILVLCFLPAALGK… has a basic residue (Arg) at position 2 and a run of 12 nonpolar residues (…LLILVLPLAALG…); thus, it appears to be a signal sequence for a secretory protein. Cleavage by the signal peptidase would occur between the G and K. 9. Translocation proceeds until a stop transfer signal associated with a hydrophobic transmembrane protein segment is recognized. The stop-transfer signal induces a pause in translocation, and the translocon changes its conformation such that the wall of this closed cylindrical structure either opens or exposes a hydrophobic path, allowing the transmembrane segment to diffuse laterally into the hydrophobic phase of the membrane.

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A-46

Abbreviated Answers to Problems

10. The maximal diameter for a spherical protein would be 6 nm (radius = 3 nm). The volume of this protein is given 4 by V = pr3 = 4/3(3.14)(3 * 10-9 m)3 = 113 * 10-21 mL. 3 If its density is 1.25 g/mL, its mass would be (1.25 g) (113 * 10-21 mL) = 141 * 10-21 g/molecule, so its

molecular weight = (6.023 * 1023)(141 * 10-21 g) = 851 * 102 g/mol = 85,100 g/mol, or about 85 kD. 11. The thickness of a phospholipid bilayer is 5 nm, so the overall channel formed by a 50S ribosome and the translocon channel is 15 nm; 15 nm , 0.35 nm per amino acid $ about 43 amino acid residues.

12.

1 1

48 CH CH CH &

CH

NH

O 76C

2

N H

2

CH2

CH2

CH2

CH2

48

O

76

C

O#

2

2

3

13. E3 ubiquitin protein ligase selects proteins by the nature of the N-terminal amino acid. Proteins with Met, Ser, Ala, Thr, Val, Gly, or Cys at the amino terminus are resistant to its action. Proteins having Arg, Lys, His, Phe, Tyr, Trp, Leu, Ile, Asn, Gln, Asp, or Glu at their N-terminus are susceptible. N-terminal Pro residues lack a free a-amino group, so such proteins are not susceptible. 14. The cell cycle relies on the cyclic synthesis and destruction of cell cycle regulatory proteins. Lactacystin inhibits cell cycle progression by interfering with programmed destruction of proteins by proteasomes. 15. The temperature-induced switch could be based on a temperature-dependent conformational change in the HtrA protein that brings the His-Ser-Asp catalytic triad into the proper spatial relationship for protease function. 16. There are many other post-translational modifications, including acylation, alkylation, amidation (at a protein’s C-terminus), biotinylation, formylation, glycosylation, isoprenylation, and oxidation. See also Table 5.5. 17. This exercise is left to the student. 18. A phosphodegron is defined as one or a series of phosphorylated residues on a substrate protein that interact directly with a protein–protein interaction domain in an E3 ubiquitin ligase, thereby linking the substrate to the ubiquitin conjugation machinery. Phosphorylation can affect the affinity of target protein binding; alternatively, phosphorylation can stimulate E2 activity. CHAPTER 33 1. a. Keq = 360,333. b. Assuming [ATP] $ [ADP], [pyruvate] must be greater than 360,333 [PEP] for the reaction to proceed in reverse. c. Keq = 0.724. d. [PEP]/[pyruvate] = 724. e. Yes. Both reactions will be favorable as long as [PEP]/ [pyruvate] falls between 0.0000028 and 724. 2. Energy charge = 0.945; phosphorylation potential = 1111 M-1. 3. 8 mM ATP will last 1.12 sec. Because the equilibrium constant for creatine-P + ADP S Cr + ATP is 175, [ATP] must be less than 1750 [ADP] for the reaction creatine-P & ADP S Cr + ATP to proceed to the right when [Cr-P] = 40 mM and [Cr] = 4 mM. 4. From Equation 20.12, ! (NADP+/NADPH) = -0.350 V; ! (NAD+/NADH) = - 0.281 V; thus, ! ! = 0.069 V, and

! G = -13,316 J/mol. If an ATP “costs” 50 kJ/mol, this reaction can produce about 0.27 ATP equivalent at these concentrations of NAD+, NADH, NADP+, and NADPH. 5. Assume that the Keq for ATP + AMP S 2 ADP = 1.2 (see legend to Figure 33.2). When [ATP] = 7.2 mM, [ADP] = 0.737 mM and [AMP] = 0.063 mM. When [ATP] decreases by 10% to 6.48 mM, [ADP] + [AMP] = 1.52 mM and thus [ADP] = 1.30 mM and [AMP] = 0.22 mM. A 10% decrease in [ATP] has resulted in a 0.22/0.063 = 3.5-fold increase in [AMP]. 6. J (the flux of F-6-P through the substrate cycle) at low [AMP] = 0.1; J at high [AMP] = 8.9. Therefore, the flux of F-6-P through the cycle has increased 89-fold. 7. Inhibition of acetyl-CoA carboxylase will lower the concentration of malonyl-CoA. Because malonyl-CoA inhibits uptake of fatty acids (see Figure 9.16), decreases in [malonyl-CoA] favor fatty acid synthesis. 8. Ethanol oxidation to acetate yields 2 NADH in the cytosol, each worth 1.5 ATPs. The acetyl thiokinase reaction consumes 2 ATP equivalents in converting acetate to acetyl-CoA (due to ATP S AMP + PPi). Combustion of acetyl-CoA S 2 CO2 in a liver cell yields a net of 10 ATPs. Therefore, the net yield of ATP from ethanol in a liver cell is 11 ATPs. Glucose S 6 CO2 in a liver cell yields 30 ATPs (see Table 19.4) or 5 ATP/C atom. Ethanol S 2 CO2 gives 11 ATPs or 5.5 ATP/C atom. 9. Palmitoyl-CoA S 8 acetyl-CoA yields 7 NADH and 7 [FADH2] = 21 + 14 = 35. Eight acetyl-CoA S palmitoyl-CoA requires 14 NADPH + 7 ATP = -63 ATP (negative sign denotes ATP consumed). The palmitoyl-CoA ∆ 8 acetyl-CoA conversion is favorable in both directions provided the free energy release is more than 1750 kJ/mol in the catabolic (acetyl-CoA forming) direction (the energy necessary to produce 35 ATP equivalents at a cost of 50 kJ/mol each) and less than 3150 kJ/mol in the anabolic (palmitoyl-CoA forming) direction (the energy released from 63 ATP equivalents). 10. Cellular respiration releases 2870 kJ/mol of glucose under standard-state conditions (and about the same amount under cellular conditions). Assuming the cell is a bacterial cell where cellular respiration produces 38 ATPs, the total cost of ATP synthesis is 38(50) = 1900 kJ/mol. Thus, the overall free energy change = -2870 + 1900 = -970 kJ/mol. Because ! G "= = -RT ln Keq, Keq = e391.4447 = 10168. This is a very large number! It will be even larger for a cell where ATP yields per glucose are less. NEL

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Abbreviated Answers to Problems

Carbon dioxide fixation leads to glucose synthesis at the cost of 12 NADPH and 18 ATP = 66 ATP equivalents. At 50 kJ/mol, the energy investment from ATP is #3300kJ/mol and the value of a glucose is + 2870 kJ/mol. Thus, ! G "= = - 430 kJ/mol = - RT ln Keq. Keq = e173.52 = 1075, which is also a very large number! 11. Glycogen phosphorylase, phosphorylase kinase, glycogen synthase, PFK-1, PFK-2, FBPase, and glucose-6-P phosphatase are liver enzymes that act specifically in glycogenolysis or gluconeogenesis; these enzymes are potential targets to control blood glucose levels. 12. From Figure 33.13: The “limit production” list would include NPY, AgRP, and ghrelin; the “raise levels” list would include cholecystokinin, leptin, insulin, melanocortins, and PYY3–36. 13. Leptin injection in ob/ob mice decreases food intake, raises fatty acid oxidation levels, and lowers body weight. Obese humans are not usually defective in leptin production. Indeed, because leptin is produced in adipocytes, obese individuals may already have high levels of leptin, so leptin injection has limited success. 14. No, although NPY is an orexic agent, it is not necessarily an obesity-promoting hormone. Mice deficient in melanocortin production, melanocortin receptors, or leptin receptors would

A-47

have an obese phenotype, because all these agents act in appetite-suppressing pathways. 15. Alcohol consumption lowers the NAD+/NADH ratio, which limits glycolysis because glyceraldehyde-3-P dehydrogenase requires NAD+, stimulates gluconeogenesis because NADH favors glyceraldehyde-3-P dehydrogenase working in the glucose synthesis direction, and limits fatty acid oxidation because b-oxidation requires NAD+. NADH is also a negative regulator of several citric acid cycle enzymes. 16. The T172D mutant of AMPK mimics the phosphorylated form in having negative charge on the side chain at position 172. 17. a. Fatty acyl-CoA. b. Transgenic mice lacking functional hypothalamic fatty acid synthase should have elevated [malonyl-CoA]. If hypothalamic levels of malonyl-CoA are high, eating should diminish, as should body fat content. Assuming physical activity is proportional to signals of nutrient availability, physical activity should increase. 18. a. Leptin-deficient mice should respond to regular leptin injections by losing weight. b. The leptin receptor. Assay wild-type and db/db mutant cells for leptin binding ability.

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Index

The letter B denotes a box; F denotes a figure; T denotes a table; bold denotes a defined or introduced term A bands, 506 A-DNA, 841 A sites, 1023 AAA+ ATPase family, 523, 938, 1101 AAA+ domains, 526F Aβ (protein), 1088B ABC transporters, 340, 342F Absolute configuration of optically active molecules, 88B Absolute zero, 58 Absorption, distribution, metabolism, and excretion, 436B Abzymes, 434 ACC. See Acetyl-CoA carboxylase Acceptor complexes, 326 Acceptor sites, 1023 Acceptor stems, 861 Accessory light-harvesting pigments, 664–665 Acetals, 200, 201F Acetaminophen, 280B, 282F Acetate units, in fatty acid synthesis, 253–255, 254F Acetates cellulose, 205 as product of hydrolysis, 67 Acetic acid, titration curve for, 44F, 45F Acetic anhydride, 65F Acetoacetate leucine, 801 Acetoacetates, 756, 801, 803 Acetoacetyl-CoA acetyltransferase, 757 Acetoacetyl-CoA thiolase, 757 Acetohydroxy acid synthase, 788 Acetone, 756 Acetyl-CoA in fatty acid synthesis, 256–257 leucine catabolism and, 801 mevalonate and, 282–283 from pyruvate, 564F Acetyl-CoA carboxylase-1, 256F Acetyl-CoA carboxylase, in fatty acid synthesis, 255–256, 256F phosphorylation of, 256–257, 256F, 256F–257F Acetyl phosphate, 62, 67F Acetylcholine, acetylcholinesterase degrading, 392 Acetylcholine receptors, 390, 391F Acetylcholine transport protein, 392 Acetylcholinesterase, description of, 392 O-Acetylserine sulfhydrylase, 788 Acid–base catalysis, 454–455 Acid dissociation constant, 42, 44T Acid hydrolysis, 111

Acidic amino acids, 80 Acids conjugate, 41 See also Amino acid entries; DNA entries; Fatty acid entries; Nucleic acids; RNA Aconitase, 600, 601 AcrA, 343 AcrB, 343, 344F, 345F Acridine orange, 845 ActD, 1049 Actin, 323, 506, 508F, 509F Actin-anchoring protein, 510B Actin gene, 998 Actinomycin D, 845 Action potentials, 387–389 Activation free energy of, 410 of replication, 937 Activation energy, 62, 444F Activation loops, 360, 1116 Activator genes, 949 Activator sites, 980 Active-site residues, 453F, 457, 457B Active sites, 405, 411, 419–420 of chorismate mutase, 467–469, 470F Active transport, 328, 335–339 calcium, 338 cardiac glycosides and, 337, 340B gastric H+,K+ATPase, 338 Actomyosin complexes, 513 Acute lymphoblastic leukemia, 783B Acute myeloblastic leukemia, 783B Acyl carrier proteins, 258 Acyl-CoA:cholesterol acyltransferase, 288 Acyl-CoA dehydrogenase, in β-oxidation, 743–745 Acyl-CoA ligase, 266, 740 Acyl-CoA oxidase, 753 Acyl-CoA synthetase, 266, 740 Acyl-enzyme intermediates, 460, 462F Acyl phosphates, 62, 67 Acyldihydroxyacetone phosphate reductase, 269 ADA complex, 991 ADA−SCID, 920, 922B Adapter molecules, 1009 ADAR (enzyme family), 1004 Adenine, 810 Adenine nucleotide pool, 1114 Adenine phosphoribosyltransferase, 879 Adenosine, 811, 812B Adenosine-5′-diphosphate, 813 metabolic integration and, 1114 Adenosine-5′-monophosphate, 813 glycogen phosphorylase and, 714 inosine monophosphate as precursor to, 873

metabolic integration and, 1114 structure, 813F Adenosine-5′-phosphosulfate, 836 Adenosine-5′-phosphosulfate-3′-phosphokinase, 790 Adenosine-5′-triphosphate, 814 metabolic integration and, 1114 Adenosine deaminase reaction, 450F Adenosine deaminases, 450F, 881, 1004 Adenovirus, 25F Adenylate kinase, 878, 1114 Adenylic acid, 813 Adenylosuccinase, 873, 876 Adenylosuccinate lyase, 873 Adenylosuccinate synthetase, 876 Adenylyl cyclase, 394, 716 modulation of, 367F ADH. See Alcohol dehydrogenase Adipocytes, 736 Adipose cells, 736 Adipose tissue, 736–739 metabolic role of, 1122–1123 ADME, 436B ADP, 813 ADP ribosylated eEF-2, 1041B Adrenaline, 82 α1-Adrenergic receptors, 384 β2-Adrenergic receptors, 357F Adrenocorticotropic hormone, 293, 350F Advanced glycation end products, 710B Aequorin, 87B Aerobes, 538 Aerobic pathway, TCA cycle as, 586 Affinity chromatography, 110 Affinity labels, 424 Agar, 207 Agaropectin, 207, 209 Agarose, 207, 209 AGEs, 710B Aggregation symmetries of globular proteins, 180T Agouti-related peptide, 1124 AICAR transformylase, 873 AIDS, 449B, 468B, 941B AIR carboxylase, 873 AIR synthetase, 873 Akt1, 748B Alanine catabolism of, 798 structure, 78F synthesis of, 786 Albuterol, 507B Alcohol, metabolic effects of, 1125B Alcohol dehydrogenase, 450, 451F, 580 Alcoholic fermentation, 562

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-2

Index

Alcohols, sugar, 197–198 Aldaric acids, 197 Aldehydes, 193 Alditols, 197 Aldol condensation, 571 Aldolase reaction, 450F, 571–572 Aldonic acids, 197 Aldose reductase, 725B Aldoses, 191 Aldosterone, 295 Aliskiren, 448B Alkaptonuria, 804B 1-Alkyl-2-Acetylglycerophosphocholine, 275 Alkylating agents, 957 All α proteins, 174, 175F All β proteins, 174, 175F ALL (leukemia), 783B All-trans-retinal, 240F Allantoicase, 884 Allantoinase, 884 Allosteric effectors, 484, 485F, 498 Allosteric enzymes, 358, 418 Allosteric inhibition, 484 Allosteric inhibitors, 484, 600 Allosteric modifier in fatty acid metabolism, 266–268 Allosteric regulation, 478–480, 483–486, 485F ATP-dependent, 1092B covalent modification and, 480, 489–490, 490F definition, 478 glycogen phosphorylase and, 716 hemoglobin and, 490–502 Allostery, 186 Allyl phenyl ether, 469F Alternative RNA splicing, 993, 1003 Altman, Sidney, 404B Alveoli, 178B Alzheimer’s disease, 1088B α-Amanitin, 982 AMD, 1059–1065, 1061B, 1061T Amethopterin, 877F Amide-linked glycosyl phosphatidylinositol, 315 Amide-linked myristoyl anchors, 315 Amide planes, in proteins, 92, 143–145 Amino acid aminotransferase, branched-chain, 788 Amino acid analyzers, 111 Amino acid catabolism, 798–805 of C-3 family, 798 of C-4 family, 798–799 of C-5 family, 800 intermediates of, 798 of leucine, 801–802 of lysine, 802 of phenylalanine and tyrosine, 803–804 of valine, isoleucine and methionine, 800–801 Amino acid derivatives, 351 Amino acid neurotransmitters, 395 Amino acid residues, 94 Amino acid sequences, 111 alignment of, 124–125 structure influenced by, 143 Amino acid sequencing chemical methods for, cleavage of disulfide bridges in, 113, 114F sequence databases and, 122 Amino acid synthesis, 773–798 of aromatic amino acids, 790–796 of aspartate family, 782–785, 783B of histidine and purine, 796–798

human dietary requirements and, 775B of α-ketoglutarate family, 777–778 metabolic intermediates in, 774T of 3-phosphoglycerate family, 788–790 of pyruvate family, 785–788 transamination in, 775–776, 776B urea cycle in, 778–782, 782B Amino acids, 75–97 acid–base properties of, 80, 82–85 catabolism. See Amino acid catabolism common in proteins, 78F–81F dietary requirements for, 775B essential, 557 in green fluorescent protein, 87B hormones as derivatives of, 351 ionic forms, 82F ionization of side chains of, 84–85 membrane locations, 310F non-essential, 775B non-polar, 77, 78F, 80 nuclear magnetic resonance and, 89–90, 89F pKa values for, 83T polar, uncharged, 78F, 80 sequencing. See Amino acid sequencing spectroscopic properties of, 87–91 structure, 11F, 76F synthesis. See Amino acid synthesis in transmembrane helices, 309 tRNA matching with, 1012–1017, 1014T Amino sugars, 199 Aminoacyl-tRNA binding, 1027, 1031B Aminoacyl-tRNA synthetases, in protein synthesis, 1010 Aminoacyl-tRNAs, 434F, 823 EF-Tu and, 1031B α-Aminoadipic acid pathway, 782 γ-Aminobutyric acid, 81, 81F, 395, 396F Aminoglycoside antibiotics, 1042 β-Aminopropionitrile, 162B Aminopterin, 877F 2-Aminopurine, 957 Aminotransferase reactions, 776B Aminotransferases, 776 branched-chain amino acid, 788 catalytic mechanism of, 429 leucine, 788 Aminotriazole, 795B AML (leukemia), 783B Ammonium biosynthesis of, 763 metabolic fate, 768–770 titration curve for, 45 in urea cycle, 782B Ammonotelic animals, 804 AMP, 813 5′-AMP, 813 AMP, See also Adenosine-5′-monophosphate AMP-activated protein kinase, 1115–1117 AMP deaminase, 881 Amphibolic pathways, 546 Amphipathic amino acids, 80 Amphipathic α-helices, 1097 Amphipathic lipids, 228 Amphipathic molecules, 37 Amphiphilic helices, 160 basic, 375–376 Amphiphilic molecules, 37, 38F AMPK, 1115, 1126 Amplification, 897

α-Amylase, 204, 706 Amylo-(1,4→1,6)-transglycosylase, 710 Amyloid-β, 1088B Amyloid plaques, 1088B Amylopectin, 204 Amylose, 204F α-Amylose, 204 Amylose, 205, 206F Anabolic processes, 543–550 Anabolic steroids, 295 Anabolism, 543 catabolism and, 544, 544F, 546, 547F divergent pathways, 546 metabolic integration and, 1110–1111 Anaerobes, 538 Anaerobic pathways, 586 Analogs therapeutic, 877B, 892B, 894B transition-state, 447, 448B–449B Anaplerotic reactions, 611 Anchoring Actin-anchoring protein, 510B of proteins to lipids, 306, 314–318 Androgenic alopecia, 293B Androgens, 243, 294 4-Androstenedione, 248B Anemia, sickle cell, 501–502 Anfinsen cages, 1089 Anfinsen, Christian B., 168–169 Angel dust, 395 Anhydride, phosphoric acid, 64–66 Animal cells, 22F, 24T, 212–213 Animals acetyl-CoA carboxylase in, 255 nitrogen excretion in, 804 pyrimidine biosynthesis in, 886–887 transgenic, 953B Anion exchange resins, 105, 106F Annealing, 908B Annexin proteins, 375 Anomeric carbon atoms, 194 Anomers, 194 ANP (peptide), 364 Anserine, 47, 47F Antagonists, 394 Anthranilate synthase, 793 Anti-inflammatory drugs, nonsteroidal, 280B–281B 2:3 Anti-terminator, 979 Antibiotics, 202–203 Antibodies catalytic activity of, 434 screening of cDNA library with, 914 Anticancer drug fluoro-substituted pyrimidines for, 894B folate analog as, 877B proteasome inhibitors in, 1103B Anticodon loops, 861, 1012 Anticodons, 862 Antifreeze glycoproteins, 215, 215F Antigen, definition, 434 Antigenic determinants, 211 Antigens, O antigens, 211 Antimicrobial agent fluoro-substituted pyrimidines for, 894B folate analog as, 877B Antiparallel beta-pleated sheet, 149, 150F, 151 Antiparallel chains, in chitin, 207 Antiports, 343 Antisnorkelling, 310, 311F NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index α1-Antitrypsin, 178B Antp, 995 2-AP (base analog), 957 AP endonucleases, 955 Apaf-1. See Apoptotic protease-activating factor 1 Apoenzymes, 407 Apoprotein, 289T Apoptosis, 653–655 Apoptosomes, 654 Apoptotic protease-activating factor 1, 654 APRT, 879 APS (adenosine-5p-phosphosulfate), 836 APS (protein), 362 Aptamers, 865 Apurinic acid, 826 araBAD operon, 975–977 AraC, 976 Arachidonates, 278 Arachidonic acid, 229, 279F fatty acid synthesis and, 266 Archaea, 17, 241 ARE-binding proteins, 1061 ARE-containing mRNAs, 1060, 1061T AREs, 1048, 1059–1065 Arginase, 779 Arginine, 79F, 777, 778–779 Arginine fingers, 526 Argininosuccinase, 778 Argininosuccinate synthetase, 778 Aromatic amino acids, 77, 79F Arrestin signalling mode, 383 Arrestins, 383 Arrhenius equation, 410 Arterial plaques, 288F Asialoglycoprotein receptor, 217 Asparaginase, 798 Asparagine, 783, 783B structure, 78F Asparagine synthetase, 783 Aspartame, 97, 793B, 804B Aspartate, 395, 782 Aspartate transcarbamoylase, 884 Aspartic acid, structure, 78F Aspartic proteases, 462, 464B, 464T, 465–466, 466F Aspartokinase, 783 Aspartokinase I, 783 Aspartokinase II, 783 Aspartokinase III, 783 β-Aspartyl-semialdehyde dehydrogenase, 785 Aspirin, 281 Associated transducers and activators of transcription, 380 Assumption, steady-state, 412 Astbury, William, 145 Asymmetric molecules, amino acids as, 76, 86–87 AT, 771 AT (enzyme) ATCase, 884 Atherosclerotic plaques, 162B Atomic fluctuations in globular proteins, 172 ATP, 3, 3F, 814 catabolism and, 546–547, 547F in cellular energy cycle, 546 chaperone-mediated protein folding and, 1092B glucose-6-phosphate utilization and, 728–729 glycogen phosphorylase and, 713 in glycolysis, 563–564, 567 human requirement for, 71–72 hydrolysis of, 69–71, 69F, 70F, 71F, 335

Keq and, 72B metabolic integration and, 1111, 1112–1114 phosphoric acid anhydride hydrolysis and, 64–66 phosphorylation and, 566F photosynthesis and, 547F, 677–680 production overview, 562F ATP:D-glucose-6-phosphotransferase, 407 ATP:GS:adenylyl transferase, 771 ATP–ADP translocase, 648 ATP–citrate lyase, 254, 610 ATP coupling, 1112–1114 ATP coupling stoichiometry, 1112–1113 ATP cycle, 547F ATP-dependent allosteric regulation, 1092B ATP derivative, sulfide synthesis from, 789–790 ATP sensors, 1092B ATP sulfurylase, 790, 836 ATP synthase inhibitors, 647 ATPase activity, 508 ATPase modules, 1101 ATPases, 1101 Atrial natriuretic peptide, 364 Atropine, 394 Attfield, Paul, 202B AU-rich elements, 1048, 1060 AU-rich mediated decay, 1059–1065, 1061B, 1061T, 1079–1080 AUBPs, 1059, 1059T, 1061–1062, 1079 Autacoids, 812B Autocatalytic excision, 433F Autogenous regulation, 977 Autophosphorylation, 360, 363 Autoregulation, 977 Autosomes, 911B Autotrophs, 538 Avian sarcoma virus, 365 Axial substituents, 196 Axonal transport, 520F Axonemal dyneins, 517 Axons, 520 Azaserine, 873 Azide, 647 b cytochromes, 633 B-DNA, 838 Baa helices, 375–376 Bacillus cereus, 333 Bacillus thuringiensis, 315B Backbones, 92, 143, 174 Bacteria cell walls, 212F classification and, 17, 19F pyrimidine biosynthesis in, 887 Bacteriophage T4 DNA ligase, 901 Bacteriophages, 23, 25F Bacteriorhodopsin, 308, 309F, 335, 341, 343, 646 Bactoprenol, 240F Baculoviruses, 914 Banting, Frederick, 185B, 1110B BAR domains, 324–325 β-Barrel structures, 311, 312F Barrier, blood–brain, 398 Base excision repair, 951, 955 Base pairing definition, 817 intrastrand, 859 Base pairs Hoogsteen, 845 Watson–Crick, 819

I-3

Base sequences, of nucleic acids, 815–816 Bases conjugate, 41 nitrogenous, 809–811 Basic amino acids, 80 Basic amphiphilic alpha-helices, 375–376 Basic Local Alignment Search Tool, 123 Basic region, 997 BCA method, 104B BCKAD complex, 801 Benzyl alcohol, 451F BER, 951 Bergström, Sune, 278B Best, Charles, 185B Beta-oxidation. See β-Oxidation Beta sheets antiparallel pleated, 149, 150F, 151 in globular proteins, 159–160 in spider silk, 151–152 Bicarbonate buffer system, 48B Bicarbonate ion, in urea cycle, 782B Bicinchoninic acid, 104B Bidirectional replication, 927 Bifunctional enzymes, 704 Bijvoet, J. M., 88B Bilayers, lipid, 302–303 Bile acids, 292–293 Bile salts, 739 Bimolecular reactions, 409, 425 Binding cooperative, 186, 484 oxygen, 498F proteoglycans and, 218–220 Binding change mechanism, 643 Binding isotherm, 134 Binding site, proteins, 135 Binding sites, ribosome, 1025 Bioinformatics, 911B Biological pest control, 315B Biomolecular dimensions, 8T Biomolecules, 5–6 high-energy, 61–69 radii of, 14T Biosphere, 539, 539F Biosynthesis anabolism as, 544–545 of bile acids, 292–293 lipid. See Fatty acid synthesis; Lipid Biosynthesis sphingolipid, 275 See also Synthesis Biosynthetic processes, 610 Biot, Jean-Baptiste, 88B Biotin carboxyl carrier protein, 255 Biotin carboxylase, 255 BiP (chaperone), 1096 Bisabolene, 240F 1,3-Bisphosphoglycerate, 67F 2,3-Bisphosphoglycerate, 498–500 Bisphosphoglycerate mutase, 575 Bisubstrate reactions, 425, 425F, 429–430, 429F, 430F BLAST program, 123–124 Bloch, Konrad, 202B, 285B Blood, 481, 498F bicarbonate buffering system in, 48B pH of, 49B sickle cell anemia and, 501–502 Blood–brain barrier, 398 Blood clotting, 481, 482F Blood pressure, 448B

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-4

Index

Blotting Northern, 908B Southern, 908B–909B Western, 908B BNP (peptide), 364 Boat conformation of sugar, 196 Bohr effect, 497 Boltzmann’s constant, 57 Bonding angles, 451F Bonds covalent, 6, 453–454, 454F double, 92, 93F, 264 glycosidic, 200, 458F, 811 high-energy, 63 ionic, 15F non-covalent, 12 See also Hydrogen bonds; Peptide bonds Bovine pancreatic ribonuclease A, 100F Bovine ribonuclease, 100F, 159F Bovine spongiform encephalopathy (BSE), 130B Boyartchuk, Victor, 317B BPG, 498–500, 498F, 499F Bradford assay, 104B Bradykinin, 350F Brain, metabolic role of, 1119 Brain natriuretic peptide, 364 Branch migration, 943, 947 Branch sites, 1001 Branched-chain amino acid aminotransferase, 788, 801 Branched-chain fatty acids, 753 Branched-chain α-keto acid dehydrogenase complex, 801 Branched structures, 7F BRCA1, 950B BRCA2, 950B Breast cancer susceptibility genes, 950B Bride of sevenless, 384B Bridges, disulfide, 113, 114F Bromoaspirin, 281F Bromocriptine, 397B–398B Bromodomains, 990 5-Bromouracil, 957 Bronchodilators, 507B Brown fat, 1123 Brown, Michael, 291 5-BU (base analog), 957 Buchanan, John, 872 Buffers, 46–48, 46F, 48B, 49F Building blocks of macromolecules, 6–10, 9F of proteins, 78F–82F Bulges, 860 α-Bungarotoxin, 394 Burst kinetics, 460, 462F Butterflies, 340B bZIP (motif), 994 C-3 family, 798 C-5 family, 800 c cytochromes, 633 C/EBP, 996 C-reactive proteins, 224, 229F C-terminal analysis, 114 C-terminal domain, 526F, 984 C-terminal ends, 111 C2H2 class, 996 C3 plants, 688 C4 plants, 688

C16 fatty acids, 263–264 Ca2+-ATPase, 338, 339F Caffeine, 812F Cages, 1089 Calbindin-9K, 375 Calciosomes, 374 Calcisomes, 374 Calcitonin, 350F Calcium-binding proteins, 374–375 Calcium carbonate, 539B Calcium ion, 373–374 Calcium-modulated proteins, 374–376 Calcium transport cycle, 338 Caldarchaeol, 24F Calmodulin, 161F, 375–376 Calmodulin-binding domain, 519 Caloric homeostasis, 1118 Caloric restriction, 1126–1128 Calorie, 55 Calorimeter, 16F, 56 Calvin–Benson cycle, 682–683, 683T, 684F, 685 cAMP, 368–369, 813 cAMP-dependent protein kinase, 315, 704, 720F cAMP receptor protein, 975 cAMP response element, 994B cAMP-response element-binding, 994B Camphene, 240F Cancer effectors of microtubule polymerization and, 519B fluoro-substituted pyrimidines for, 894B folate analog for, 877B oncogenes and, 371B–372B proteasome inhibitors for, 1103B telomeres and, 857B Canola oil, 231 CAP, 974–975 Cap structures, 999 Capsids, 23 Capsules, bacterial, 314 Carbamoyl-phosphate synthetase I, 768, 777 Carbamoyl phosphate synthetase II, 884 Carbohydrate recognition domains, 223 Carbohydrate-responsive element-binding protein, 730 Carbohydrates, 189–227 in diet, 557B glycoproteins and, 213–218 See also Glycoprotein monosaccharides, 191–200 See also Monosaccharide oligosaccharide, 200–203 polysaccharide, 203–213 See also Polysaccharide proteoglycans and, 218–222, 219F structural code of, 222–224 Carbon anomeric, 194 reduction of, 548F Carbon cycle, 539, 539F Carbon dioxide, in photosynthesis, 680–689 Carbon dioxide fixation, 660 Carbon monoxide, 647 Carbonic anhydrase, 17F β-Carbonyl group, in fatty acid synthesis, 260–261 Carbonyl phosphate, 697 2-Carboxy-3-keto-arabinitol, 681 γ-Carboxyglutamic acid, 81, 81F Carboxylase, biotin, 255 Carboxypeptidase, 114–115

Cardiac glycosides, 198, 337, 340B Cardiolipin, 235F, 236 Cardiotonic steroids, 337 CARDs. See Caspase-recruitment domains Carnauba wax, 239 Carnitine, 741–742, 742B Carnitine acyltransferase I, 741 Carnitine acyltransferase II, 741 β-Carotene, 7FS Carrier proteins acyl, 258 biotin carboxyl, 255 ubiquitin, 1098 Cartilage, proteoglycans in, 221–222, 221F Cascades clotting, 242B, 459, 482F enzyme, 353 phosphorylase, 719 Caspase-recruitment domains, 654 Caspases, 654 Cassettes, expression, 920–921 Catabolic processes, 543–550 Catabolism, 543 anabolism and, 544, 544F, 546, 547F convergent pathways, 544–546 fatty acid. See Fatty acid catabolism hydrogen and electrons released in, 548F metabolic integration and, 1109, 1111 stages of, 545F tricarboxylic acid cycle and. See Tricarboxylic acid cycle Catabolite activator protein, 974–975 Catalase, naming of, 406 Catalysis, 409 glycogen synthase and, 709, 711 in isomerization of glucose-6-phosphate, 568 ribonucleotide reductase and, 890–891 rotational, 644 See also Enzymes, mechanisms of action Catalytic power, 404, 405 chorismate mutase, 466–467 Catalytic sites, 889 Catalytic triads, 459 Catechol-O-methyl-transferase, 397B–398B Catecholamines, 389, 397B–398B, 398–399 CATH database, 164–165 Cation exchangers, 105, 106F, 107F Cationic free radicals, 667 Caveolae, 325, 326F Caveolins, 325, 326F -CCA (sequence), 823 CCR5 receptor, 384 CCT, 1091 CDKs, 936 cDNA libraries, 907 screening of, 914 cDNAs, 907 CDP-diacylglycerol, 269–270, 271F, 273F CDP-ethanolamine, 270–271 Cech, Thomas R., 404B Celebrex, 281F Cell bodies, 386 Cell body, of neurons, 386 Cell–cell communication, 390F Cell growth, 220–221 Cell membranes, 10, 21 composition of, 301 Cell signalling, 385F Cell surface polysaccharides, 212–213 NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index Cell walls, 19 bacterial, 210, 212F of gram-positive bacteria, 212, 212F Cellobiose, 201 Cells, 2 animal, 22F, 24T, 212–213 buffering system in, 46 eukaryotic. See Eukaryotes excitable, 386 glial, 386 membranes of. See Membranes prokaryotic. See Prokaryotes Cellular energy, 546 Cellular lipidomics, 246 Cellulase, 207 Celluloid, 210B Cellulose, 203, 205–207, 206F, 210B dietary, 558 Cellulose acetates, 205 Centrifugation, 110, 554F, 849B Ceramide, 373 Ceramide-activated protein kinase, 373 Ceramides, 237, 275–277 Cerebrosides, 275–277 Cerezyme, 216B Cesium chloride centrifugation, 849B Cetyl palmitate, 239B CF1CF0–ATP synthase, 678 cGMP, 813 Chain elongation of fatty acids, 263–265 of RNA transcripts, 968–969 Chain termination method, 834–836 Chain termination protocol, 835–836 Chains in chitin, 207 hydrocarbon, membranes and, 305 peptide, initiation of, 1024–1026 polypeptide, fragmentation of, 117T Chair conformation of sugar, 196 Channel proteins, 331F Channels ligand-gated ion, 391–392 oligomeric ion, 391–392 voltage-gated ion, 387 Channon, H. J., 285B Chaperones, 152, 176, 1092B Hsp70, 1088 Chaperonins, 176, 1088–1091 Chargaff, Erwin, 817 Chargaff’s rules, 817T Charged tRNAs, 1010 Charges, on transported species, 329 CHD1, 992 Checkpoints in DNA replication, 936 Chelation, 996 Chemical forces, weak, 12, 13T Chemical kinetics, 408 Chemical methods, of amino acid sequencing. See Amino acid sequencing Chemical mutagens, 957 Chemical shift, 89–90, 89F, 90F Chemical synapses, 389 Chemical synthesis, of nucleic acid, 856–858 Chemiosmotic coupling, 640–641 Chemotherapy fluoro-substituted pyrimidines for, 892B folate analog for, 896B proteasome inhibitors in, 1103B

Chemotrophic organisms, 61 Chemotrophs, 538 Chi sites, 945–946 Chimeric constructs, 899 Chimeric plasmids, 899–904 Chiral centres of monosaccharides, 192 naming of, 91B Chiral molecules amino acids as, 76, 86–87 naming of, 86 Chitin, 203, 207 ChlD1, 674 ChlD2, 674 Chlorogenic acid, 701B Chlorophyll a, 7FS Chlorophylls, 663 Chloroplasts, 10, 22, 23F, 661–662, 1097 Cholecystokinin, 1124 Cholesterol, 243, 243–244F, 247B elevated, 291–292 lovastatin and, 286B statin effects on, 286B structure, 7FS, 282F synthesis of, 282–288, 287F Cholesterol ester transfer protein, 288 Cholesterol esters, 288 Cholic acid, 292F Choline plasmalogens, 237F Choline sphingomyelin, 238F Cholinergic synapses, 389–390 Chondroitin, 209 Chorionic gonadotropin, 350F Chorismate mutase, 466–472, 469F, 470F, 471F, 472F, 793 Chorismate synthase, 793 Chorismic acid, 790 ChREBP, 730 Chromatin, 853–855 Chromatin-remodelling complexes, 990 Chromatography in amino acid separation, 105, 111 gas, 556F Chromodomain, helicase, DNA-binding protein, 992 Chromodomains, 990 Chromophore, 87B, 87F Chromosomal proteins, non-histone, 853 Chromosome pairing, 942 Chromosomes, 22 DNA of, 819 eukaryotic, 853–856 replication of ends, 939–940 telomeres and, 857B, 940B Chylomicrons, 288, 739 Chymotrypsin, 459–464, 463F biological function, 128 esters and, 461F in polypeptide fragmentation, 116 size of molecule, 94T structure, 101F, 460F, 461F α-Chymotrypsin, 481 Chymotrypsinogen, 59F, 60F, 460F, 481, 482F cis-Complexes, 327 cis Double bonds in fatty acid synthesis, 264 cis-Elements, 1048, 1057 Citrate, 254 Citrate synthase alpha-helix of, 160, 161F as dimer, 600

I-5

Citrate synthase reaction, 600 Citric acid cycle, 592 See also Tricarboxylic acid cycle Claisen rearrangements, 467, 469F Clamp loaders, 932 Clathrate structures, 37, 37F Clathrin-mediated endocytosis, 383 ClC channels, 334–335 Cleavage of disulfide bridges, 113, 114F oligosaccharide, 217F Clefts, synaptic, 386, 389, 392 Cloned fusion protein, 915T Cloned genes, 912–917, 915T Cloning, 897, 898–904 Cloning sites, 899 Cloning vectors, plasmids as, 899 Closed promoter complex, 965 Closed quaternary structures, 184 Closed systems, 54, 55F Clostripain, 116 Clot, blood, 481 Clotting factors, 242B, 481 Clustered channels, 389 CMC, 302 5′-CMP, 813 Co-chaperonins, 1090 Co-inducers, 971 Co-repressors, 971 Coated pits, 289 Coated vesicles, 289 Coaxial stacking, 861 Cobratoxin, 394 Cocaine, 397B–398B Codon–anticodon pairing in protein synthesis, 1017–1019, 1017T, 1018T, 1055F, 1055T Codons, 862, 965B definition, 1010 multiple, 978 nonsense, 1011, 1067–1068 premature termination, 1049, 1067–1068 Coenzyme A, 258F, 599B Coenzyme Q, 605, 628–633 reduction of, 628 Coenzyme Q–cytochrome c reductase, 628 Coenzymes, 407, 550 Cofactors, determination of rate, 478 Cofactors, enzyme, 407 Cognate tRNA molecules, 1013 Cognition, 994B Cohesin, 856 Coiled coils, 153, 155B Coils, 160 Colchicine, 519B Collagen, structure, 156 Collagen-related diseases, 162B Collective motions in globular proteins, 172 Colligative properties of water, 39 Collip, James, 185B Colony hybridization experiments, 906 Combinatorial exons, 1003 Combinatorial libraries, 905B Committed step in fatty acid synthesis, 255 Communication, cell–cell, 390F Compartmentalization, metabolism and, 553–555, 555F Compartments in cells, 10 of chloroplasts, 661–662

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-6

Index

Competing resonance, 65F, 66 Competitive inhibition, 419–421, 421B, 421F Competitive inhibitors, 419, 422F Complement regulatory factor, 221 Complementarity, structural, 14–15, 15F, 134 Complex III, 633 Complex IV, 635–637, 647 Complex lipid synthesis, 269–277 γ-Complexes, 932 Complexin, 326 Compulsory substrates, 427–428 Concentration, hydrolysis of ATP and, 70–71, 71F Concentration gradients, 327 Condensation, of acetyl-CoA, 259–260 Condensin, 856 Configuration of monosaccharide, 192 of protein, 102, 103F Conformation, 2 protein, 102, 103F Conformational changes allosteric regulation and, 484–485, 486F in globular proteins, 172 of hemoglobin, 496F ligand-induced, 135 in phosphofructokinase tetramer, 479F of sugar, 458F Conjugate acids, 41 Conjugate bases, 41 Conjugated proteins, 132–133 Consensus sequences, 967 Constitutive exons, 1003 Constitutive expression, 972 Constitutive splicing, 1003 Contraction, muscle. See Muscle contraction Contrahelicases, 934 Converter enzymes, 480 Cooperative binding, 186, 484 Cooperativity, 186, 495B Copper, 628 CoQ, 605, 628–633 CorA family of membrane channels, 333, 334F Core, 985 Core oligosaccharides, 214F Core polymerase, 964 Cores, hydrophobic, 174 Corey, Robert, 146 Cori, Carl Ferdinand, 702B Cori cycle, 702B, 702F Cori, Theresa Radnitz, 702B Corticosteroids, 294–295 Cortisol, 243, 244F, 295, 721 Cotton, 205 Coumadin, 242B Coupling constant, 89 Coupling stoichiometry, 1112–1113 Covalent amide bonds, 77F Covalent bonds, 6, 453–454, 454F Covalent catalysis, 453–454, 454F Covalent electrophilic catalysis, 454 Covalent intermediates, 453 Covalent modification, 480, 480F, 486–490, 490F Covalently modified enzyme intermediates, 428–429 COX, 280B–281B, 281F CP43 (subunit), 674 CP47 (subunit), 674 Cpn60s, 176 CPS-I, 777

CPS-II, 884–887, 887B CR (caloric restriction), 1127 Creatine, 427F Creatine kinase, 68F, 427 Creatine phosphate, 62, 427F Creatine supplements, 1121B CREB, 994B CREB-type transcription factor, 994B Creutzfeldt-Jakob disease, 130B Crick, Francis H. C., 818 Cristae, 623 Critical micelle concentration, 302 Crixivan, 467F Cronin, John, 90B Cross-bridges, 506 Cross-talk, 1092 Crosslink in gram-positive cell wall, 211F Crossovers, 173 Crotonases, 745 CRP, 975 Cruciform structures, 845 CTD, 984 CTP, 814, 887 CTP synthetase, 887 Curare, 394 Curves oxygen-binding, of hemoglobin, 498F oxygen saturation, 498F, 500F substrate saturation, 411 Cx type, 996 Cyanide, 647 Cyanobacteria, 21 Cyanogen bromide, 116, 117F CycC/CDK8, 990 3′,5′-Cyclic AMP, 813 See also cAMP Cyclic AMP. See cAMP Cyclic AMP–dependent protein kinase, 487, 488F Cyclic electron transfer pathways, 671 3′,5′-Cyclic GMP, 813 Cyclic hemiketals, 194 Cyclic phosphonate ester, 435F Cyclic structures, 7F Cyclic symmetry, 181, 182F Cyclin-dependent protein kinases, 936 Cyclins, 936 Cyclohexadienone intermediate, 469F Cyclooxygenase, 278, 280B–281B Cyclophilin A, 451, 453F Cysteine, 788 catabolism of, 798 structure, 79F Cystic fibrosis, 922B Cytidine, 811 Cytidine 5′-monophosphate, 813 Cytidylic acid, 813 Cytochrome a, 633 Cytochrome b5, 265 Cytochrome b5 reductase, 265 Cytochrome b557, 763 Cytochrome bc1 complex, 633 Cytochrome c amino acid sequences of, 125 backbone structures of, 174F as mobile electron carrier, 636 phylogenetic tree for, 125, 126F size of molecule, 94T Cytochrome c oxidase, 628, 637 Cytochrome P-450, 753, 755

Cytochromes, 628 Cytoplasmic calcium ion, 374F Cytoplasmic dyneins, 517, 518F Cytoplasmic membrane, 299 Cytosine, 810 Cytosine methylation, 841 Cytoskeletons, 22, 322, 515–516, 516F Cytosolic acetyl-CoA, 254–255 Cytosolic NADH, 650–653 Cytosolic ribosome, 1022–1023, 1023T Cytotoxic cancer drugs, 342F α-D-Fructose-1,6-bisphosphate, 198F β-D-Lactose, 201 D loops, 861 α-D-Ribose-5-phosphate, 198F D1 (subunit), 674 D2 (subunit), 674 DAG, 245, 269–271, 271F, 372 DAHP synthase, 791 Dark reactions, photosynthetic, 663 Database, amino acid sequence, 122 DB (domain), 916 DCE, 437F dCMP deaminase, 892 DEAD-box ATPases/helicases, 1002 DEAD/H-box, 990 Deadenylation, 771, 1055 Deadenylation-independent mRNA decay, 1057 Debranching enzymes, 706 Decarboxylase, pyrophosphomevalonate, 285 Decarboxylation, in fatty acid synthesis, 259–260 5′→3′ Decay, 1056 3′→5′ Decay, 1057 Decay, AU-rich mediated, 1059–1065, 1061B, 1061T, 1079–1080 Decay-accelerating factor, 215 Decoding centres, 1021, 1027 Decorin, 218, 219F Degenerate code, 1011 Degenerate oligonucleotides, 906 DegP (protein), 1103 Degradation acetylcholine, 392F Edman, 113 lipoproteins, 289, 290F protein, 216–218, 1098–1105, 1104B See also Catabolism Dehydration synthesis, 200 Dehydrogenase β-aspartyl-semialdehyde, 785 dihydroorotate, 884 glycerol-3-phosphate, 269 isopropylmalate, 788 oxidative catabolism and, 547, 548F 5-Dehydroquinate dehydratase, 793 Dehydroquinate synthase, 793 Deisenhofer, Johann, 291 Deletions, 956 Denaturation of chymotrypsinogen, 59F, 60F of DNA, 847 of protein, 16, 166–168 Denatured DNA, 847 Dendrites, 386 Denitrifying bacteria, 763 Density, DNA, 849B Dental plaque, 205 2-Deoxy-α-D-ribose, 198 NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index 2-Deoxy-streptamine, 1042 Deoxy sugars, 198 Deoxycholic acid, 243, 244F Deoxyhemoglobin, 495F, 497F Deoxyribonucleic acid. See DNA entries Deoxyribonucleosides, 811 Deoxyribonucleotides, formation of, 888–891 Depolarized membranes, 387 Deramaxx, 281F Dermatan sulfate, 209 Desaturase, 274 Design, of drugs, 426B Designer enzymes, 435 Desipramine, 398B Desmolase, 248B, 293 Desolvation, 445 Destabilization electrostatic repulsion and, 64, 66 in enzyme-substrate complex, 445–447, 445F Destabilization of ES, 445, 445F Detergents, 303F Detoxication, 755 Dextrans, 205 Dextrorotatory behaviour, 86 D-Galactosamine, 199 DGCR8, 1072 D-Glucosamine, 199 N-acetyl-D-Glucosamine, 207 DHFR gene, 998 Diabetes mellitus, 185B, 197 cataracts in, 725B glucokinase and, 567 gluconeogenesis inhibitors and, 701B glycation end products in, 710B ketone bodies in, 757B metabolic syndrome and, 748B mouse model of, 1122B Diacylglycerol, 245, 269–271, 271F, 372 Diacylglycerol acyltransferase, 269 Dialysis, 103 Diastase, 201 Diastereomers, 193 Dicarboxylic acids, 753, 755 Dicer, 1072 cis-1,2-Dichloroethylene, 437F Dideoxy method, 834–836 Dideoxy sequencing gels, 836 Dideoxynucleotides, 835 Dielectric constants, 35, 36T 2,4-Dienoyl-CoA reductase, 753 Diet amino acids, 775B calorie-restricted, 1126–1128 fatty acids and, 736–739, 752B high-fat, 557B, 748B lipids, 558 Dietary fibre, 558 Digestive serine protease, 459 Digitalis, 340B Dihedral symmetry, 181, 182F Dihydrofolate reductase gene, 998 Dihydroorotase, 884 Dihydroorotate dehydrogenase, 884 Dihydrotestosterone, 293B Dihydroxy acid dehydratase, 788 Dihydroxyacetone phosphate, 269, 272 3,4-Dihydroxyphenylalanine, 399 Dimers hemoglobin, 493F

hormones and, 363, 364 of identical subunits, 466 Dimethylallyl pyrophosphate, 283 Dipeptide, 94 DIPF, 394, 395F Diphosphatidylglycerol, 235, 235F Diphtheria toxin, 1041B Dipoles, 14, 148 Direct-reversal repair systems, 951 Directed enzyme evolution, 436 Directional character, 512 Directional cloning, 901 Disaccharides, 200–202 Discriminator bases, 1015 Dissociation Henderson–Hasselbalch equation and, 43 of histidine–imidazole group, 47 Dissociation constants, 134 Disulfide bridges, 113, 114F Diterpenes, 240 D,L naming system, 86 DNA, 815–819, 824–830 buoyant density of, 849B chemical synthesis, 856–858 in chromosomes, 819 cloning and. See Cloning definition, 815 directed changes in heredity and, 920–922 double helix of, 5F, 817–819 genetic recombination and, 941–950 higher complexity structures of, 850–853 nanotechnology, 820B primary structure, 5F, 11F quadruplexes, 846 recombinant. See Recombinant DNA repair of, 950–955 replication. See DNA replication restriction endonucleases and, 827–830, 829T–830T RNA, differences from, 824, 826 secondary structure. See DNA, secondary structure DNA:protein interactions, 980 DNA chips, 905B DNA-dependent RNA polymerases, 963 DNA double helix/helices, 817 DNA footprinting, 966, 968B DNA glycosylase, 955 DNA gyrases, 852, 928 DNA helicases, 506 DNA libraries, 904–912, 905B, 912–917 cDNA, 907, 914 combinatorial, 905B microarrays and, 910 Southern hybridization and, 906–907 DNA metabolism, 926–962 breast cancer susceptibility genes, 950B chromosome ends, 939–940 DNA polymerases, 930–936, 930T, 932T, 934T, 935B, 939T DNA repair, 950–955 DNA replication, 927–929, 934T eukaryotic cells and, 936–939 genetic recombination, 941–950 “knockout” mice, 949B mutation, molecular basis of, 955–959 replication, recombination, and repair, 948B RNA genomes, 940–941 Szostak, Jack W. and, 927B

I-7

telomeres, 857B, 940B transgenic animals, 953B DNA microarrays, 910 DNA multiplexes, 845–846 DNA polymerase α, 938 DNA polymerase β, 938 DNA polymerase γ, 938 DNA polymerase δ, 938 DNA polymerase ε, 938 DNA polymerase I, 930–932 DNA polymerase III, 930, 932–933 DNA polymerase III holoenzyme, 932–933, 932T DNA polymerases, 834, 928, 930–936, 930T, 932T, 934T, 935B, 939T DNA replication, 927–929 bacterial, 930 in chain termination method, 834 semidiscontinuous, 928–929 unwinding of helix in, 928 DNA, secondary structure, 837–850 A-form DNA, 841, 843T conformational variation in polynucleotide strands, 837–838 denaturation and renaturation, 847–850 density and, 849B dynamism of double helix, 843–845 novel structures, 845–847 stable conformations of double strands, 838–840 Watson–Crick base pairs, 838–839 Z-DNA, 841–843, 843T DNA strand exchange reactions, 946 DNA synthesizers, 857 DNA tweezers, 820B DnaB, 928 DnaG, 933 DnaJ (chaperone), 1089 DnaK (chaperone), 1089 DNases, 827 Docosahexaenoic acid, 267B Dodson, G., 185B Dolichol phosphate, 240F Dolichols, 241 Domains PDZ, 1103 protein, 162–163 Dopa decarboxylase, 399 Dopamine, 398 2-DOS, 1042 Double-bond character of peptide bond, 92, 93F Double-displacement catalytic mechanisms, 429, 429F, 430F Double-displacement reactions, 255, 425, 428–429, 429F, 430F Double helix/helices, 5F in solution, 845 unwinding of, 928 Downstream elements, 966 Downstream promoter elements, 985 DPEs, 985 DRB, 1049 Drosha, 1072 Drug design, 426B, 435 Drugs, 408 carbohydrate-based, 216B enzymes and, 408 fluoro-substituted analogs as, 892B, 894B folate analog, 877B nonsteroidal anti-inflammatory, 280B–281B protease inhibitor, 449B, 467F, 468B

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-8

Index

DSBs, 951 DSC (method), 1105B dsDNA, 943–944 Duodenum, 737 Duplexes, hybrid, 850 dUTPase, 891 Dynamite, 210B Dyneins, 506, 523F Dystrophin, 510B–511B Dystrophy, muscular, 510B–511B E. coli, 20F DNA polymerases, 930–932, 930T elongation factor of, 1027T flagellar motor of, 529F–532F glutamine synthetase in, 770–773 E. coli glutamine synthetase, 770–773 E. coli glutaminyl-tRNA synthetase of, 1016 E sites, 1023 E1 helicase, 527F–528F E1 (protein), 526 E1 (enzyme), 1098, 1099F E2 (enzyme), 1098, 1099F E3-binding protein (E3BP), 595 E3 (enzyme), 1098, 1099F Eating behaviour, hormones controlling, 1124–1126 4EBP, 1038 Eccles, J. C., 387 Edman degradation, 113 Edman’s reagent, 113 EF-G, 1030, 1031B EF hand proteins, 375 EF-Ts, 1027 EF-Tu, 1026, 1031B Effector molecules, 478 Effectors, 352, 370, 375–379 Efflux pumps, 341F EGF, 358, 887B EGF receptor, 359 Egg whites, 167F Eglin C, 460F Ehlers–Danlos syndrome, 162B Eicosanoids, 231, 245, 269 synthesis of, 278–281 Eicosapentaenoic acid, 267B eIF4F complex, 1035 eIFs, 1035 EJC, 1066, 1069 Elaidic acid, 231F Elastase, 116, 128, 178B chymotrypsin and, 461F chymotrypsinogen and, 460F Elastin, 178B Electrical charge, in amino acid separation, 105 Electrical potentials, 387 Electrical synapses, 389 Electrochemical potential, 329 Electrogenic transport, 335 Electrolytes acid dissociation constants for, 44T strong, 41 weak, 42 Electron “holes”, 667 Electron transfer flavoprotein, 745 Electron transfer potential, 64T Electron-transferring flavoprotein, 628 Electron transport. See Oxidative phosphorylation Electron-transport chain, 621, 627–640 Electron-transport pathways, 592

Electrophilic catalysis, covalent, 454 Electrophilic centres, 454, 454F Electrophoresis, 108–110, 834–835, 908B–909B Electrophoretic migration, 834 Electrospray ionization mass spectrometry, 119 Electrostatic destabilization, 447 Electrostatic effects, 445 Electrostatic interactions, 141F, 839 Electrostatic repulsion, 64, 65F, 66F Element, cAMP response, 994B β-Eliminations, 801B ELISA, 184 Elongation, 1023 of C16 fatty acids, 263–265 of RNA transcripts, 968–969 Elongation cycle, 1026–1027 Elongation factor G, 1030 Elongation factor Ts, 1027 Elongation factor Tu, 1026 Elongation factors, E. coli, 1027T Elongator, 993 Embden, G., 562 Embden–Meyerhof pathway, 562 Emphysema, 178B Enalapril, 448B Enantiomers amino acids as, 86, 86F description of, 193 Endergonic processes, 59 Endoamylase, 205 Endopeptidases, 116 Endoplasmic reticulum, 22 signal peptidase and, 1096 Endoproteases, 456F Endorphins, 352, 399 Endosomes, 289, 383 Endosymbionts, 539B Endothelial relaxing factor, 500B Endothelins, 399 Endothelium, 500B Enediol intermediate, 569F Enediolate transition state, 450F Energy, free, of activation, 410 Energy cycle, 546–547 Energy diagrams, for chemical reactions, 410F Energy dispersion, 57 Energy flow, in biosphere, 539 Energy homeostasis, 1118 Energy-shuttle molecule, ATP as, 62–63 Energy transduction, 548, 665 Engelman, Donald, 305 Enhancers, 985, 986 Enkephalins, 399 Enol phosphates, 61 Enolase, 577 3-Enolpyruvylshikimate-5-phosphate synthase, 793 Enoyl-CoA hydratase, 745 Enoyl-CoA isomerase, 752 Enthalpy, 55–57, 56, 56F Entropy, 4, 57 absolute zero and, 58 in enzyme-substrate complex, 445F, 446F hydrolysis and ionization and, 66 loss of, 445 negative, 58B Environment, water’s role in, 33F, 50 Enzyme cascades, 353, 716 Enzyme catalysis active-site residues, 453F, 457B

enzyme-substrate complex destabilization effects on, 445–447, 445F, 446F Enzyme-catalyzed reactions equations defining, 411–418, 412F kinetics of, 425–431 mathematically defined rate of, 408–410 Michaelis constants, 413T ratios for, 416T substrate saturation curve for, 411F Enzyme kinetics, 408 Enzyme-linked immunosorbent assays, 184 Enzyme regulation, 477–504 allosteric. See Allosteric regulation covalent modification and, 480, 480F, 486–490, 490F factors influencing, 477–483 isozymes, 481–483, 483F other mechanisms, 481 zymogens and, 481 Enzyme–substrate complexes, 411, 444 binding energy in, 445 entropy loss and destabilization in, 445–447, 445F, 446F intrinsic binding energy of, 445 NACs and, 451F Enzyme–substrate dissociation constant, 412 Enzymes, 403–441 allosteric, 358, 418 buffers and, 47F classification, 406–407, 406T cofactors, 407, 407T converter, 480 defining characteristics, 404–407 definition, 403 description of, 17 designer, 435 inhibition of, 419–424, 420T, 421B, 421F–423F Km values for, 513T mechanisms of action. See Enzymes, mechanisms of action membrane-associated allosteric, 358–359 pH and, 419F rate acceleration and, 443T regulatory, 404 See also Enzyme regulation ribozymes as, 432–434, 433F specificity, 405, 431–432 temperature and, 419F transition-state stabilization and, 444–445, 444F Enzymes, mechanisms of action, 450–476 acid–base catalysis, 454–455 of aspartic proteases, 462, 464B, 464T, 465–466, 466F of chorismate mutase, 466–472, 470F, 471F, 472F covalent catalysis, 453–454 destabilization of enzyme–substrate complexes, 445–447, 445F, 446F hydrolytic mechanism of lysozyme, 457–458 low-barrier hydrogen bonds and, 455–456, 456F, 463F metal ion catalysis, 70, 407T, 456, 456F near-attack conformations and, 450–453, 451F, 468–469 rate acceleration magnitudes and, 442–444 of serine proteases, 459–462, 463F, 464B transition-state analogs, 447–450 transition-state stabilization, 444–445, 444F Epalrestat, 725B NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index Epidermal growth factor, 220, 887B Epidermal growth factor receptors, 358, 358F–359F Epidermal growth factor repeats, 289 Epimers, 193 Epinephrine, 351, 398 glucagon vs., 719–721 glycogen and, 719 structure and function, 81F, 82 Equatorial substituents, 196 Equilibrium, 57 in hydrolysis of ATP, 69–71 Equilibrium constant, 40 ERF, 500B Erucic acid, 231 Erythromycin, 203F, 1042 Escherichia coli. See E. coli ESI-MS, 119 Essential amino acids, 557, 775B Essential fatty acids, 229, 265, 558 Essential light chain, 508 Esters cholesterol, 288 enzymes and, 435F, 461F sugar, 198 Estradiol, 243, 244F, 294 Estrogens, 294 ESTs, 907 ETF, 745 Ethanol, pyruvate metabolism and, 564F Ethanolamine, 272 Ether glycerophospholipid, 236–237, 236F Ethidium bromide, 845 Eudesmol, 240F Eukarya, 17, 19F Eukaryotes cell structure, 21–23 cytosolic ribosomes of, 1022–1023, 1023T DNA replication in, 936–939 evolution and, 18 fatty acid synthesis in, 261–263 gene transcription in, 981–993, 984T glycerolipid synthesis in, 272 phospholipids and, 272 processing and delivery of transcripts to ribosomes, 997–1005 protein synthesis in, 1034–1044, 1037T, 1042T Eukaryotic cells, 8, 21–23 Eukaryotic gene transcripts, 823 Eukaryotic initiation factors, 1035, 1037T Eukaryotic peptide chain initiation, 1034–1038 Evolution of aminoacyl-tRNA synthetases, 1014 enzyme design and, 436 related proteins and, 127 RNA and, 823B Evolved coupling stoichiometry, 1113 Exchange reactions, 429–430 Excitable cells, 386 Excitatory amino acid neurotransmitters, 395 Excitatory neurotransmitters, 395 Excretion, nitrogen, 804 Exercise, 718B, 748B Exergonic processes, 59 Exit sites, 1023 Exon junction complex, 1066, 1069 Exons, 997 Exosomes, 1057, 1058B ExPASy molecular biology server, 110, 122

Exporters, ABC, 340 Expressed sequence tags, 907 Expression constitutive, 972 gene, 87, 283, 350F unified theory of, 1080–1083 See also Transcription protein, 913, 914 RNA, 913 Expression cassettes, 920–921 Expression vectors, 912–914 Extra loops, 862 Extracellular matrix, 213 Extrinsic pathways, 481 Extrinsic proteins, 304 Eye, diabetic cataract in, 725B F-2,6-BPase, 704 F-actin, 508 f-Met-tRNAifMet, 1024 F0 unit, 642 F1 unit, 642 Facilitated diffusion, 329–333 FACT, 991 Factor-binding centres, 1030, 1032 Factor, clotting, 242B σ Factors, 964 Facultative anaerobes, 538 FAD/FADH2, 598B Familial hypercholesterolemia, 291 Families, of secondary structures, 165 Faraday’s constant, 64, 329 Farnesyl, 315 Farnesyl groups, 316F Farnesyl pyrophosphate, 283 Farnesyl transferase, 317B Fast foods, 232B Fast skeletal muscle troponin T genes, 1003 Fasting state, 261, 705 Fat-free mice, 1122B Fatty acid catabolism, 735–760 breakdown of fatty acids, 739–749 carnitine deficiency and, 742B dietary antioxidants and, 752B dietary intake and adipose tissue, 736–739 exercise and, 748B ketone bodies and, 742B, 756–757, 757B metabolic fuel, 736T odd-carbon fatty acids, 749–751 other pathways in, 753–755 Refsum’s disease, 755B unsaturated fatty acids, 752–753 vitamin B12 and, 751B Fatty acid synthase, 261–263 Fatty acid synthesis acetate units in, 253–255 acetyl-CoA carboxylase in, 255–257, 256F–257F acyl carrier proteins in, 258 arachidonic acid and, 266 cis double bond in, 264 condensation of acetyl-CoA in, 259–260 cytosolic acetyl-CoA in, 254–255 elongation in, 263–264 in eukaryotes, 261–263 malonyl-CoA in, 255 NADPH in, 253 reduction of β-carbonyl group in, 260–261 Fatty acid thiokinase, 266, 740 ω3 Fatty acids, 267B

ω6 Fatty acids, 267B Fatty acids branched-chain, 753 C16, 263–264 essential, 229, 265, 558 in fast foods, 232B function, 229–232, 230T on megasynthases, 261–263 polyunsaturated, 229 docosahexaenoic acid as, 267B regulation of, 266–268 structure, 229–232, 230T See also Fatty acid catabolism; Fatty acid synthesis Fatty acyl, 741 Fatty acyl synthase I, 258 Fatty acyl synthase II, 258 Fe-protein, 765 Feedback inhibition, 483 Feedback regulation, 483 Fehling’s solution, 196 FeMo-cofactor, 766 Fermentation, 580 Ferredoxin–NADP+ reductase, 668, 671 Fetal hemoglobin, 499–500 FGAR amidotransferase, 873 Fibres, muscle, 506 Fibrils, 157 Fibrin, 481, 482F Fibrinogen, 481, 482F Fibroblast growth factor, 220 Fibroin, 156 Fibronectin, 219 Fibrous proteins, 101, 102F FIH-1. See Hydroxylase factor–inhibiting HIF Finasteride, 293B Fingers (DNA polymerase), 935, 936F First-order reactions, 408, 409F Fischer convention, 88B Fischer, Emil, 88B, 431 Fischer projections, 191F, 192 Flagella, 21 Flagellar rotor, 527–532, 529F–532F Flagellin, 530 Flavin coenzymes, 598B Flavodoxin, 160, 161F Flavoproteins, 628 Flexible rods, 216 FliD (protein), 531 Flippases, 320 Floppases, 320 Fluid mosaic model, 304 Fluorescence, 665 Fluoro-substituted analogs, 892B, 894B Fluoroacetate in tricarboxylic acid cycle, 602 5-Fluorouracil, 894B Folate analogs, 877B Folded dimers, 363 Folding. See Protein folding Foldosomes, 1091 Folds, of secondary structures, 165 Follicle-stimulating hormone, 350F Food pyramid, 3F Fool’s gold, 612B Forces weak, 12–14, 13T quaternary protein structure and, 183 454 Technology, 836 Fractional saturation, 494B

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-9

I-10

Index

Fragmentation of polypeptide chain, 117T Frameshift mutations, 958 Free energy of activation, 405F, 410, 444F equilibrium and, 58 Fructokinase, 581 D-Fructose, 192F Fructose, 193, 199B in glycolysis, 581 Fructose-1,6-bisphosphatase, 447, 685, 696, 699 Fructose-1-phosphate aldolase, 582 Fructose-2,6-bisphosphatase, 704 Fructose-2,6-bisphosphate, 570F, 699, 704 Fructose bisphosphate aldolase, 571–572 α-L-Fucose, 198F Fumarase, 47F Fumarate, 605–606, 779, 798, 803–804 Fumonisin, 246 Functional genomics, 179B, 911B Functional homology, 176 Furanose, 194, 195F Fusion protein expression, 914 Fusion proteins, 914–915, 915T G-actin, 508 G-protein–coupled receptor kinases, 383 G-protein–coupled receptors, 269, 309, 355–357, 356, 367, 380 G proteins, 717 family of, 1023 muscarinic receptors and, 394 phospholipase C activation by, 372–373 as signal transducers, 367–368, 370, 370T G-quadruplexes, 846 Gab-1 (protein), 362 GABA, 81, 81F, 395, 397F Galactokinase, 583 Galactose, 159F, 583 Galactose-1-phosphate uridylyltransferase, 583, 584F Galactosemia, 583 β-Galactosidase, 585B, 971 β-Galactosyl-1,3-α-N-acetylgalactosyl-serine, 214F Galactosylceramide, 277F Galectin-1, 224F Galectins, 223, 224F Gangliosides description of, 238, 275–277 GM1, 238F GM2, 277F GAPs (protein), 370, 380, 382F GAR synthetase, 873 GAR transformylase, 873 Gas chromatography, 105, 556F Gaseous neurotransmitters, 389 Gases, nerve, 394 Gastrin, 350F Gates, 330–333 ∆Gb, 445 GCS-100, 216B 5′-GCTGGTGG-3′, 946 GDI, 368 GEF, 367, 1027 Gel dideoxy sequencing, 836 Gel electrophoresis, 834 Gel filtration chromatography, 107–108 Gel phase, 320 Geminin, 938 Gene chips, 910 Gene expression, unified theory, 1079–1083

Gene expression responses, 349 Gene fusion systems, 915T Gene regulatory proteins, 994–997 Gene replacement therapy, 920 Gene silencing, 823–824 Gene therapy, 883B Gene transcription. See Transcription General acid–base catalysis, 454–455 General recombination, 942, 943 General transcription factors, 985, 987 Genes definition, 18, 926 numbers needed for organisms, 19T regulatory, 972 structural, 972 transcription and, See also Transcription in transgenic animals, 953B tumor-suppressor, 371B–372B whimsical names for, 383B Genetic code, 1009–1012 protein synthesis and, 1010–1012, 1011T, 1012B Genetic engineering, 897 Genetic recombination, 941–950 Genomic libraries, 905–906 Genomics, 179B, 540 Geraniol, 240F Geranylgeranyl, 315, 316F GFP, 87B, 87F, 915 Ghrelin, 1124 Gibberellic acid, 240F Gibbs free energy, 58 GimC, 1091 Globular proteins, 101, 102F, 159–186 aggregation symmetries, 180T folding of, 172–174 helices and sheets in, 159–160, 161F model of, 172–174 packing considerations in, 160–162 motion in, 171–172 Globules, molten, 169 GlpF glycerol channel, 334–335 Glucagon, 350F, 719–721 D-Glucaric acid, 197 Glucocorticoid response element, 986 Glucocorticoids, 295, 721 Glucogenic amino acids, 557 Glucogenic intermediates, 798 Glucokinase, 565–568, 567, 568F Gluconeogenesis, 693–705, 694 glucose monitors, 695B glycolysis vs., 581, 696 inhibitors of, 701B overview, 693–696 phosphoenolpyruvate carboxykinase, 694B regulation of, 701–705 unique reactions in, 696–701 D-Gluconic acid, 197F Gluconic acids, 197 Gluconolactonase, 722 D-δ-Gluconolactone, 197F Glucose, 432F absolute configuration of, 88B binding of by yeast hexokinase, 567F breakdown of, 405F combustion, 16F disaccharide of, 159F in honey, 199B insulin and, 712, 717–719 pentose phosphate pathway and, 722–730 phosphorylation of, 566F

α-D-Glucose-1-phosphate, 198F, 205F Glucose-6-phosphatase, 696, 699 Glucose-6-phosphate, 568–569 glycogen phosphorylase and, 713 glycolysis and, 566F, 567F isomerization of, 568 Glucose-6-phosphate dehydrogenase, 722 Glucose oxidase, 199B Glucose oxidation, 405F Glucose phosphate isomerase, 568 α(1→6) Glucosidase, 706 Glucosylceramide, 277F D-Glucuronic acid, 197 Glutamate amino acid catabolism and, 800 GABA conversion of, 397F as neurotransmitter, 395 Glutamate:aspartate aminotransferase, 430F Glutamate:oxo-glutarate amino transferase, 769 Glutamate–aspartate aminotransferase, 430F, 776 Glutamate dehydrogenase, 768 Glutamate-dependent transamination, 774F Glutamate synthase, 769 Glutamic acid, 78F, 85F Glutamine description of, 768 structure, 78F synthesis of, 769–770 Glutamine phosphoribosyl pyrophosphate amidotransferase, 873 Glutamine synthetase, 768, 795B regulation of, 770–773 size of molecule, 95T Glutaminyl-tRNA synthetase, E. coli, 1016 Glycans, 203 L-Glyceraldehyde, 86 D-Glyceraldehyde, 86 Glyceraldehyde, configuration of, 88B Glyceraldehyde-3-phosphate dehydrogenase, 574 Glycerokinase, 269 Glycerol, 432F glycolysis and, 585 Glycerol-3-phosphate acyltransferase, 269 Glycerol-3-phosphate dehydrogenase, 269 Glycerol kinase, 586 Glycerol phosphate, 212 Glycerolipids, 269–270 sn-Glycerophosphate dehydrogenase, 628 Glycerophosphate dehydrogenases, 586, 651 Glycerophosphate shuttle, 651 Glycerophospholipids, 234F, 234F–235F, 235F–236F, 269 Glycinamide ribonucleotide synthetase, 873 Glycination, 82 Glycine, 395 in green fluorescent protein, 87B structure, 78F synthesis of, 788 titration of, 84F Glycine hydrochloride, 82 Glycine oxidase, 788 Glycine receptors, 398F Glycocholic acid, 292F Glycoconjugates, 189 Glycogen, 203, 205, 705–721 catabolism of, 705–708 regulation of metabolism of, 711–721 synthesis, 708–711 Glycogen phosphorylase, 205, 707, 712, 716 Glycogen phosphorylase reaction, 712–713 NEL

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Index Glycogen synthase, 709, 711 Glycogen synthase D, 711 Glycogen synthase I, 711 Glycogen synthase kinase 3, 719 Glycogenin, 709 Glycolysis, 405F, 537, 561–590 coupled reactions in, 563–564 essential features, 562 during exercise, 718B first phase, 563F, 564–573 cleavage by fructose bisphosphate aldolase, 571–572 first phosphorylation by hexokinase or or glucokinase, 565–568 isomerization of glucose-6-phosphate, 568–569 second phosphorylation by phosphofructokinase, 569–570 triose phosphate isomerase, 572–573 hypoxic stress and, 586–587 metabolic pathway for glucose, 405F NADH and pyruvate in, 580–581 phosphoglucoisomerase, 568–569, 570B pyruvate metabolism and, 564F reactions and thermodynamics, 566T regulation by cells, 581 second phase, 563F, 573–579 steady-state concentrations of metabolites in erythrocytes, 566T substrates other than glucose, 581–586 von Gierke’s disease, 719B Glycolytic pathway, 563F Glycomic dimension, 222 Glyconjugates, 222 Glycopeptide transpeptidase, 424–425, 424F Glycophorin, 307 Glycoprotein peptidase, 424F Glycoproteins, 213–215, 214F Glycosaminoglycans, 209, 218–220, 221F Glycosides, 198–200, 337, 340B Glycosidic bonds, 200, 458F, 811 Glycosphingolipids, 237, 373 Glycosyl phosphatidylinositol anchors, 316F, 318 Glyoxal, 437F Glyoxylate, 686 Glyoxylate cycle, 615–618 Glyoxysomes, 616, 753 Glyphosate, 795B GM-CSF, 1060 GMK, 216B GMP, 813 5′-GMP, 813 GMP, See also Guanosine 5′-monophosphate GMP synthetase, 876 GOGAT, 769 Goldstein, Joseph, 291 Golgi apparatus, 22 of animal cell, 23F of plant cell, 23F GPCR kinases, 383 GPCR signals, 367–368 Gradients, ion, in neuron, 387 Gram-negative bacteria, 209, 210F, 212F, 213F, 314F Gram-positive bacteria, 209, 210F, 212, 212F, 315 Grana, 661 Granum (thylakoid), 661 Gratuitous inducers, 971 GRE, 986 Green fluorescent protein, 87B, 87F, 915

GRKs, 383 GroEL, 176 GroES–GroEL complexes, 1090 Group transfer potential, 63, 64T Growth factor receptors, 358 Growth factors, 220–221 Growth hormone, 350F GrpE (protein), 1089 GSK3, 719 GTFs, 985, 987 GTP, 814 hydrolysis, 64, 1031 molecular motors and, 517F GTP-binding proteins, 355, 717 GTPase-activating proteins, 370, 380, 382F GU-rich elements, 1058, 1065 Guanidino group, 778 Guanidino phosphates, 62, 68–69, 427F Guanine, 810 Guanine deaminase, 881 Guanine-nucleotide dissociation inhibitor, 368 Guanine-nucleotide exchange factor, 367, 1027 Guanosine, 811 Guanosine 5′-monophosphate, 813 Guanylate kinase, 878 Guanylic acid, 813 Guanylin, 363, 364F Guanylyl cyclases, 355, 357–358, 362–363, 366–367 Guanylyl transferase, 999 Guide strands, 1072 Guncotton, 210B GW182, 1072 Gyrases, DNA, 852 H bonds. See Hydrogen bonds H-DNA, 846 H. marismortui, 434F H zones, 506 H+-drug antiporter, 343 H+, K+-ATPase, 338, 340F H+/2e– ratio, 640 H1 (histone), 853 H2A (histone), 853 H2B (histone), 853 H3 (histone), 853 H4 (histone), 853 Hairpin-encoding DNA, 1076B Hairpin loops, 527F–528F Hairpins, 173 Half-cell reactions, 624 Half-lives, 1048 Halobacterium halobium, 308, 335, 341, 343 Halophiles, 19F Halorhodopsin, 341 Handedness, 146B of helices, 153, 157, 173, 532, 841 of polypeptide chains, 173F Hanes–Woolf plots, 417, 417F Hatch–Slack pathway, 687 HATs, 991 Haworth, Norman, 194 Haworth projections, 195–196 Hb. See Hemoglobin HDACs, 992 HDL, 288 Heart cardiac glycosides and, 198, 337, 340B metabolic role of, 1122 Heart myocytes, 213 Heat capacity, 58

I-11

Heat denaturation of DNA, 847 Heat shock element, 986 Heat shock proteins, 176, 1088 Heat shock transcription factor, 987 Heat-stable enterotoxin, 364 Heavy chains, 508 Heavy isotopes, 552 Hedgehog genes, 384B α-Helical barrels, 314 Helical twist, 840 Helical wheels, 160, 161F, 376 Helicases, 506, 522, 524T, 525F, 526, 527F–528F, 928 Helix/Helices, 100F, 145–148 amphiphilic, 160 Baa, 375–376 beta form, 145 double. See Double helix in globular proteins, 159–160, 161F α-helix basic amphiphilic, 375–376 from calmodulin, 161F from citrate synthase, 161F from flavodoxin, 161F in proteins, 145–148 in spider silk, 151–152 unwinding of, 928 Helix 2, 995 Helix 3, 995 Helix behaviour of amino acids, 149T Helix capping, 148, 149F Helix-turn-helix, 994 α-Helix, transmembrane, 309 Heme, 491, 492F Heme iron, 493F, 496F Heme-regulated inhibitor, 1039 Hemiacetals, 193–194, 200F Hemicellulose, 558 Hemiketals, 194, 199, 200F Hemoglobin, 490–502 2,3-bisphosphoglycerate and, 498–500 dimers of, 493F dissociation of oxygen from, 497–498 heme iron in, 493, 496F, 497B myoglobin and, 490–495 nitric oxide and, 500B oxy and deoxy forms of, 496 oxygen binding of, 491F, 494–495B, 496B, 498F pathological sequence variants of, 129T sickle-cell anemia and, 501–502 size of molecule, 95T structure, 94, 101F, 125, 127F, 490–493, 492F, 496–497 subunit motion of, 495F Hemolymph, 202B Henderson-Hasselbalch equation, 43 Henderson, Richard, 309 Heparin, 209 HEPES, 49F Heptad repeat pattern, 155B Herbicides, 795B Heredity, 926 directed changes in, 920–922 Heterochromatin protein 1, 992 Heteroduplex DNA, 942 Heterogeneous nuclear RNA, 820, 999 Heterologous interactions, 181, 181F Heterologous probes, 907 Heteromultimeric proteins, 94, 112, 180 Heteropolysaccharides, 203

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-12

Index

Heterotetramers, 180 Heterotrophs, 538 Hexokinase, 432F, 565–568, 567, 567F, 568F size of molecule, 95T Hexokinase I, mammalian, 568F Hexose monophosphate shunt, 722 Hexoses, abundance of, 191–192 hGH, 953B HGPRT, 879, 880B HIF, 586 High-density lipoproteins, 288 High-energy biomolecules, 61–69 High-energy bond transfer potential, 64T High-energy bonds, 63 High-energy intermediates, 470B, 573 High-energy phosphate compounds, 61, 62T High-fat diet, 557B, 748B High-performance liquid chromatography, 105, 110 High-pressure liquid chromatography, 110 High-spin state, 497B High-throughput DNA sequencing, 836–837 Higher-order RNA regulons, 1081 Histamine, 81F, 82 Histidine evolution and, 800B structure, 79F synthesis of, 796 Histidine–imidazole group, 44T, 47, 50 Histone acetyltransferases, 991 Histone deacetylase complexes, 992 Histone-modifying enzymes, 990 Histones, 819, 853 HIV-1, 184, 941B HIV-1 protease, 449B, 465–466, 465F, 467F HIV mRNA, 467F HLA-A2, 308F HMG-CoA lyase, 758 HMG-CoA reductase, 282, 284F HMG-CoA synthase, 282–283 hnRNA, 820, 823, 999 Hodgkin, Alan, 387 Holliday junctions, 943, 947 Holliday model, 942 Holoenzymes, 407 Homeobox domain, 995 Homeostasis, 17, 1118 Homodimers, 94, 469F Homodisaccharides, 201 Homogentisate dioxygenase, 804B Homoglycans, 203 Homologous proteins, 122 Homologous recombination, 941, 942–943 Homolytic cleavage, 750 Homomultimeric proteins, 94, 179–180 Homopolysaccharides, 203 Homoserine acyltransferase, 785 Homoserine dehydrogenase, 785 Homoserine kinase, 785 Honey, 199B Hoogsteen base pairs, 845 Hop diffusion, 322 Hormone-sensitive lipase, 737 Hormones, 349 definition, 351 eating behaviour and, 1124–1126 eicosanoids as, 278 fatty acid synthesis regulated by, 268–269 glycogen synthesis regulated by, 352, 717–718

peptide, 351 polypeptide, 352 signal-transducing receptors responding to, 354F, 355–366 steroid, 244F, 351 HP1, 992 HREs, 586 HSE, 986 Hsp, 1088 Hsp40 (chaperone), 1089 Hsp60 (chaperone), 176, 1088 Hsp70 (chaperone), 176, 1088, 1092B–1093B Hsp90 (chaperone), 1088 HSTF, 987 HTH, 994 HtrA proteases, 1103 Human genome, 133 Human Genome Project, 911B Human growth hormone, 953B Human immunodeficiency virus, 941B Huntingtin, 1088B Huntington disease, 1088B Huxley, Andrew, 387 Hyaluronates, 209, 221–222 Hyaluronic acid–binding domain, 222 Hyaluronic acids, 203 Hyatt, Isaiah, 210B Hyatt, John, 210B Hybrid duplexes, 850 Hybridization nucleic acid, 850 screening of genomic library by, 906 Southern, 906–907, 908B–909B Hydration, 35–36, 39–40 Hydration shells, 35, 36F Hydride ions, 547, 548F Hydrocarbon chain in membrane bilayer, 305 Hydrogen acceptors, 663 Hydrogen bonds, 13–14, 13T in DNA, 839, 845–847 length and functional groups, 13F low barrier, 455–456, 456F in parallel pleated sheets, 149, 150F with polar solutes, 36 in protein secondary structure, 142, 147F in water, 34–35 Hydrogen donors, 663 Hydrogen ions, 39 Hydrogenation, partial, 231 Hydrolysis of ATP, to ADP, 66F entropy factors from, 66 enzymes and, 455F GTP, 1031 of phosphoric acid anhydrides, 64–66 of RNA, 826, 827 Hydronium ions, 39 Hydropathy, 309T, 310F Hydropathy index, 309 Hydropathy plots, 309 Hydrophilic amino acids, 80 Hydrophobic amino acids, 80 Hydrophobic collapse, 169 Hydrophobic cores, 174 Hydrophobic effect, 153 Hydrophobic interaction chromatography, 110 Hydrophobic interactions, 10, 13T, 14, 36–37, 38F, 142, 471F, 839 Hydrophobic lipids, 228

Hydrophobic proteins in bacterial cell wall, 211 L-Hydroxyacyl-CoA dehydrogenase, 745 Hydroxyapatite, 159 β-Hydroxybutyrate dehydrogenase, 757 β-Hydroxybutyrates, 756 7α-Hydroxycholesterol, 292F β-Hydroxydecanoyl thioester dehydrase, 264 Hydroxyeicosanoic acid, 278 Hydroxyethyl-TPP, 595 Hydroxyl ions, 39 7α-Hydroxylase, 293 Hydroxylase factor-inhibiting HIF, 586 Hydroxylysines, 81, 81F, 159F p-Hydroxyphenylpyruvate dioxygenase, 804 17-α-Hydroxyprogesterone, 248B Hydroxyproline, 81, 81F 17-β-Hydroxysteroid dehydrogenase, 248B 17-β-Hydroxysteroid dehydrogenase 3, deficiency, 248B Hypercholesterolemia, familial, 291 Hyperchromic shift, 847 Hyperpolarization, 395 Hyperuricemia, 883 Hyperventilation, 49B Hypothalamus, 1126 Hypothesis lock-and-key, 431 wobble, of codon–anticodon pairing, 1017 Hypoventilation, 49B Hypoxanthine, 810 Hypoxanthine-guanine phosphoribosyltransferase, 879 Hypoxia, 586 Hypoxia inducible factor, 586 Hypoxia responsive elements, 586 I-739,749, 317B I bands, 506 Ibuprofen, 281, 281F Ice, 34–35 Identical subunits, protein, 181 L-Iduronic acid, 197 IFs, 1025 Imidazole, 45, 45F Immunoglobulin, 183F, 184, 184B, 434 Immunoglobulin G, 12F Immunoglobulin module, 163F Immunoprecipitation, 916–917 IMP, 873 IMP cyclohydrolase, 873 IMP dehydrogenase, 876 Importers, ABC, 340 In vitro mutagenesis, 919–920 Indirect readout, 995 Indole-3-glycerol phosphate synthase, 795 Induced dipoles, 14 Induced fit, 567, 568F Induced fit hypothesis, 431–432 Inducers, 971 Inducible operons, 975 Induction, 478, 971 Inflammation, 224 Influx pumps, 341F Information in DNA, 819 metabolic maps and, 539–542, 540F–541F Infrared region, 88 Inhibited velocity, 419 Inhibition, competitive, 419–421, 421B, 421F NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index Inhibitors allosteric, 484 amino acid biosynthesis, 795B of enzyme activity, 419–424, 420T, 421B, 421F–423F HIV-1 protease, 467F metabolic reaction sequence and, 550F monoamine oxidase, 397B–398B protein synthesis, 1040–1044 Inhibitory neurotransmitters, 395–396 Initiation, 1023 peptide chain, 1024–1026 Initiation factors, 1024, 1025 Initiator (site), 985 Initiator aminoacyl-tRNA, 1023 Initiator tRNA, 1024 Inner mitochondrial membrane, 622 acylcarnitine transport across, 741 Inner mitochondrial membrane translocon, 1097 Inorganic precursors, 9F Inosine-5′l-monophosphate, 873 Inositol-1,4,5-trisphosphate, 245, 372 Inr (site), 985 Insects chitin of, 199, 203, 207 trehalose of, 202B Insertion sequences, 950 Insertions, 956 Insulin, 350F, 481, 481F, 1110B eating behaviour and, 1126 glycogen synthesis and, 712, 717–719 as polypeptide hormone, 352 size of molecule, 94T structure, 111F, 185B Insulin-like growth factor 1, 384 Insulin receptor substrates, 361, 363F Insulin receptor tyrosine kinase, 363F Insulin receptors, 358, 359F, 361–363 Insulin resistance, 701B Integral membrane proteins, 307–314, 328 Integral proteins, 304 Interactions, electrostatic, 471F Intercalating agents, 845 Intercalation, 959 Interconvertible enzymes, 480 Intermediary metabolism, 537 Intermediate-density lipoproteins, 288 Intermediates, 537 acyl carrier proteins and, 258 acyl-enzyme, 460, 462F amphibolic, 546 2-carboxy-3-keto-arabinitol as, 681 high-energy, 470B, 573 lariat, 1001 metabolic integration and, 1110–1111 Intermembrane space, 622 Internal energy, 55 Internal loops, 860 International unit, 415 Interneurons, 386 Intersubunit disulfide bridges, 183 Intervening sequences, 820 Intracellular buffering system, 46–47 Intracellular calcium-binding proteins, 374–375 Intracellular nucleotidases, 880 Intracellular second messengers, 368T Intramolecular tunnels, 796B Intrasteric control, 378, 487 Intrastrand base pairing, 859

Intrasubunit disulfide bonds, 183 Intrinsic binding energy, 445 Intrinsic pathways, 481 Intrinsic proteins, 304 Intrinsically unstructured proteins, 176–177 Introns, 997 Invariant residues, 125 Invertase, 202, 588 Inverted repeats, 845 Ion channels ligand-gated, 355, 357, 391–392 oligomeric, 355, 391–392 voltage-gated, 387 Ion exchange chromatography, 105 Ion gradients, 387 Ion product of water, 40 Ion selectivity, 333 Ionic bonds, 15F Ionic interactions, 13T, 14, 142 Ionization of amino acid side chains, 84–85 entropy factors from, 66 stabilization of hydrolysis products by, 65 of water, 39–40 Ionization constants, 42 Ions hydrating, 35 hydride, 547, 548F IP3, 245, 372, 374 IPTG, 971 Iron:vanadium cofactor, 766 Iron, heme, 493F Iron–sulfur cluster, 601 Iron–sulfur proteins, 628 Irreversible enzyme inhibition, 423–424, 424F Irreversible inhibitors, 419, 423–424, 424F IRS (proteins), 361 Islets of Langerhans, 717 Isoacceptor tRNAs, 1017 Isocaproic aldehyde, 248B, 294F Isocitrate, in tricarboxylic acid cycle, 600–601 Isocitrate dehydrogenase, regulation of, 615 Isocitrate lyase, 615–617 Isoelectric focusing, 109 Isoelectric point, 85, 104, 109 Isolated sulfite oxidase deficiency, 418B Isolated systems, 54, 55F Isoleucine catabolism of, 800 structure, 79F synthesis of, 785, 787–788 Isologous interactions, 181, 181F Isomaltose, 201, 205 Isomerization of citrate by aconitase, 601 of glucose-6-phosphate, 568–569 of proline residues, 172, 173F Isopentenyl pyrophosphate, 283 Isoprene, 240, 241B Isopropylmalate dehydratase, 788 Isopropylmalate dehydrogenase, 788 Isopropylmalate synthase, 788 Isopycnic centrifugation, 849B Isoschizomers, 828 Isotopic tracers, 551–552, 551T, 552F Isozymes, 378, 481–483, 483F ISs, 950 ITC (method), 1105B IXa (clotting factor), 481

I-13

JAK/STAT signalling pathway, 380 Janus protein kinase, 380 JHE. See Juvenile hormone esterase Joule, 55 Junctions, 860 synaptic, 395 Juvenile hormone, 449B Juvenile hormone esterase, 449B Kallikrein, 481 kcat, 415, 415T kcat/Km ratio, 415–416 KcsA, 330, 332–333 KcsA potassium channel, 332F Kendrew, John, 153 Keq, metabolic integration and, 1113 Keratan sulfate, 209 Keratin α-, 153–154 β-, 156 Ketals, 199, 200F α-Keto acids, 775–776 Keto–enol tautomeric shifts, 810 Keto–enol tautomerization, 469F β-Ketoacyl-ACP dehydratase, 260 β-Ketoacyl-ACP reductase, 260 β-Ketoacyl-ACP synthase, 259–260 β-Ketoacyl-CoA intermediates, 746 Ketogenesis, 756 Ketogenic amino acids, 557 Ketogenic intermediates, 798 α-Ketoglutarate, 800 Ketone bodies, 557, 756–757, 757B Ketoses, 191, 192F 3-Ketosphinganine reductase, 275 3-Ketosphinganine synthase, 275 β-Ketothiolase, 282, 746 Keys, 431 Kidneys, gluconeogenesis, 695–696 Kilocalorie, 55 Kinases homoserine, 785 mitogen-activated protein, 377B receptor tyrosine, 357–359, 372–373, 373F tyrosine, 355 Kinesin 1, 517, 518F Kinesin–microtubule complex, 523F Kinesins, 506, 517, 518F, 520–522, 521F, 522F, 523F Kinetics burst, 460, 462F chemical, 408 definition, 408 of enzyme-catalyzed reactions, 425–431 ping-pong, in fatty acid synthesis, 255 zero-order, 411 Km, 413, 414T KNF model, 485 Knobs, synaptic, 386 “Knockout” mice, 949B Knoop, Franz, 739 Koshland–Nemethy–Filmer sequential model, 485, 486F Koverall, 48B Krebs cycle, 591 See also Tricarboxylic acid cycle Krebs, Hans, 591 Kretsinger, Robert, 375 Kusumi, Akihito, 322 Kw, 40

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I-14 L-DOPA,

Index

82F, 398 L0 state, 322 Labels, affinity, 424 lac operon, 971 lac repressor, 973, 974B α-Lactalbumin, 128 Lactase, 202, 585, 585B Lactate, from pyruvate, 564F Lactate dehydrogenase, 483, 483F, 581 Lactic acid fermentation, 562 Lactic acidosis, 719B δ-Lactone, 435F Lactose, structure, 201 Lactose intolerance, 585, 585B Lactose synthase, 585B Lagging strands, 929 LamB protein, 313 Lambda receptor, 313 Lamellae, 21, 660 Landscape, energy, 170, 171F Langdon, Robert, 285B Lanolin, 239 Lanosterol, 240F, 241, 286–288 Lariats, 1001, 1005 Lateral heterogeneity, of membranes, 318 Lathyrism, 162B α-Latrotoxin, 390 LBHBs, 455–456 LC1., 508 LC2, 508 LCAD, 743 LCAT, 289 LDH, 483, 483F, 581 LDL, 215, 288, 289, 291 Ld state, 320 Leader peptidase, 1094 Leader peptides, 1093 Leading strands, 929 Leading substrates, 425, 427–428 Lecithin, 235 Lecithin:cholesterol acyltransferase, 289 Lectins, 222–224, 222T Left-handed DNA, 841 Leloir pathway, 583–584 Lemieux, Raymond Urgel, 190B Leptin, 1126 Lesch–Nyhan syndrome, 880B Lethal synthesis, 892B Leucine catabolism of, 801 structure, 78F synthesis of, 788 Leucine aminotransferase, 788 Leucine zipper-basic region, 994 Leukosialin, 215 Leukotriene, 278 Levers, 1092B Levinthal’s paradox, 169 Levorotatory behaviour, 86 LHC1, 676 LHCs, 668, 676 Libraries, DNA. See DNA libraries Licensing of replication origins, 937 Life times, scale of, 18T Ligand-gated channels, 330, 391–392 Ligand-gated ion channels, 355, 391–392 Ligand-induced conformational changes, 135 Ligand-induced dimerization, 358F, 359 Ligands, 15, 134

Light activation, 685 Light chains, 508 Light energy, chlorophyll capturing, 663–667 Light-harvesting complexes, 668, 676 Light-induced change in chloroplast compartments, 661–662 Light reactions, photosynthetic, 662 Light, ultraviolet amino acid absorbing, 88 pyrimidine dimer repair and, 955 Lignins, 558, 791 Limit dextrins, 706 Limonene, 240F Linear aliphatic structures, 7F Linear introns, 1005 Linear molecular motors, 506 Lineweaver–Burk double reciprocal plots, 416–417, 417F of competitive inhibition, 421F of non-competitive inhibition, 422F, 423F non-linear, 418 of uncompetitive inhibition, 423F Link proteins, 222 1→4 Linked glucose units, 201 Linkers, 901 Linking numbers, 851 Linoleic acid, 229 γ-Linoleic acid, 229 1-Linoleoyl-2-palmitoylphosphatidylcholine, 236 Lipase, 289 Lipid-anchored proteins, 306 Lipid anchors, 316F Lipid biosynthesis bile acids and, 292–293 of complex lipids, 269–277 CDP-diacylglycerol and, 269–271, 271F ceramides and, 275–277 glycerolipids and, 269–270 phosphatidylethanolamine, 270–271 platelet-activating factor and, 275 sphingolipid and, 275 fatty acid. See Fatty acid synthesis steroid hormones and, 293–295 Lipidomics, 246 Lipids, 228–298 as biological signals, 244–246 in diet, 558 fatty acids, 229–232, 230T See also Fatty acid entries glycerophospholipids, 233F, 234F–235F, 235F–236F sphingolipids, 237, 238F spontaneously formed structures of, 302–303, 302F steroids, 243–244, 243F, 244F terpenes, 240–242, 240F–242F, 241B triacylglycerols, 232, 233F waxes and, 239F See also Lipid biosynthesis Lipoic acid, 599B Lipopolysaccharide, 211, 213F Lipoprotein complex, 288 Lipoprotein lipase, 289 Lipoproteins, 215, 289–292, 289F human, 288T Liposomes, 303 Liquid crystalline phase, 320 Liquid-disordered state, 320 Liquid-ordered state, 322

Lithium ions, 375B Liver cortisol and, 721 gluconeogenesis, 695–696 metabolic role of, 1123–1124 pentose phosphate pathway and, 722 Lock-and-key model, 15F, 431 Locks, 431 Lomonskaya, Olga, 345 Long-chain acyl-CoA dehydrogenase, 743 βαβ-Loops, 173 Loops, 860 extra, 862 internal, 860 reactive-centre, 178B variable, 862 Loss, of light energy, 665 Loss of entropy, 445 Lovastatin, 286B Low-barrier hydrogen bonds, 455–456, 456F, 463F Low-density lipoprotein receptors, 289–291, 291F Low-density lipoproteins, 215, 288, 289, 291 Low-spin state, 497B Luciferase, 836 Luteinizing hormone, 350F Lyase, ATP-citrate, 254 Lycopene, 240F Lymphocyte homing receptor, 220 Lysine catabolism of, 803 structure, 79F synthesis of, 779, 783 titration, 85F Lysis, cell, 26 Lysogeny, 26 Lysolecithin, 245B Lysophosphatidic acid, 245 Lysosomal acid lipases, 289 Lysosomes, 22 Lysozyme, 15F, 47F, 128, 457–458, 458F M discs, 506 M lines, 506 Macrolide antibiotics, 1042 Macromolecular synthesis, 1110 Macromolecules, 2 metabolites and, 6–10, 8, 9F Magnesium ion, ATP hydrolysis and, 70 Major grooves, 839 Major histocompatibility antigen, 308F Makhatadze, George, 143 Malate–aspartate shuttle, 651–652 Malate synthase, 615, 617, 618 Malathion, 394 MALDI-TOF mass spectrometry, 119 Male pattern baldness, 293B Malic enzyme, 611, 751 Malignancy. See Cancer Malonate, 422F Malonyl-CoA, 254 Malonyl-CoA-acetyl-CoA-ACP transacylase, 163F, 258 Maltase, 202 Maltoporin, 313 Maltose, 201, 430 Mammals CPS-II activation in, 887B polyunsaturated fatty acids and, 265–266 Mannan, 207F NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index Mannitol, 198 Mannose, 583 α-Mannosyl-serine, 214F MAP kinase, 887B MAPKs, 377B Maple syrup urine disease, 801–802 Maps, metabolic, 539–542, 540F–541F Marfan syndrome, 162B Markers, 911B Mass spectrometry, 118–122, 555, 555F Mathematically defined enzyme-catalyzed reactions, 408–410 Matrix, 623 nuclear, 853 Matrix-assisted laser desorption ionization-time of flight mass spectrometry, 119 Mb. See Myoglobin MC4R, 1126 MCAD, 743 MDR ATPase, 342F Mechanical work, 55 Mechanism-based inhibitors, 892B Mechanosensation, 325 Mechanotransduction, 325 MED proteins, 988 Mediator (complex), 987, 988 Medium-chain acyl-CoA dehydrogenase, 743 Megasynthases, 261 Melanocortin-4 receptor, 1126 Melanocortins, 1124 α- and β-Melanocyte–stimulating hormones, 1124 Melting temperatures, 321, 847 Melville, Herman, 239B Membrane-associated allosteric enzymes, 358–359 Membrane-associated processes, 622 Membrane channel proteins, 329–333 Membrane proteins, 101, 102F, 304–318, 305F bacteriorhodopsin as, 308, 309F dynamic insertion, 312F with single transmembrane segment, 307–308 Membrane rafts, 322, 323F Membrane–skeleton fence model, 322 Membrane–spanning supramolecular complexes, 670 Membrane thickness, 304 Membranes, 299–348 asymmetry, 318 curvature, 323–325 depolarized, 387 fluid mosaic model of, 304 lateral diffusion, 322–323 lipids in water and, 302–303 motions in, 319–320 oxidative phosphorylation and, 622–623 proteins of. See Membrane proteins transport across, 327–328 Menten, Maud L., 411 Menthol, 240F Merrifield, Bruce, 130 Mesosomes, 21 Messenger ribonucleoproteins, 1051 Messenger RNA. See mRNA Messengers, second, 368–374, 368F, 368T Met-aminopeptidase, 1093 Metabolic channeling, 886–887 Metabolic experiment, isotopic tracers in, 551–552, 551T, 552F Metabolic integration ATP coupling and, 1112–1114

caloric restriction and, 1126–1128 cellular energy status, 1114–1115 eating behaviour and, 1124–1126 energy balance regulation, 1115–1117 in multicellular organisms, 1118–1124, 1118T systems analysis, 1109–1112 Metabolic maps, 539–542, 540F–541F Metabolic pathways compartmentalization of, 553–555, 555F glycolysis, 405F multi-enzyme systems in, 543F protein-centric view, 542F Metabolic responses, 349 Metabolical integration, 1109–1132 Metabolism, 537–560 anabolic and catabolic processes in, 543–550 cellular, 16F, 17 experimental elucidation, 550–554 maps of, 539–542, 540F–541F metabolic diversity, 538–539 metabolomes and, 555–556, 555F Metabolites, macromolecules and, 6–10, 9F Metabolomes, 555 Metabolomics, 555, 555F Metabolons, 542 Metal activated enzymes, 456 Metal ion catalysis, 70, 407T, 456, 456F Metal response element, 986 Metalloenzymes, 456 Metallothionein, 987 Meteorite, Murchison, 90B Metformin, 701B, 1117 Methanogens, 19 Methionine catabolism of, 800 structure, 79F synthesis of, 783, 785 Methionyl-tRNAifMet formyl transferase, 1024 Methotrexate, 877F Methyl-D-glucoside, 200F Methyl-directed pathway, 954 Methyl-GTP cap, 1034 Methylmalonyl-CoA epimerase, 749 Methylmalonyl-CoA mutase, 750 Mevalonate, 282–284, 283F, 285B Mevinolin, 286B Mevinolinic acid, 286B Mice fat-free, 1122B “knockout”, 949B transgenic, 953B Micelles, 37, 38F, 302, 302F Michaelis constant, 413 Michaelis, Lenore, 411 Michaelis–Menten equation, 411–418, 412F, 413, 484F Micro-RNAs, 824, 1049, 1071 Microarrays, DNA, 910 Microdomains, 322 Microtubule-binding protein tau, 1088B Microtubules, 184F, 515, 517F, 519B, 520F, 523F Mimicry, molecular, 1031B Mineralocorticoids, 295 Miniband units, 854 Minimum gene set, 18 Minor grooves, 839 Minus end, of microtubule, 515 -35 region, 967

I-15

miRNA duplex, 1072 miRNA/miRNA*, 1072 miRNAs description of, 824, 1071 in post-transcriptional gene regulation, 1049, 1071–1079 Mirtrons, 1072 Mismatch repair, 951, 954 Mitchell’s chemiosmotic hypothesis, 639 Mitochondria, 22F of animal cell, 22F of eukaryotic cell, 22 function, 10 of plant cell, 22F Mitochondrial diseases, 611B Mitochondrial inner membrane, 741 Mitochondrial outer membrane translocon, 1097 Mitochondrial protein, 1097 Mitogen-activated protein kinases, 377B Mitogens, 887B Mitosis, 22 Mixed-function oxidase, 293 Mixed non-competitive inhibition, 422, 423F MMR, 951 Mobile electron carriers, 629 Mobile elements, 950 MoCo, 763–764 Model, Koshland–Nemethy–Filmer, 485, 486F Modifications allosteric regulation and, 490F reversible and non-reversible, 353 Modified standard state, 60 Modules, 162–164 MoFe-protein, 765 Molar absorptivity, 96 Molecular activity, of enzyme, 415 Molecular chaperones, 176, 1088, 1089 Molecular disease, sickle cell anemia, 501–502 Molecular evolution, 125 Molecular mimicry, 1031B Molecular motors, 505–536 DNA unwinding and, 522–527 flagellar rotors and, 527–532, 529F–532F microtubules and, 515–522, 519B muscle contraction and. See Muscle contraction Molecularity, of reaction, 408 Molten globules, 169 Molybdenum cofactor, 763 Monoamine oxidase, 397B–398B Monoamine oxidase inhibitors, 397B–398B Monocytes, 279 Monolayers, lipid, 302, 302F Monomeric proteins, 94 Monosaccharides, 190, 191–200 classification of, 194 derivative forms of, 196–200 Monoterpenes, 240 Monounsaturated fatty acids, 229 Morpheeins, 503 Motion of active-site residues, 453F in globular proteins, 171–172 in membranes, 319–320 Motor domains, 521F Motor neurons, 386, 387F Motor proteins, 505 MRE, 986 Mristoyl group, 366

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-16

Index

mRNA, 816, 1023–1034 HIV, 467F RBPs and, 1052B–1053B, 1062–1065, 1064B recognition and alignment of, 1025 sequence information in, 820–821 surveillance systems, 1066 translation in protein synthesis, 820–821, 1023–1034, 1025T, 1027T mRNA decay, 1055–1059, 1059T, 1077–1078 mRNA half-life, 1049, 1052B–1053B mRNA turnover, 1048, 1049–1054, 1054T mRNPs, 1051 α- and β-MSH, 1124 Mucins, 213 Mucopolysaccharides, 203 Multi-enzyme systems, 542–543 Multi-spanning membrane proteins, 308 Multicellular organisms, 1118–1124, 1118T Multidrug resistance, 340 Multifunctional polypeptide, 886 Multilamellar vesicles, 302F, 303 Multimeric proteins, 94 Multiple codons, 978 Multiple sclerosis, 435 Multiplexes, 845–846 Multisubstrate reactions, 430–431 Muramic acid, 199 Murchison meteorite, 90B Murein, 210 Muscarinic receptors, 390, 394–395 Muscle, metabolic role of, 1119–1122, 1121B Muscle contraction, 505–515 actin and myosin in, 506–511 calcium release and, 506 Duchenne muscular dystrophy, 510B–511B optical trapping experiments, 514B P-loops, 512B sliding filament model of, 512–515, 512F, 513F smooth muscle effectors, 507B Mutagenesis, 919–920 Mutant form of human sulfite oxidase, 418B Mutant protein, 178B Mutarotation, 194 Mutations, 955 metabolism affected by, 551 molecular basis of, 128, 955–959 MWC model, 484 Myo-inositol, 198 Myocytes, heart, 213 Myofibrils, 506, 508F Myoglobin hemoglobin compared with, 125, 127F, 490–495 oxygen binding in, 491F, 498F size of molecule, 94T structure, 125, 492F Myosin, 506–507, 509F, 510F conformational change and, 515F, 521F size of molecule, 95T Myosin heads, 508 Myosin V, 518F Myristoyl, 366 Myristoyl-CoA:protein N-myristoyltransferase, 315 N-(5′-phosphoribosyl)-anthranilate isomerase, 795 N-Acetylneuraminic acid, 238 N (face), 629 N-Formyl-Met-tRNAifMet, 1024F N-linked glycoproteins, 214F N-linked saccharide, 213

N-myristoyl groups, 316F N-myristoylation, 315 N-terminal ends, 111 Na+, K+,-ATPase, 335–337 NAC (chaperone), 1088 NACs (conformations), 450, 451F, 468–469, 471F NAD+, in catabolism, 483F, 547–548, 548F NAD+-dependent dehydrogenases, 428 NADH glycolysis and, 483F as inhibitor of citrate synthase, 600 NADH–coenzyme Q reductase, 628 NADPH, 3, 3F in anabolism, 548–549, 549F in fatty acid synthesis, 253 glucose-6-phosphate and, 728–729 metabolic integration and, 1111 NADPH-specific glyceraldehyde-3-phosphate dehydrogenase, 682 NADPH-specific malate dehydrogenase, 687 NaK channel, 333, 334F Naming of chiral centres, 91B of chiral molecules, 86 of enzymes, 406–407 of genes, 384B of polysaccharides, 203 of proteins, 384B Nanodevices, 820B Nanotechnology, 820B Nascent chain-associated complex, 1088 Nascent polypeptides, 1087 Natively unfolded protein, 176 Natriuretic hormones, 363 ncRNAs, 823 NDPs, 814 Near-attack conformations, 450, 451F, 468–469 Negative cooperativity, 485 Negative entropy, 58B Negative regulation, 972 Negentropy, 58B NER, 951 Nerve gases, 394 Neuraminic acid, 199 Neurodegenerative disorder, 1088B Neurofibrillary tangle, 1088B Neurofibrillary tangles, 1088B Neuroglia, 386 Neurological disorders, 397B–398B, 1088B Neurons, 386, 387F Neuropeptide Y, 1124 Neurotransmission, 349 Neurotransmission pathways, 386–400 Neurotransmitters, 389, 389T, 395 catecholamine, 398–399 Neutral pH, 41 Neutrality, 41 Neutrophils, 279 NFTs, 1088B NHEJ, 951 Nick translation, 932 Nicolson, G. L., 304, 306 Nicotinamide coenzymes, 597B Nicotinic acetylcholine receptors, 391–392 Nicotinic receptors, 390 Nitrate assimilation, 762–763 Nitrate reductase, 763, 764 Nitric oxide, 366, 500B Nitrifying bacteria, 763

Nitrite reductase, 763 Nitrocellulose, 210B Nitrogen, 761–807 amino acids. See Amino acid catabolism; Amino acid synthesis ammonium and, 768–770 atmospheric, 5 E. coli glutamine synthetase and, 770–773 excretion, 804 inorganic forms acquired, 762–768 Nitrogen and amino acid metabolism, 761 Nitrogen balance, 557 Nitrogen cycle, 763F Nitrogen fixation, 762, 764–768 Nitrogenase, 764–766 Nitrogenase reaction, 766–767 Nitrogenase reductase, 765 Nitrogenous bases, 809–811 Nitrophenolate, 462F NMD, 1049, 1066–1070 NMDA receptors, 396F NMR. See Nuclear magnetic resonance NMR (spectroscopy) See Nuclear magnetic resonance spectroscopy NMT, 315 Nobel, Alfred, 210B Nomenclature. See Naming Non-coding RNAs, 823 Non-competitive inhibition, 421–422, 422F, 423F Non-covalent bond, 12 Non-cyclic photophosphorylation, 679 Non-essential amino acid, 775B Non-helical polypeptides, 311 Non-histone chromosomal proteins, 819, 853 Non-homologous DNA end-joining, 951 Non-homologous recombination, 941 Non-identical subunits, protein, 181 Non-linear Lineweaver–Burk plots, 418 Non-polar amino acids, 77, 78F, 80 Non-receptor tyrosine kinase, 365–366 Non-reducing ends, 201 Non-steroid hormones, 350F Non-steroidal anti-inflammatory drugs, 280B–281B Non-stop decay, 1049, 1070 Non-template strands, 965B Nonsense codons, 1011, 1067–1068 Nonsense mediated decay systems, 1049, 1066–1070 Nonsense suppression, 1019 Norepinephrine, 351, 398, 398B Normal stop codons, 1067 Northern blotting, 908B NPY/AgRP-producing neurons, 1124 NRI, 773 NRII, 773 NSD, 1049, 1070 NTPs, 814 Nuclear area, of prokaryotic cells, 21 Nuclear magnetic resonance (NMR) amino acids characterized by, 89–90, 89F spectroscopy, 89–90, 552–553, 553F, 555, 556F Nuclear magnetic resonance spectroscopy, 89–90, 552–553, 553F, 555, 556F Nuclear matrix, 853 Nuclear pre-mRNA splicing, 1000–1001 Nucleases, 826–827 Nucleic acids, 809, 815–830, 833–870 base sequence of, 815–816 chemical synthesis of, 856–858 NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index classes of, 816–817, 824–825 definition, 815 DNA. See DNA hybridization of, 850 hydrolysis of, 825–830 primary structure, 11F, 834–837 RNA. See RNA secondary structure. See DNA, secondary structure; RNA, secondary and tertiary structures Nucleolus, 1022 Nucleophilic centres, 454, 454F Nucleoprotein filaments, 946 Nucleoside 5′-diphosphates, 814 Nucleoside 5′-triphosphates, 814 Nucleoside diphosphate kinase, 604, 699, 879 Nucleoside diphosphates, 813–814 Nucleoside triphosphates, 813–814 Nucleosides, 811–812 Nucleosomes, 819 chromatin and, 853–854 Nucleotide excision repair, 951, 955 Nucleotides, 808–815, 809 nitrogenous bases, 809–811 nucleic acids and. See DNA; RNA nucleosides and, 811–812 structure and chemistry of, 11F, 812–815 Nucleotides, synthesis and degradation, 871–896 analogs, therapeutic, 877B, 892B, 894B deoxyribonucleotide formation and, 888–891 Lesch–Nyhan syndrome, 880B mammalian CPS-II and, 887B of purines, 872–884 of pyrimidines, 884–888 severe combined immunodeficiency syndrome and, 883B tetrahydrofolate and 1-carbon units, 875B of thymines, 891–894 Nucleus/nuclei of eukaryotic cell, 22 function, 9 of plant cell, 23F steroid hormones and, 294 Nutrients, 537 Nutrition, 557 See also Diet O antigens, 211 O-linked saccharides, 213, 214F, 215F Obligate aerobes, 538 Obligate anaerobes, 538 Obligate coupling stoichiometry, 1112 Obligatory substrates, 427–428 Octadecanoic acid, 229 Odd-carbon fatty acids, 749–751 OECs, 670 Okazaki fragments, 929 Oleic acid, 229 Oleoyl alcohol, 239F Oleoyl stearate, 239F Oligo(α1,4→α1,4)glucanotransferase, 706 Oligomeric ion channels, 355, 391–392 Oligomers, 180 Oligomycin, 647 Oligonucleotides gene chips and, 910 phosphoramidite chemistry and, 857 Oligopeptides, 94 Oligosaccharides, 190, 200–203 cleavage of, 216–217 N-linked, 216

residue, 200 synthesis, 190B OMP decarboxylase, 886 Oncogenes, 370, 371B–372B Oncogenic viruses, 365 One international unit, 415 1:2 Pause, 979 Open heterologous quaternary structures, 184 Open promoter complexes, 966 Open systems, 54, 55F Operational codes, 1015 Operators, 971 Operon hypothesis, 972 Operons, 1080–1082 araBAD, 975–977 definition, 971 Optical activity, 86 Optically active molecules, 88B ORCs, 937 Order, for reactant, 408 Ordered reactions, 425 Ordered single-displacement mechanisms, 428 Organelles, 2, 8–10 Ori sequence, 899 Origin recognition complexes, 937 Origins of replication, 899, 927 Ornithine, 777 Ornithine transcarbamoylase, 777 Orotate phosphoribosyltransferase, 886 Orthologous proteins, 123 Osmotic pressure, 39 Osteoblasts, 338 Osteoclasts, 338, 341F Ouabain, 198, 337 Outer membrane, 622 Ovalbumin, 167F Ovalbumin gene, 998 Overall activity sites, 889 Oxidase, mixed-function, 293 ω-Oxidation, 753, 755 Oxidation α-oxidation, of branched-chain fatty acids, 753 β-oxidation. See β-Oxidation prostaglandins and, 278 β-Oxidation, 739–753 4-step cycle in, 741–746 energy yield of, 746–747 fatty acid activation, 740–741 fatty acyl groups and, 741 metabolic water and, 748–749 of odd-carbon fatty acids, 749–751 of unsaturated fatty acids, 752–753 Oxidative cleavage of disulfide bridges, 114F Oxidative phosphorylation, 548, 592, 621–658 apoptosis and, 653–655 cytosolic NADH and, 650–653 electron-transport chain and, 627–640 as membrane-associated process, 622–623 P/O ratio for, 649–650 proton gradient and, 641–649 reduction potentials and, 622–623 thermodynamics of, 640–641 Oxidized donors, 663 2,3-Oxidosqualene lanosterol cyclase, 286 Oxygen in metabolism, 538 in myoglobin, 498F

I-17

Oxygen cycle, 539, 539F Oxygen-evolving complexes, 670 Oxygen saturation curves, 498F, 500F Oxymyoglobin, 492 Oxytocin, 507B P, 629 P (face) P-Bodies, 1054T P-cluster, 766 P-loops (NTPases), 512B p-Nitrophenylacetate, 455F, 461F P/O ratios, 649–650 P proteins, 467 P-Selectins, 223 P sites, 1023 P680 (reaction centre), 667 P700 (reaction centre), 667, 675 P870 (reaction centre), 672 PA700 (regulator), 1101 PAF, 236, 237B Pairing base, 817, 859 chromosome, 942 codon–anticodon, 1017–1019, 1017T, 1018T, 1055F, 1055T Pairs diastereomeric, 193 enantiomeric, 193 Palindromes, 845, 972 Palm (DNA polymerase), 935, 936F Palmitate synthesis, 259F Palmitic acid, 229, 231 Palmitoleoyl-ACP, 264F Palmitoyl-CoA in fatty acid synthesis, 256–257 sphingolipid synthesis and, 275 Pancreatic ribonuclease A, bovine, 100F Papain, 116 Papillomavirus E1 protein, 526F PAR-CLIP, 1064B–1065B Parallel beta-pleated sheets, 149 Parallel chains, in chitin, 207 Paralogous proteins, 123, 125 Parkinson’s disease, 631B, 1088B Partial hydrogenation, 231 Partition properties, of amino acid mixtures, 105 Parvalbumin, 375 Passenger strands, 1072 Passive diffusion, 328–329, 330F Pasteur, Louis, 88B Patch recombinants, 943 Path independence, 55 Pathways metabolic. See Metabolic pathways methyl-directed, 954 neurotransmission, 386–400 of palmitate synthesis, 259F pentose phosphate, 722–730 signal transduction, 351–355, 354F transduction, 374F Pauling, Linus, 146, 150B, 444 1:2 Pause, 979 Pawson, Anthony, 351B PCNA (protein), 938 PCP, 395 PCR, 917–920 PCR-based mutagenesis, 920 PDC, 595

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-18

Index

PDZ domains, 1103 Pelouze, Theophile, 210B Penicillin, 424–425, 424F Pentaglycine chains, 210 Pentose phosphate pathway, 722–730 Pentoses, 811 Pentraxins, 224 PEP. See Phosphoenolpyruvate PEP carboxykinase, 612, 696, 699 PEP carboxylase, 611, 687 Pepsin, 47F, 116, 406, 465F Pepstatin, 473F Peptidases, 1094, 1096 Peptide bonds, 76 handedness of, 146B of protein, 91–93, 91F, 92F Peptide chain initiation of, 1024–1026 in eukaryotes, 1035–1038 Peptide hormones, 351 Peptide mass fingerprinting, 121–122 Peptide neurotransmitters, 389 Peptides atrial natriuretic, 364 leader, 1093 polypeptide chains of, 94 solid-phase synthesis of, 130, 131F vasoactive intestinal, 399 Peptidoglycan, 209–214, 210F Peptidyl site, 1023 Peptidyl transfer, 1027 Peptidyl transferase, 434F, 1021 Peptidyl transferase centres, 1027 Peptidyl transferase reactions, 433–434, 434F Peripheral proteins, 304 Periplasmic spaces, 211 Perkin condensation, 600 Permanent dipoles, 14 Peroxisome proliferator-activated receptor, 748B Peroxisome proliferator-activated receptor δ Peroxisomes, 22, 753 Perutz, Max, 146, 153 PEST sequences, 1100 PET. See Positron emission tomography PFK-2, 704 PGC-1 (gene regulator), 1128 pH, 40–46, 41T ATP hydrolysis and, 69, 69F blood and, 49B, 498F buffers and, 46–49, 47F denaturation of DNA and, 847–848 enzymatic activity and, 418–419, 419F of glycine solution, 84 pH gradients, 338 Pharmaceuticals, 408 Phase transitions, 321–322 PHDs, 586 Phencyclidine, 395 Phenol, 435F Phenylalanine catabolism of, 803 structure, 79F synthesis of, 793 ultraviolet light absorbed by, 88 Phenylalanine-4-monooxygenase, 793 Phenylalanine aminotransferase, 793 Phenylalanine hydroxylase, 793 Phenylketonuria, 793B, 804B Phenylketonurics, 804B

Pheophytin, 670 Phorbol esters, 378 Phosphatase, naming of, 406 Phosphate compounds, high-energy, 61, 62T Phosphate esters, 198 Phosphate system, 46–47 Phosphatidic acid, 234F, 235, 269 Phosphatidylcholine, 235F, 373 Phosphatidylethanolamine, 235, 270–271, 272 Phosphatidylglycerol, 235F Phosphatidylinositol, 235F, 269, 372 Phosphatidylinositol-4,5-bisphosphate, 372 Phosphatidylinositol-4-P, 372 Phosphatidylserine, 272 Phosphinothricin, 795B Phosphocreatine shuttle, 68F Phosphodiesterases, 826 Phosphoenolpyruvate formation of, 67, 68F hydrolysis of, 61 Phosphoenolpyruvate carboxykinase, 694B Phosphoethanolamine transferase, 271 Phosphofructokinase, 479–480, 479F, 569–570, 569F Phosphoglucoisomerase, 568, 569F, 570B Phosphoglucomutase reaction, 470B, 583 6-Phosphogluconate dehydrogenase, 722 Phosphogluconate pathway, 722 Phosphoglucose isomerase, 568 2-Phosphoglycerate, 68F 3-Phosphoglycerate, 67F, 680 3-Phosphoglycerate dehydrogenase, 788 3-Phosphoglycerate family of amino acids, 788–791 Phosphoglycerate kinase, 575 3-Phosphoglycerate kinase, 682 Phosphoglycerate mutase, 576 Phosphoglycolohydroxamate, 450F Phosphohistidine, 604 Phospholipase A1, 245F Phospholipase A2, 245F Phospholipase A2, 371F Phospholipase C, 371F, 372–373, 394 Phospholipase C-β, 373F Phospholipase D, 371F Phospholipases, 245, 245B, 245F, 372 Phospholipids, 245B, 245F asymmetry, 319F eukaryotes synthesizing, 272 glycerophospholipids, 233–236 motion and, 320F phase transitions, 322T Phosphomannoisomerase, 583 Phosphopentose epimerase, 724 Phosphopentose isomerase, 724 Phosphoprotein phosphatase-1, 712, 716 Phosphoprotein phosphatases, 480 Phosphoramidite chemistry, 857 Phosphorelay system, 377B Phosphorescence, 88 Phosphoribosylanthranilate transferase, 795 Phosphoribosyltransferases, 879 Phosphoribulose kinase, 683 Phosphoric acid hydrolysis of anhydrides of, 64–66 titration curve for, 45, 46F Phosphoric anhydrides, 61, 65F Phosphorolysis, 713 Phosphoryl group transfer reaction, 63F Phosphorylase, backbone structures of, 174F

Phosphorylase a, 716 Phosphorylase b, 716 Phosphorylase cascade, 719 Phosphorylation of acetyl-CoA carboxylase, 256–257, 256F of glucose, 566 oxidative. See Oxidative phosphorylation reversible, 486 Phosphorylation–dephosphorylation cycles, 266–268, 378 Phosphorylation potential, 1115 Phosphotriesterase, 166F Photochemical apparatus, 1112 Photolyase, 954 Photolysis, 668 Photophosphorylation, 673 Photoreactivating enzyme, 954 Photorespiration, 686 Photosynthesis, 659–692 ATP synthesis in, 677–680 Calvin cycle series, 683T carbon dioxide in, 680–689 limited fixation of, 686–689 organic molecules from, 680–686 general properties, 660–663 photosystems in, 667–671 quantum yield, 677 reaction centres in, 671–677 solar energy and, 663–667 Photosynthetic reaction centres, 671–677 Photosynthetic units, 667 Photosystem I, 667 Photosystem II, 667 Photosystems, 667–671 Phototrophic organisms, 61 Phototrophs, 538, 1112 Phylloquinone, 675 Phylogenetic tree, cytochrome c, 125, 126F Phytanic acid α-hydroxylase, 753 Phytanic acid α-oxidase, 753 Phytoalexin, 1128 Phytol, 240F, 664, 753 PI, 235F, 269, 372, 375B PI-88, 216B PIC, 990 PIDs, 352, 353 α-Pinene, 240F Ping-pong reactions. See Double-displacement reactions PIP, 372 PIP2, 372 PIR server, 122 Pitch, 147, 838 Pitocin, 507B Pits, coated, 289 Pizzarello, Sandra, 90B pKa, 83T PKU, 793B Planar structures, 7F Planar transition states, 447F Plant cells, 25T Plants isoprene emitted from, 241B photosynthesis in. See Photosynthesis Plaques amyloid, 1088B arterial, 288F atherosclerotic, 162B Plasm, 898 NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index Plasma membrane receptors, 350F Plasma membranes, 21, 299, 301F Plasmalogens, 237, 272–275 Plasmids, 850–851, 898 Plastids, 21, 661 Plastocyanin:ferredoxin oxidoreductase, 671 Plastoquinol:plastocyanin oxidoreductase, 670 Plastoquinone, 670 Platelet-activating factor, 236, 237B, 380 in lipid synthesis, 275 Pleated sheets, β-, 149–151 Pleated strands, 100F Plus end, of microtubule, 515 PNP, 881 Point mutations, 956–957 Points, isoelectric, 85, 104, 109 pol (enzymes), 930 Polar bears, 233B Polar, uncharged amino acid, 78F Polarity, of water, 35 Poly(A) polymerase, 1000 Poly(A) tails, 821, 1000, 1034 3′-Polyadenylylation, 1000 Polyadenylylation signal, 1000 Polyamino acids, 148 Polycistronic mRNA, 970n2 Poly(Gly-Pro-Pro), 158F Polyhedrin, 914 Polylinkers, 901 Polymerase chain reactions, 917–920 Polymerases, universal mechanism, 935B Polymerization, of Hb S, 501F Polynucleotide fragments, 834–835 Polynucleotides, 815 Polyol pathways, 725B Polypeptide chains fragmentation of, 117T history, 150B right-handed twist, 173F Polypeptide hormones, 352 Polypeptides, 77, 94 backbone, 92, 174F Hsp70 chaperones and, 1089 structure, 11F Polyprenols, 241 Polyprotic acids, NDPs and NTPs as, 814 Polyribosomes, 1034 Polysaccharides, 190, 203–213 in bacterial cell walls, 209–217, 211F, 212F cell surface, 212–213 function, 203–210 naming of, 203 storage, 204–205 structural, 205–213 structure, 11F Polysomes, in protein synthesis, 1034 Polyunsaturated fatty acids, 229 docosahexaenoic acid as, 267B in mammals, 265–266 Porin, 622 Porin proteins, 313 Portal vein system, 717–718 Positive inside rule, 309 Positron emission tomography, 582B Post-transcriptional gene regulation, 993, 1048–1085 AU-rich mediated decay, 1059–1065, 1061B, 1061T, 1079–1080 cellular processes maintaining RNA integrity, 1066–1071

exosomes, 1057, 1058B miRNAs as regulators in, 1071–1079 mRNA decay, 1055–1059, 1059T, 1078–1079 mRNA half-life, 1052B–1053B mRNA turnover, 1049–1054, 1054T RIP-Chip and PAR-CLIP and, 1064B–1065B siRNA and shRNA, 1076B–1077B unified theory of gene expression, 1079–1083 Post-transcriptional processing of mRNA, 999–1000 Post-transcriptional RNA operons, 1081 Post-translational modifications of protein, 86, 132, 993, 1091 Postsynaptic cells, 389 Potassium, action potential and, 387–389 Potassium channels, 330–333 Potentiality, thermodynamic, 404 Potentials action, 387–389 electrical, in neuron, 387 group transfer, 63, 64T proton transfer, 64T PP1, 712, 716 PP2A, 730 pp60v-src, 365 PPARγ (receptor), 1128 PPT, 795B Pre-initiation complex, 990 Pre-miRNAs, 1072 Pre-mRNAs, 999 Pre-RCs, 937 Precursors inorganic, 9F to GMP and AMP, 873 to plasmalogens, 272 zymogens as, 481 Prefoldin, 1091 Pregnenolone, 248B, 293, 294F Premature-termination codons, 1049, 1058, 1067–1068 Prephenate, 469F Prephenate dehydratase, 793 Prephenate dehydrogenase, 793 Prepro-hormones, 352 Prepro-opiomelanocortin, 352 Preproalbumin, 1095 Prereplication complexes, 937 Pressure, osmotic, 39 Presynaptic cells, 389 Pri-miRNAs, 1072 PriA (protein), 949 Pribnow boxes, 967 Primary active transport, 335 Primase, 933 Primed synthesis method, 835 Primers, 834, 930 Priming reactions, in glycolysis, 565, 565F Prions, 26, 28–29, 130B Privalov, Peter, 143 Pro α-2 collagen gene, 998 Pro-enzymes, 459 Pro-proteins, 1093 prob, 906 Probes definition, 850 techniques using, 906–907, 908B Processes, of neurons, 386 Processing, of transcripts, 997 Processing bodies, 1051

I-19

Processive movement, 524 Processivity, 930 Product formation, rate of, 413 Products, 443 accumulation of, 478 Proenzymes, 481 Progesterone, 243, 244F, 248B, 294 Progestins, 244F Proinsulin, 481, 481F Projections Fischer, 191F, 192 Haworth, 195–196 Prokaryotes, 17, 19F features of, 21, 21T gene transcription in, 963–970, 965B, 968B regulation, 970–981, 974B, 974T peptide chain initiation in, 1024–1026 Prokaryotic promoters, 966–967 Prolactin, 350F Proline, 78F, 447F, 800 conformations, 453F synthesis of, 777 Proline racemase, 447 Proline racemase reaction, 447F Proline switches, 1092B Prolyl hydroxylases, 586 Prolyl isomerase, 453F Promoters, 901, 964, 968B in eukaryote, 985 Propeller twist, 840 β-Propellors, 291 Propionyl-CoA carboxylase, 749 Prostaglandin endoperoxide H synthase, 278, 280F Prostaglandins, 269, 278, 278B Prosthetic groups, 87B, 133, 407, 628 Protease, 116F, 406 aspartic, 462, 464B, 464T, 465–466, 466F HIV-1, 449B, 465–466, 465F, 467F Protease inhibitors, 449B, 468B Proteasome activator-700 kD, 1101 Proteasome inhibitors, 1103B Proteasomes, 1098, 1100 Protein:protein interactions, 980 Protein Data Bank, 122 Protein denaturation, thermodynamic parameters, 57T Protein deposit, diseases with, 1088B Protein expression, 913, 914 Protein folding, 1086–1093 chaperone-mediated, 1092B–1093B diseases of, 179B of globular proteins, 159–162, 172–174 Hsp70 chaperones for, 1089 mechanism of, 169–170 N-linked oligosaccharides in, 218B thermodynamics, 170–171 unfolding, 59–60, 60F Protein interaction domains, 352, 353 Protein isoforms, 1003 Protein kinase A, 369, 376, 377F, 487–488, 488F Protein kinase C, 372, 378 Protein kinases classification of, 487T covalent modification and, 480 description of, 487–488 mitogen-activated, 377B Protein modules, 162–164 Protein phosphatase 2A, 730 Protein–protein interactions, 135, 916

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-20

Index

Protein structure, classes, 165, 175F Protein synthesis, 1009–1047 amino acid matching with tRNA, 1012–1017, 1014T codon–anticodon pairing and, 1017–1019, 1017T, 1018T diphtheria toxin and, 1041B in eukaryotic cells, 1034–1044, 1037T, 1042T genetic code and, 1010–1012, 1011T, 1012B messenger RNA and, 820–821, 1023–1034, 1025T, 1027T molecular mimicry, 1031B ribosome structure and assembly, 180, 1019–1023, 1020T, 1023T Sonenberg, Nahum and, 1010B Protein synthesis inhibitors, 1040–1044, 1042T Protein translocation, 1093, 1094 Protein triage, 1004B Protein tyrosine phosphatase SHP-2, 379 14-3-3 Proteins, 384B Proteins α/β proteins, 174, 175F α + β Proteins, 174, 175F acetylcholine transport, 392 actin-anchoring, 510B amino acid sequences of, databases of, 122 annexin, 375 biological functions, 133–135 biotin carboxyl carrier, 255 calcium-binding, 374–375 cAMP receptor, 975 classification schemes for, 164–166 conjugated, 132–133 degradation of, 216–218, 1098–1105, 1104B domains of, 162–163 EF hand, 375 extrinsic, 304 in fatty acid synthesis, 255 fibrous, 101, 102F flexibility and motion, 171–172 folding of. See Protein folding fusion, 915T G proteins. See G proteins globular. See Globular proteins heat shock, 176, 1088 hydrophobic, 211 integral, 304 intrinsic, 304 intrinsically unstructured, 176–177 link, 222 location targeting, 1093 membrane. See Membrane proteins multimeric, 94 mutant, 178B peripheral, 304 post-translational processing, 1092 purification, 102–105 ras, 370 receptor, 386 scaffolding, 324, 381 shifting of, 171–172 size of molecules of, 94T surface of, 160F targeting of molecules by globular proteins. See Globular protein marginal stability of, 171 unfolding of, 59–60, 60F viral transforming, 365

whimsical names for, 384B See also Protein synthesis Proteins, structure and function, 98–139 amino acid sequences, 122–129 amino acid techniques, 105–111 conjugated proteins, 132–133 isolation and purification, 102–105 levels of organization, 99 polypeptide synthesis, 130–132 primary structure determination, 111–122 quaternary symmetry of, 181, 182F weak forces in, 183 secondary, 100F, 140–152 amide plane in, 92 beta-pleated sheets in, 149–151 beta-turns in, 152 shape and solubility classes, 101 tertiary, 99, 101F, 128F, 129F, 152–180 Proteoglycan cell growth and, 220–221 function, 218–222 Proteoglycan–hyaluronate aggregates, 222 Proteoglycans, 218–222 Proteolytic enzymes, 117T digestive, 481 Proteomes, 133 Proteomics, 540, 911B Prothrombin, 242B Proto-oncogenes, 370, 371B–372B Protofilaments, 531 Proton gradients, 641–649 Proton pumps, 341F Proton symports, 343 Proton transfer potential, 64T Proton transport, 343 Protoporphyrin IX, 492F Proviruses, 941 Prozac, 397B–398B PrP, 28–29 PS decarboxylase, 272 PS1. See Photosystem I PS2. See Photosystem II PsaA, 675 PsaB, 675 PsaC, 675 PsaD, 675 Pseudoknot formation, 861 Pseudosubstrate sequences, 378, 487, 1116 Pseudouridine, 822, 862 PSI complexes, 668 PSII complexes, 668 PTCs (codons), 1049, 1058 PTCs (peptidyl transferase centres), 1027 Pterins, 875B PTRO, 1081 Pulse-chase experiments, 1052B–1053B Pure non-competitive inhibition, 422, 422F Purine nucleoside cycle, 881–882 Purine nucleoside phosphorylase, 881 Purine riboside, 450F Purine ring, 810F, 872F Purines, 796, 872–884 catabolism of, 880–884 in nucleic acid structure, 809–811 salvage of, 879 synthesis of, 872–879 Purple patches, 343 Pyranose, 194, 195F

Pyranose ring, 569F Pyrimidine dimer repair, 954 Pyrimidine ring, 810F Pyrimidines fluoro-substituted, 894B in nucleic acid structure, 809–811 synthesis and degradation, 884–888 Pyroglutamic acid, 81, 81F Pyrophosphomevalonate decarboxylase, 285 Pyrrole-2-carboxylate, 447F [Delta]-1-Pyrroline-2-carboxylate, 447F Pyrrolysine, 81, 81F Pyruvate, 68F in glycolysis, 483F glycolytic production of, 564F Pyruvate carboxylase, 611 in gluconeogenesis, 694 Pyruvate carboxylase reaction, 696 Pyruvate decarboxylase, 580 Pyruvate dehydrogenase complex, 490F, 595 Pyruvate dehydrogenase kinase, 490F, 613 Pyruvate dehydrogenase phosphatase, 490F, 615 Pyruvate family of amino acids, 788 Pyruvate kinase, 71F, 577–579 Pyruvate-Pi dikinase, 688 PYY3-36, 1126 Q cycle, 635 Q-SNAREs, 328F Qa-SNARE, 326, 327F Qbc-SNARE, 326, 327F Quadruplexes, 846 Quality control, protein, 1103, 1104B Quantum yield of photosynthesis, 667, 677 Quaternary structure, of protein, 99, 179–185 R (conformational state), 496 R (pathway), 1115 R-SNARE, 326, 327F, 328F R state, 715 R1 (protein), 889 R2 (protein), 889 Radiation, ultraviolet, 88 Ramachandran plot, 144, 145F Random coil, 160 Random reactions, 425, 427, 427F RANTES, 384, 385F Rapid axonal transport, 520F Rapid initial bursts, 460 Ras, 317B ras gene, 370 Ras protein, 370 Rat cartilage proteoglycans, 219F Rat growth hormone, 953B Rate, 408 factors influencing, 478 Rate constant, 408 Rate laws, 408–409 Rate-limiting steps, 460 Rate of enzyme catalysis, 408–410 Rate of product formation, 413 Ratio, kcat/Km, 415–416 Rational drug design, 426B RBPs, 1049, 1052B–1053B, 1062–1065, 1064B Reactants rate laws and, 408–409 and transition states, 443 NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index Reaction glycogen synthase, 709, 711 priming, 565, 565F Reaction centres, photosynthetic, 667, 671–677 Reaction half-cells, 624 Reaction stoichiometry, 1112 Reactions bimolecular, 409, 425 second-order, 408–409 Reactive-centre loop, 178B Reactivity, specificity and, 432, 432F Reagent, Edman’s, 113 Real-time polymerase chain reaction, 917–919 Reannealing, 849 RecA (protein), 523, 943, 946 RecB (protein), 943 RecBCD (enzyme complex), 943–944 RecC (protein), 943 RecD (protein), 943 Receptor guanylyl cyclase, 357–358 Receptor protein, cAMP, 975 Receptor proteins, 352, 386 Receptor signal transduction, 376–379 Receptor superfamilies, 355, 356F Receptor tyrosine kinases, 357–358, 372–373, 373F Receptors acetylcholine, 390, 391F asialoglycoprotein, 217 G-protein-coupled, 355–356 LDL, 289–291 lymphocyte homing, 220 muscarinic, 394 nicotinic acetylcholine, 391F nitric oxide, 366–367 signal-transducing, 352, 354F, 355–366 TMS, 357–358 Reciprocal control, 702 Recognition matrix, 995 Recombinant DNA, 897–925 cloning and, 898–904, 912–917, 915T cystic fibrosis and ADA-SCID, 922B directed changes in heredity and, 920–922 DNA libraries and, 904–912, 905B, 912–917 Human Genome Project, 911B polymerase chain reaction and, 917–920 Smith, Michael and, 898B Southern blotting, 908B–909B Recombinant DNA molecules, 897 Recombinant DNA technology, 897 Recombinant plasmids, 899 Recombinase, 946 Recombination, genetic, 941–950 Recombination-dependent replication, 948B Red blood cell, sickle cell anemia and, 501–502 Red wine, polysaccharide in, 208B Redox couples, 624 Reduced acceptors, 663 Reducing ends, 201 Reducing sugars, 197 Reductases, 5α-, 293B Reduction potentials, 64T, 622–623 Reductive citric acid cycle, 612B Reductive cleavage, of disulfide bridges, 114F Reentrant loops, 311 Reference half-cells, 624 Refsum’s disease, 755B Regular structures, of protein, 99, 102F Regulation allosteric. See Allosteric regulation

by enzymes, 404, 405–406 of glycogen synthase, 709, 711 metabolic, 17, 547F of phosphofructokinase, 479–480 Regulators of G-protein signalling, 380–382 Regulatory elements, 985 Regulatory enzymes, 404, 418, 483 Regulatory genes, 972 Regulatory light chain, 508 Regulatory proteins, 971 Regulatory RNAs, 823 Regulons, 1080–1082 Relaxed DNA, 851 Relaxed form, of hemoglobin subunits, 496 Release factor, 1032 Relenza, 216B Renaturation of DNA, 849 Rep helicase, 524, 525F Repeats, inverted, 845 Replication, DNA, 834, 927–930 Replication factor C, 940 Replication factories, 935 Replication fork restart, 949 Replication forks, 927 Replication licensing factors, 937 Replication protein A, 938 Replicator activator proteins, 937 Replicators, 899 Replicons, 936 Reporter genes, 915 Repressible operons, 975 Repression, 478, 971 Repressors lac, 973, 974B trp, 977 Repulsion, electrostatic, 64, 65F, 66F Reserpine, 397B–398B RESID database, 132 Residues, 200 amino acid, 94 oligosaccharide, 200 sialic acid, 217F Resonance, 65–66 Resonance energy transfer, 665 Respiratory acidosis, 49B Respiratory alkalosis, 49B Response elements, 985, 986–987 cAMP, 994B Restriction endonucleases, 827–830, 829T–830T Restriction enzymes, 827 Restriction fragment size, 828 Resveratrol, 1128 Reticulum endoplasmic, 22 sarcoplasmic, 338, 339F, 507F Retina, docosahexaenoic acid in, 267B Retrograde translocation, 1097 Retroviruses, 940, 941B Reuptake, of neurotransmitters, 395 Reverse transcriptases, 907, 940–941 Reversible covalent modification, 480F Reversible inhibitors, 419 Reversible phosphorylation, 486 RF, 1032 RF-1, 1032 RF-2, 1032 RF-3, 1032 RFC, 940 rGH, 953B

I-21

RGS, 380 RGS modules, 381 RGS proteins, 380–382 Rhamnogalacturonan II, 208B α-L-Rhamnose, 198F ρ, 969 Rho termination factor, 969 Ribbon structures of proteins, 163F Ribbons, extended, 206 Ribitol, 198 Ribitol phosphate, 212 Ribonuclease bovine, 100F, 159F unfolding of, 168F Ribonuclease A, 159F size of molecule of, 94T Ribonucleic acid. See mRNA; RNA Ribonucleoprotein particles, 1000 small nuclear, 823 Ribonucleosides, 811 Ribonucleotide reductase, 888, 890–891 Ribonucleotides, 813 Ribose-5-P, 728–729 Ribose-5-phosphate pyrophosphokinase, 872 Ribose zippers, 861 Ribosomal proteins, 1020 Ribosomal RNA, 821 Ribosomal subunits, 1032 Ribosome binding site, 1025 Ribosome nascent chain:SRP complex, 1095–1097 Ribosome recycling factors, 1032 Ribosomes anatomy of, 1021 assembly of, 1019–1023 catalytic power of, 433–434 definition, 820, 1019 eukaryotic, 1022–1023, 1023T life cycle of, 1034F prokaryotic, 1019–1021 protein synthesis, 180 structure and assembly, 180, 821, 1019–1023, 1020T, 1023T Riboswitches, 867 Ribothymidylic acid, 822 Ribozymes, 404B, 432–434, 1004 Ribulose-1,5-bisphosphate, 680 Ribulose-5-phosphate kinase, 682 Ribulose bisphosphate carboxylase/oxygenase, 680 Right-handed DNA, 841 Rigour-like state, 514 Ring purine, 810F pyrimidine, 810F RIP-Chip, 1064B–1065B RNA, 858–867 catalytic properties, 404B, 432–433 definition, 815 DNA, differences from, 824, 826 early evolution and, 823B as genetic material, 941B hydrolysis of, 826, 827 integrity maintenance, 1066–1071 messenger. See mRNA ribosomal, 816 ribozymes, 404B, 432–434 roles in cells, 819–824 secondary structures, 858, 862, 863–865 tertiary structures, 862, 865 types, 816–817

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-22

Index

RNA-binding proteins, 1049 RNA-directed DNA polymerase, 940 RNA editing, 993, 1004 RNA-EMSA, 1062–1065 RNA expression, 913 RNA genomes, 940–941 RNA-induced silencing complex, 1072 RNA interference, 824, 1071 RNA polymerase holoenzyme, 964 RNA polymerase I, 982 RNA polymerase II, 982 RNA polymerase III, 982 RNA polymerases, in eukaryotes, 982 RNAi, 824, 1071 RNase P, 404B RNases, 827 RNC-SRP, 1095–1097 RNPs, 1000 Robinson, Robert, 285B Rosanoff convention, 88B Rosanoff, M. A., 88B Rossman folds, 526 Rotating molecular motors, 506 Rotational catalysis, 644 Rotors, 642 Rough endoplasmic reticulum, 22F Rous sarcoma virus, 365 RPA, 940 rRNA, 821, 863–865, 1019–1020 (R,S) system, 86 Rsk1, 386 RT-PCR, 917 Rubisco, 680 Rubisco activase, 681 RuBP. See Ribulose-1,5-bisphosphate Ruminants, 206–207 RuvA (protein), 943, 947 RuvB (protein), 943, 947 RuvC (protein), 943, 947 RuvC resolvase, 947 5S rRNA, 1019 16S ribosomal RNA, 1019, 1027 19S caps (regulator), 1101 19S regulator, 1100 20S proteasome, 1100 23S rRNA, 1019, 1027 26S proteasome, 1100 30S initiation complex, 1025 30S subunit, 1019 43S preinitiation complex, 1035 50S subunit, 434F, 1019 70S initiation complex, 1025 70S ribosome:RF-1:RF-3-GTP:termination signal, 1032 S-Adenosylmethionine synthase, 785 S-palmitoyl groups, 316F 7S RNA, 1095 S-shaped curves, 483 S0 state, 320 Saccharides, 213, 214F, 215F Saccharomyces cerevisiae, 317B SAGA complex, 991 SAICAR synthetase, 873 Salivary α-amylase, 204 Salmonella, 211 Salt bridges between subunits of hemoglobin, 497F SAM (protein processing), 1097 SAM (S-adenosylmethionine), 785

Sample half-cells, 624 Samuelsson, Bengt, 278B Sanger, Frederick, 112 Sanger’s chain termination method, 834–836 Saponification, 232 Sarcolemma, 506 Sarcoma virus, Rous, 365 Sarcomeres, 506 Sarcoplasmic reticulum, 338, 339F, 506, 507F Sarin, 394, 395F Saturated fatty acids, 229 Saturation behaviour, 329 Saturation curves, 134, 411, 411F oxygen, 498F, 500F Saturation effects, 411 Sav1866, 341 Saxitoxin, 393B SCAD, 743 Scaffolding proteins, 324, 381 Schiff base linkages, 343F Schultze, Major E., 210B SCID, 920 SCOP database, 164–165, 175F Scramblases, 320 Scrapie, 130B Scurvy, 162B SDS-polyacrylamide gel electrophoresis, 108 Sec, 1012B Sec61 complex, 1096 SECIS elements, 1012B Second genetic code, 1012 Second messengers, 353, 368–374, 368F, 368T, 716 Second-order reactions, 409 Secondary active transport, 335, 343–345 Secondary structure. See DNA, secondary structure; Protein, structure Secreted polypeptide hormones, 352 Sedimentation coefficients, 821 Sedoheptulose-1,7-bisphosphatase, 683 “Seed elements”, 1071 Seed regions, 1076 Selectable markers, 899 Selectins, 223 L-Selectins, 223 Selectivity filters, 330–333 Selenocysteine, 81, 81F, 1012B Self-splicing, 433 Semiconservative replication, 927 Sensors, 981 Sensory neurons, 386 Sequence-independent detection, 918 Sequence-specific probe binding assays, 919 Sequence-specific transcription factors, 995 Sequence variants, hemoglobin, 129T Sequencing amino acid. See Amino acid sequencing DNA, high-throughput, 836–837 Sequential model, 485 Sequential reactions, 425 Serglycin, 219F Serine catabolism of, 798 ethanolamine and, 272 in green fluorescent protein, 87B in lipid synthesis, 272 sphingolipid synthesis and, 275 structure, 78F synthesis of, 788 Serine acetyltransferase, 788

Serine dehydratase, 798, 801B Serine hydroxymethyltransferase, 788 Serine phosphatase, 788 Serine proteases, 128, 459–462 Serotonin, 81F, 82 Serpin, 178B Serum albumin, 737 Serum cholesterol, 448B Sesquiterpenes, 240 Sevenless, 384B Severe combined immunodeficiency syndrome, 883B Sex hormones, 294 SF1, 523, 525F SF2, 523, 525F SF3, 525F SH modification in cleavage of disulfide bridges, 114F SH2 domains, 351B Shc (protein), 362 β-Sheets. See Beta sheets Shift, hyperchromic, 847 Shikimate dehydrogenase, 793 Shikimate kinase, 793 Shikimate pathway, 791 Shine-Dalgarno sequence, 1025 Short-chain acyl-CoA dehydrogenase, 743 Short interspersed elements, 993 shRNA, 1076B–1077B Shuttle systems, 650 Shuttle vectors, 904 Sialic acids, 199, 217F, 238 Sickle cell anemia, 501–502 Side chain, amino acid, 84–85 Sigma factors, 964 Sigmoid curves, 483, 484F Signal peptidase, 1094, 1096 Signal receptors, 1094 Signal recognition particles, 1094, 1095 Signal sequences, 132, 1093 Signal transducers, 370 Signal-transducing receptors, 354F, 355–366 Signal transduction, 352–355 Signal transduction pathways, 315, 352–355 Signal transduction units, 368 Signalling pathways, 353 Signals, GPCR, 367–368 Signalsomes, 353 Silencers, 985 Silencing, gene, 823–824 Silk, spider, 151–152 Simple proteins, 132 Simple sugars, 190 See also Monosaccharides SINEs, 993 Singer, S. J., 304, 306 Single-displacement bisubstrate mechanism, 425F Single-displacement reactions, 425–428, 425F, 427F Single particle tracking, 322 Single polycistronic mRNA, 970 Single-strand assimilation, 947 Single-strand damage repair, 951 Single-strand uptake, 947 Single-stranded DNA-binding proteins, 928 Single transmembrane segment, 307–308, 307F–308F Single-transmembrane segment catalytic receptors, 355 SIR2, 1127–1128 NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index siRNAs, 824, 1076B–1077B Siroheme, 764 SIRT1, 1127–1128 Sirtuin 1, 1127–1128 Size exclusion chromatography, 107 Skeletal muscle, 507F, 508F, 1119–1122, 1121B See also Muscle contraction Sld2, 937 Sld3, 937 Sliding clamps, 932 Sliding contacts, 493, 493F Sliding filament model, 512–517 Small interfering RNA, 824 Small nuclear ribonucleoprotein particles, 823 Small nuclear RNAs, 816, 823 Small nucleolar RNAs, 824 Small RNAs, 816–817, 823 Small ubiquitin-like protein modifiers, 1101–1102 SMC proteins, 855–856 Smith, Michael, 898B Smooth endoplasmic reticulum, 22F Smooth muscle effectors, 507B Smooth muscles, 507B Snake venom, 245B SNAP-25, 326 SNARE motifs, 326 SNAREs, 326, 327F SNF2, 990 Snorkelling, 310, 311F snoRNAs, 824 snRNPs, 823, 1002 Soaps, 232 Sodium, action potential and, 387–389 Sodium channel toxin, 393B Sodium dodecylsulfate-polyacrylamide gel electrophoresis, 108 Sodium pump, 335–337 Solar energy, 663–667 Solid-ordered state, 320 Solid-phase synthesis, of peptide, 130, 131F Solubility, of globular protein, 104F Soluble protein activator, 285 Soluble protein, unfolding of, 59–60 Solutes colligative properties and, 39 DNA denaturation and, 847 water properties and, 38 Solution, double helix in, 845 Solvents industrial, 437F water, 35–39, 50 Soma, 386 Somatic recombination, 942 Somatostatin, 350F Son of sevenless, 384B Sonenberg, Nahum, 1010B Sonic hedgehog, 384B Sorbitol, 198 Sørensen, Søren, 40 Sorting and assembly complex, 1097 Southern blotting, 908B–909B Southern, E. M., 908B Southern hybridization, 906–907, 908B–909B Specific acid–base catalysis, 454–455 Specific activity, 104, 415 Specific linking difference, 852 Specific phospholipases, 370–371

Specificity, enzyme, 405, 431–432, 432F Spectrin, 323 Spectroscopy amino acid properties, 87–91 nuclear magnetic resonance, 89–90, 552–553, 553F, 555, 556F Spermaceti, 239B Sphingolipids, 237, 238F, 269, 275, 276F Sphingomyelinase, 373 Sphingomyelins, 238, 275, 277F, 373 Sphingosine, 238 Sphingosine-1-phosphate, 246 Spider webs, 151–152 Splice recombinants, 943 Spliceosomes, 1002 Splicing, 999–1003 alternative, 993, 1003 lariat intermediate and, 1001 nuclear pre-mRNA, 1000–1001 RNA, 433F Squalene, 240F, 241, 283–285, 284F, 285B Squalene monooxygenase, 285 SR (sarcoplasmic reticulum), 338, 339F, 506, 507F Srb/Med (complex), 987 sRNAs, 824 SRP54, 1095 SRPs (signal recognition particles), 1094, 1095 SRs (signal receptors), 1094 SSB proteins, 928 Stabilization, transition-state, 444F Stacking, coaxial, 861 Stage 1 (aerobic catabolism), 544 Stage 2 (aerobic catabolism), 544 Stage 3 (aerobic catabolism), 545 Stalks, 642 Standard reduction potential, 624 Standard state, 56, 60 Stanozolol, 295 Staphvax, 216B Staphylococcal protease, 116 Staphylococcus aureus, 313 Starch, 203–205 Starch phosphorylase, 204, 205F State function, 55 Statins, 286B, 448B Statistical thermodynamics, 57 Stators, 642 Steady state, 4, 1115 Steady-state assumption, 412 Stearic acid, 229, 239F 1-Stearoyl-2-oleoyl-phosphatidylcholine, 236, 236F Stearoyl-CoA desaturase, 265, 265F Stearyl palmitate, 239F Stem-loop structures, 859, 969 Steroid hormones, 351 action of, 351 synthesis of, 293–295, 294F Steroid receptors, 987 Steroids anabolic, 295 structure and function, 243–244, 245F, 248B Stoeckenius, Walter, 341 Stoichiometry, 1112–1113 Stomata, 689 Stop-transfer sequences, 1096 Stop-transfer signals, 1094 Storage polysaccharides, 204–205 Strand exchange, 943 Strand invasion, 942

β-Strands, 173F, 311 Streptomyces lividans, 330, 332F Streptomycin, 203F Stroma, 660 Strong electrolytes, 41 Strong polyprotic acids, 814 Structural analogs, 431 Structural Classification of Proteins database, 164–165, 175F Structural complementarity, 14–15, 15F, 134 Structural genes, 972 Structural genomics, 179B Structural polarity, 11 Structural polysaccharides, 205–213 Structural strain, 445 Strychnine, 396 Substrate-binding sites, 419–420 Substrate cycles, 705 Substrate-level control, 703 Substrate-level phosphorylation, 575, 604 Substrate saturation curves, 411, 411F Substrate specificity sites, 889 Substrates binding of, 411 definition, 405 determination of rate, 478 leading, 425, 427–428 obligatory, 425, 427–428 suicide, 424 Subtilisin, 116 Subunits motion in hemoglobin, 495F protein, 99 ribosomal, 1032 Succinate, structure, 422F Succinate–coenzyme Q reductase, 628 Succinate dehydrogenase, 421–422, 422F as FAD-dependent, 605–606 Succinate thiokinase, 604 N-Succinyl-5-aminoimidazole-4-carboxamide ribonucleotide, 874F Succinyl-CoA, 604 amino acid catabolism and, 800 Succinyl-CoA synthetase, 604 Sucrase, 202 Sucrose, 190B, 201–202, 430 Sucrose phosphorylase, 588 Sugar acids, 196–197 Sugar alcohols, 197–198 Sugar code, 222 Sugar esters, 198 Sugar nucleotides, 708 Sugars amino, 199 deoxy, 198 exchange reaction of, 430 simple, 190 structure, 11F See also Monosaccharides; Polysaccharides Suicide substrates, 424, 892B Sulfate, sulfide synthesis from, 789–790 Sulfatide, 238 Sulfide synthesis, 789–790 Sulfite oxidase assimilation of, 790 wild-type, 418B Sulfometuron methyl, 795B Sumoylation, 991 Supercoils, 851–853

NEL Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

I-23

I-24

Index

Superfamilies, of secondary structure, 165 Superfamily 1, 523 Superfamily 2, 523 Superhelix density, 852 Suppressor tRNAs, 1019 Suppressors, 1019 Supramolecular complexes, 8, 9F SWISS-2DPAGE database, 110 SWISS-PROT protein sequence database, 122 Switch complexes, 532 Switches, 1092B Symmetry, 181, 182F Symmetry model, 484 Symports, 343 Synapses, 386, 389, 390 Synapsis, 942 Synaptic bulbs, 386 Synaptic clefts, 386, 389, 392 Synaptic junctions, 395 Synaptic knobs, 386 Synaptic terminals, 386 Synaptotagmin, 327 Synchrony, 213 Syndecan, 218, 219F Synonymous codons, 1011 Synovial fluid, 209 Synthesis amino acid. See Amino acid synthesis cholesterol, 282–288 dehydration and, 200 of eicosanoids, 278–281 fatty acid. See Fatty acid synthesis protein. See Protein synthesis See also Biosynthesis Synthetic combinatorial libraries, 905B Synvinolin, 286B Systems analysis, 1109–1112 Systems biology, 555 Systems, thermodynamic, 53 Szent-Györgyi, Albert, 512–513 Szostak, Jack W., 927B T (conformational state), 496 T state, 715 T-tubules, 506 TA (domain), 916 Tabun, 394, 395F TAFIIs, 988 TAFs, 988 Tails, myosin, 510F Tamiflu, 216B, 449B Tandem enzymes, 704 Tandem mass spectrometry, 120–121 Tangle, neurofibrillary, 1088B TATA-binding protein, 988 TATA boxes, 985 τ (protein), 1088B Taurocholic acid, 292 Taut form, of hemoglobin subunits, 496 Tautomerization, 67 Taxol, 519B Tay-Sachs disease, 292 TBP, 988 TBP–associated factors, 988 TCA cycle, 592 See also Tricarboxylic acid cycle TcC loops, 861 TcC stem-loops, 862 Teichoic acids, 212

Telomerase, 857B Telomeres, 817, 857B, 940B Temperatures enzymatic activity and, 419 melting, 321, 847 Template DNA, description of, 927 Template strands, 965B Templates, 834, 927 Tense form, of hemoglobin subunits, 496 Terminals, synaptic, 386 Termination, 1024 Termination codons, 1011 Termination sites, 969 3:4 Terminator, 979 Terpenes, 240–242, 241B Tertiary structure, of protein. See Proteins, structure Testosterone, 243, 244F, 248B, 294F, 350F Tetrahedral oxyanion transition states, 464B Tetrahydrofolate, 875B Tetrahymena, 433F Tetraloops, 860 Tetrodotoxin, 393B TF (chaperone), 1088 TFIIA, 988 TFIIB, 988 TFIID, 988, 991 TFIIE, 988 TFIIF, 988 TFIIH, 988 TFIIIA, 983 TFIIIB, 983 TFIIIC, 983 Thermal cyclers, 917 Thermal denaturation, of DNA, 847 Thermoacidophiles, 19 Thermodynamic potential, 71, 403 Thermodynamics, 53–74 ATP hydrolysis and, 69–71 coupled processes and, 71 enthalpy and, 55–57, 56, 56F first law of, 54–55 high-energy biomolecules and, 61–69, 62T parameters of, 61T of protein denaturation, 57T second law of, 57–58 third law of, 58 Thermogenin, 649B, 1123 Thermolysin, 116, 456F THF, 875B Thiamine pyrophosphate, 597B Thick filaments, 508–509 Thin filaments, 508 Thioester linkages, 599B Thioester-linked fatty acyl anchors, 315 Thioesters, 62 Thioether-linked prenyl anchors, 315, 318 Thiolase, 283, 746 Thioredoxin, 889 Thioredoxin reductase, 889 Third-base degeneracy, 1011 -35 region, 967 3:4 Terminator, 979 Threonine, 79F, 783 synthesis of, 785 Threonine deaminase, 788 Threonine synthase, 785 Threshold cycles, 919 Thrombin, 128, 481, 482F

Thromboxane, 278 Thumb (DNA polymerase), 935, 936F Thylakoid lumen, 662 Thylakoid membranes, 660 Thylakoid spaces, 662 Thylakoid vesicles, 660 Thymidine, 811 Thymidylate synthase, 893 Thymines, 810 synthesis and degradation, 891–894 Thyroid hormones, 351 TICs (chloroplast membranes), 1097 Tight turn, in protein, 152 TIM barrel, 166 TIM22 (translocon), 1097 TIM23 (complex), 1097 Titration curve, 44, 44F Tm, 321 1-TMS, 355, 357 7-TMS, 357 7-TMS proteins, 357 TMS receptors, 357–358 Tnsertion sequences, 950 TOCs (chloroplast membranes), 1097 TolC, 343 Tolrestat, 725B TOM (translocon), 1097 TonEBP, 162 Top-of-power strokes, 513 Topoisomerases, 851 Toroidal supercoiled DNA, 853 Tosyl-L-phenylalanine chloromethyl ketone, 473F Toxic shock syndrome, 237B Tracers, isotopic, 551–552, 551T, 552F TRAM (subunit), 1096 trans-Complexes, 326, 328F trans-Elements, 1048, 1057 2,3-trans-enoyl-ACP reductase, 260 Trans-hydration of fumarase, 606 Transaldolase, 727 Transamination reaction, 775–776, 776B Transcript start sites, 966 Transcription, 809, 963–1008 associated transducers and activators of, 380 CREB-type transcription factors and, 994B in eukaryotes, 981–993, 984T processing and delivery of eukaryotic transcripts to ribosomes, 997–1005 in prokaryotes, 963–970, 965B, 968B regulation of, 970–981, 974B, 974T recognition of specific sequences, 994–997 steroid hormones and, 294 Transcription activators, 992 Transcription attenuation, 978 Transcription factors, 983 heat shock, 987 Transcription start sites, 966 Transcriptional activators, 980 Transcriptome, 911B Transcriptomics, 540 Transcripts, 965B Transducers, 352, 1092B–1093B Transduction energy, 665 of receptor signals, 367–368 Transduction pathways, 374F signal, 352–355, 354F NEL

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.

Index Transfection, 953B Transfer, peptidyl, 1027 Transfer potential, 63–64, 64T Transfer protein, cholesterol ester, 289 Transfer RNA, 816, 1012–1017, 1014T Transferase, peptidyl, 1021 Transformation experiment, bacterial, 902 Transforming growth factor β, 220 Transgenic animals, 953B Transient receptor potential channels, 333 Transition-state analogs, 447, 448B–449B Transition-state stabilization, 444F Transition-state theory, 445 Transition states, 409, 444 esters and, 435F intermediates vs., 444n1 NACs and, 451F stabilization and, 446F Transition temperatures, 321–322 Transitions (mutations), 956 Transketolase, 726 Translation, 809, 963, 1019 Translation repression, 1077 Translesion DNA synthesis, 954 Translocases, 522, 525F, 741 Translocation, 1021, 1030 protein, 1093, 1094 Translocon inner chloroplast membranes, 1097 Translocon outer chloroplast membranes, 1097 Translocons, 1094 7-Transmembrane segment proteins, 357 Transmembrane segments, single, 307–308 Transpeptidation, 1027 Transport electron. See Oxidative phosphorylation membrane, 327–328 Transposable elements, 950 Transposition, 941 Transposition events, 950 Transposons, 950 Transverse asymmetry, 318 Transverse tubules, 506, 507F Transversions, 956 Tranylcypromine, 398B Treadmilling, 515, 517F Tree of life, 19F Trehalose, 202B Triacontanol palmitate, 239F Triacylglycerol lipase, 737 Triacylglycerols, 269, 288, 736 formation of, 272F structure and function, 232–233 Triage, 1004B Triantennary oligosaccharides, 217 TriC, 1091 Tricarboxylic acid cycle, 591–620 acetate-based modification, 615–618 acetyl-CoA and, 600–604 anaplerotic reactions and, 611–613 cleavage of 2-Carbon Compound, 593–594 diagram of, 594F diets and, 557B energetic consequences of, 607–609 fluoroacetate blocking, 602 and intermediates for biosynthesis, 610–611 oxaloacetate, 604–607 pyruvate and, 595–600 regulation of, 613–615

Trigger factor, 1088 Trioleoylglycerols, 232 Triose kinase, 582 Triose phosphate isomerase, 166 backbone structures of, 174F in glycolysis, 572–573 Trioses, 191 Tripartite complexes, 343, 344F Triple helix of collagen, 157–159 Triplex DNA, 846 Tristearin, 233F Tristearoylglycerol, 232 Triterpenes, 240 Triton X-100, 302F tRNA, 816, 822–823 protein synthesis and, 1012–1017, 1014T structure, 861–863 secondary, 862 tertiary, 862 tRNAiMet, 1035 Trojan horse inhibitor, 602 Trojan horse substrates, 424, 892B Tropocollagen, 156 Tropomyosin, 506, 508 Troponin, 506 Troponin C, 376, 1119 Troponin complexes, 508 trp operon, 971 trp repressor, 977 Trypsin elastase and, 461F naming of, 406 in polypeptide fragmentation, 115, 116F as serine protease, 128 Trypsinogen, 460F Tryptophan structure, 79F synthesis of, 793–796 ultraviolet light absorbed by, 88 Tryptophan synthase, 795 D-Tubocurarine, 394 Tubulin, 87F, 184, 515, 517F Tumour promoters, 378 Tumour-suppressor genes, 371B–372B Tumours. See Cancer Tunneling, 466F, 796 Tunnels drug transport, 344F intramolecular, 796B Turnover numbers, 415, 415T β-Turns, 152 Tus protein, 934 Twist, 840, 851 Two-dimensional gel electrophoresis, 109 Two-state transition, 167 2:3 Anti-terminator, 979 Tyk2, 380 Type 1 diabetes, 757B Type 2 diabetes, 701B, 710B, 725B, 748B, 757B Type II restriction enzymes, 827 Tyrosine catabolism of, 803 catecholamine transmitters and, 398–399 in green fluorescent protein, 87B structure, 79F synthesis of, 793 ultraviolet light absorbed by, 88

Tyrosine aminotransferase, 793 Tyrosine hydroxylase, 399 Tyrosine kinase pp60c-src, 378–379 Tyrosine kinases, 355, 357–358, 359–360, 488 insulin receptor, 363F nonreceptor, 365–366 phospholipase C activation by, 372–373 U (pathway), 1115 U-turns, 860 UASs, 986 Ubiquinone, 628 Ubiquitin, 1098 Ubiquitin-activating enzyme, 1098, 1099F Ubiquitin-carrier protein, 1099 Ubiquitin-protein ligase, 1099 Ubiquitination, 991, 1098, 1101 UCP1, 649B UDP–glucose, 708 UDP–glucose-4-epimerase, 583 UDP–glucose pyrophosphorylase, 584, 708 Ultracentrifugation, 110 Ultrafiltration, 103 Ultraviolet light amino acid absorbing, 88 pyrimidine dimer repair and, 955 UMP, 813 5′-UMP, 813 UMP, See also Uridine 5′-monophosphate UMP synthase, 886 Uncharged amino acids, 78F, 80 Uncompetitive inhibition, 422, 423F Uncouplers, 647 Uncoupling protein 1, 649B Undecaprenyl alcohol, 240F Unfolded protein, 176 Unfolding of protein, 59–60, 60F Unilamellar vesicles, 302F, 303 Unimolecular reactions, 408 Unsaturated fatty acids, 229, 265, 752–753 3′ Untranslated regions, 1059 Unwin, Nigel, 309 Unwinding of helices, 928 UP, 967 Up-frameshift proteins, 1066, 1069 UPFs, 1066, 1069 Upstream activation sequences, 986 Upstream elements, 966, 967 UQ, 628 Uracil, 810 Urate oxidase, 883 Urea, 804 Urea cycle, 777, 778–782, 780F, 782B Urease, 884 Ureido group, 778 Ureotelic animals, 804 Uric acid, 804, 810, 872 structure, 872F Uricotelic animals, 804 Uridine, 811 Uridine 5′-monophosphate, 813F Uridylic acid, 813 Uroguanylin, 363, 364F Uronic acids, 197 UTP, 814 V-type ATPase, 392 Vacuolar ATPases, 338 Vacuoles, 22

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I-25

I-26

Index

Valine catabolism of, 800 structure, 78F synthesis of, 787–788 van der Waals contact distance, 13 van der Waals interaction energy profile, 14F van der Waals interactions, 12, 13F, 13T DNA and, 839 ubiquity of, 142 Vancocin, 216B Vane, John, 278B Van’t Hoff plot, 56 Variable loops, 862 Vasoactive intestinal peptide, 399 VDAC, 653 Vectors, expression, 912–914 Velocity, 411F, 419 in Michaelis–Menten equation, 412–413, 412F of reaction, 408 Versican, 218, 219F Very long-chain acyl-CoA dehydrogenase, 743 Very-low-density lipoproteins, 288, 289 Vesicles coated, 289 membranes and, 325–327 multilamellar, 302F, 303 unilamellar, 302F, 303 Viagra, 426B, 426F du Vigneaud, Vincent, 130 VIIa (clotting factor), 481 Vinblastine, 519B Vinca alkaloids, 519B Vincristine, 519B Viral neuraminidase inhibitors, 449B Viral transforming proteins, 365 Viroids, 26–28 Viruses Rous sarcoma, 365 structure and function, 23, 25F, 26

Viscose, 208B Vitamin B12, 751B Vitamin C deficiency, 162B Vitamin K epoxide reductase, 242B Vitamin K reductase, 242B Vitamins, 549, 549T Vitellogenin gene, 998 VLCAD, 743 VLDL, 288, 289 Voltage-dependent anion channel, 653 Voltage-gated channels, 330, 387 von Euler, Ulf, 278B von Gierke’s disease, 719B Wall, cell, bacterial, 209–217, 212F Warfarin, 242B Water, 32–52 buffers and, 46–48 environmental aspects, 33F, 50 lipid structures formed in, 302–303 pH and, 40–46, 41T properties of, 33–39, 50 colligative, 39 hydrogen bonding and, 34–35 ionization, 39–40, 39F solvent, 35–39 structure, 34F Water:plastoquinone oxidoreductase, 670 Watson–Crick base pairs, 839–840 Watson–Crick postulate of double helix, 818 Watson, J. D., 818 Waxes, 239–240, 239B, 239F WD40 repeat domains, 654 Weak electrolytes, 42 Weak forces chemical, 12, 13T quaternary protein structure and, 183 Webs, spider, 151–152 Western blotting, 908B Wheels, helical, 160, 161F, 375–376

White adipose tissue, 1128 Whole genome sequencing, 18 Wild-type human sulfite oxidase, 418B Wobble, 1017 Wobble position, 1017 Woodward, R. B., 285B Writhe, 851 Wza, 314 X chromosomes, 911B Xa (clotting factor), 481 Xanthine, 810 Xanthine oxidase, 881, 882 XIa (clotting factor), 481 XIIa (clotting factor), 481 XPA (protein), 955 Xylitol, 198 α-Xylosyl-threonine, 214F Xylulose-5-phosphate, 730 Y chromosomes, 911B YACs, 904 Yeast aldolase reaction, 450F Yeast artificial chromosomes, 904 Yeast hexokinase, 567F Yields, 406F Z-DNA, 841–843, 843T Z lines, 506 Z-variant of protein, 178B Zero-order kinetics, 411 Zinc finger, 994 Zippers, ribose, 861 Zn-finger (motif), 994, 995–996 Zwitterion, 76, 82F Zymogens, 459, 481, 1093

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litre

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Icons and Colours in Illustrations The following symbols and colours are used in this text to help in illustrating structures, reactions, and biochemical principles. Elements: = Oxygen

= Nitrogen

= Phosphorus

= Sulfur

= Carbon

= Chlorine

Inorganic phosphate and pyrophosphate are sometimes symbolized by the following icons: P

P P

Inorganic phosphate (Pi)

Pyrophosphate (PPi)

Sugars:

Glucose

Galactose

Mannose

Fructose

Ribose

Nucleotides: = Guanine

= Cytosine

= Adenine

= Thymine

= Uracil

Amino acids: = Non-polar/hydrophobic

= Polar/uncharged

= Acidic

= Basic

Enzymes:

+ = Enzyme activation = Enzyme inhibition or inactivation

E = Enzyme

= Enzyme

Enzyme names are printed in red.

In reactions, blocks of color over parts of molecular structures are used so that discrete parts of the reaction can be easily followed from one intermediate to another, making it easy to see where the reactants originate and how the products are produced. Some examples: O –O

P

+

OH

NH3

Hydroxyl group

Amino group

COO–

–O

Phosphoryl group

Carboxyl group

Red arrows are used to indicate nucleophilic attack. These colors are internally consistent within reactions and are generally consistent within the scope of a chapter or treatment of a particular topic.

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! Organisms resemble their parents. (see page 4) Clockwise from top:

Stavroula Andreopoulous and her mother, Agni; Imed Gallouzi, his wife Kerrie-Ann Trudeau, and their son Jackson; and William Willmore, his wife Azine, and their daughter Kianna

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Common Abbreviations Used by Biochemists A Ab Ag Ac-CoA ACh ACP ADH ADP AIDS AMP ALA ATCase atm ATP BChl bp BPG BPheo C cal CaM cAMP CAP cDNA CDP CDR Chl CM CMP CoA or CoASH CoQ cpm CTP D d dd DAG DEAE DHAP DHF DHFR DNP Dopa DNA ds ℰ E4P EF EGF EPR ER ESI-MS ℱ FAB FAD FADH2 FBP FBPase Fd fMet FMN F-1-P F-6-P

adenine or the amino acid alanine antibody antigen acetyl-coenzyme A acetylcholine acyl carrier protein alcohol dehydrogenase adenosine diphosphate acquired immunodeficiency syndrome adenosine monophosphate δ-aminolevulinic acid aspartate transcarbamoylase atmosphere adenosine triphosphate bacteriochlorophyll base pair bisphosphoglycerate bacteriopheophytin cytosine or the amino acid cysteine calorie calmodulin cyclic 39,59-adenosine monophosphate catabolite activator protein complementary DNA cytidine diphosphate complementarity-determining region chlorophyll carboxymethyl cytidine monophosphate coenzyme A coenzyme Q counts per minute cytidine triphosphate dalton or the amino acid aspartate deoxy dideoxy diacylglycerol diethylaminoethyl dihydroxyacetone phosphate dihydrofolate dihydrofolate reductase dinitrophenol dihydroxyphenylalanine deoxyribonucleic acid double-stranded reduction potential erythrose-4-phosphate elongation factor epidermal growth factor electron paramagnetic resonance endoplasmic reticulum electrospray ionization mass spectrometry Faraday’s constant antibody molecule fragment that binds antigen flavin adenine dinucleotide reduced flavin adenine dinucleotide fructose-1,6-bisphosphate fructose bisphosphatase ferredoxin N-formyl-methionine flavin mononucleotide fructose-1-phosphate fructose-6-phosphate

G G GABA Gal GDP GFP GLC Glc GMP G-1-P G-3-P G-6-P G6PD GS GSH GSSG GTP h h Hb HDL HGPRT HIV HMG-CoA hnRNA HPLC HX Hyl Hyp I IDL IF IgG IMP IP3 IPTG IR ITP J k k Km kb kD L LDH LDL Lys M m mL mm Man Mb miRNA mol mRNA MS mV N n NAD! NADH

guanine or the amino acid glycine Gibbs free energy γ-aminobutyric acid galactose guanosine diphosphate green fluorescent protein gas-liquid chromatography glucose guanosine monophosphate glucose-1-phosphate glyceraldehyde-3-phosphate glucose-6-phosphate glucose-6-phosphate dehydrogenase glutamine synthetase glutathione (reduced glutathione) glutathione disulfide (oxidized glutathione) guanosine triphosphate hour Planck’s constant hemoglobin high-density lipoprotein hypoxanthine-guanine phosphoribosyltransferase human immunodeficiency virus hydroxymethylglutaryl-coenzyme A heterogeneous nuclear RNA high-pressure (or high-performance) liquid chromatography hypoxanthine hydroxylysine hydroxyproline inosine or the amino acid isoleucine intermediate density lipoprotein initiation factor immunoglobulin G inosine monophosphate inositol-1, 4, 5-trisphosphate isopropylthiogalactoside infrared inosine triphosphate joule kilo (103) rate constant Michaelis constant kilobases kilodaltons liter lactate dehydrogenase low density lipoprotein lysine molar milli (10"3) milliliter millimeter mannose myoglobin micro RNA mole messenger RNA mass spectrometry millivolt Avogadro’s number nano (10"9) nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide

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NADP! NADPH NAG NAM NANA ncRNA nm NMR O P p Pi PAGE PBG PC PCR PE PEP PEPCK PFK PG 2-PG 3-PG PDGF PGI PGK PGM Pheo PIP2 PK PKU PLP Pol PPi PQ PRPP PS PTH Q QH2 R

nicotinamide adenine dinucleotide phosphate reduced nicotinamide adenine dinucleotide phosphate N-acetylglucosamine N-acetylmuramic acid N-acetylneuraminic acid noncoding RNA nanometer nuclear magnetic resonance orotidine phosphate or the amino acid proline pico (10"12) inorganic phosphate polyacrylamide gel electrophoresis porphobilinogen plastocyanin or the phospholipid phosphatidylcholine polymerase chain reaction phosphatidylethanolamine phosphoenolypyruvate PEP carboxykinase phosphofructokinase prostaglandin 2-phosphoglycerate 3-phosphoglycerate platelet-derived growth factor phosphoglucoisomerase phosphoglycerate kinase phosphoglucomutase pheophytin phosphatidylinositol-4,5-bisphosphate pyruvate kinase phenylketonuria pyridoxal-5-phosphate polymerase pyrophosphate ion plastoquinone 5-phosphoribosyl-1-pyrophosphate photosystem or the phospholipid phosphatidylserine phenylthiohydandoin coenzyme Q, ubiquinone or the amino acid glutamine reduced coenzyme Q (ubiquinol) gas constant or the amino acid arginine

r RER RF RFLP RNA RNAi rRNA R-5-P Ru-1,5-P Ru-5-P S s s SAM SDS siRNA SM snafu snRNA snoRNA sRNA snRNP SRP T T TCA TDP THF TMP TMV TPP TTP U UDP UDPG UMP UTP UV V Vmax VLDL X XMP Xu-5-P yr YAC

revolution rough endoplasmic reticulum release factor restriction fragment length polymorphism ribonucleic acid RNA interference ribosomal RNA ribose-5-phosphate ribulose-1,5-bisphosphate ribulose-5-phosphate Svedberg unit or the amino acid serine second sedimentation coefficient S-adenosylmethionine sodium dodecylsulfate small interfering RNA sphingomyelin situation normal, all fouled up small nuclear RNA small nucleolar RNA small RNAs small nuclear ribonucleoprotein signal recognition particle thymine or the amino acid threonine absolute temperature tricarboxylic acid cycle thymidine diphosphate tetrahydrofolate thymidine monophosphate tobacco mosaic virus thiamine pyrophosphate thymidine triphosphate uracil uridine diphosphate UDP-glucose uridine monophosphate uridine triphosphate ultraviolet volt or the amino acid valine maximal velocity very low density lipoprotein xanthine xanthine monophosphate xylulose-5-phosphate year yeast artificial chromosome

Abbreviations for Amino Acids Alanine

Ala

A

Leucine

Leu

L

Arginine

Arg

R

Lysine

Lys

K

Asparagine

Asn

N

Methionine

Met

M

Aspartate

Asp

D

Phenylalanine

Phe

F

Cysteine

Cys

C

Proline

Pro

P

Glutamate

Glu

E

Serine

Ser

S

Glutamine

Gln

Q

Threonine

Thr

T

Glycine

Gly

G

Tryptophan

Trp

W

Histidine

His

H

Tyrosine

Tyr

Y

Isoleucine

Ile

I

Valine

Val

V

Copyright 2012 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it.