Textbook of Biochemistry 9788194746317

Table of contents :
Cover
Half Title
Title
Copyright
Preface
Contents
Chapter 1: Introduction to Biochemistry
1.1 Introduction
1.2 Characteristic Features of the Living Organisms
1.3 Molecules of Life
1.4 Diversity in the Living World
1.5 Basic Unit of Life: Cell
1.6 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 2: Water: The Unique Solvent
2.1 Introduction
2.2 Structure and Properties of Water
2.3 Weak Non-covalent Interactions in Aqueous Solutions
2.4 Ionization of Water
2.5 The pH Scale
2.6 Ionization of Acids and Bases
2.7 Henderson-Hasselbalch Equation
2.8 Acid-base Titrations
2.9 Buffers
2.10 Physiological Buffer Systems
2.11 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 3: Carbohydrates
3.1 Introduction
3.2 Monosaccharides and Disaccharides
3.3 Nomenclature and Classification of Monosaccharides
3.4 Physical Properties of Monosaccharides
3.5 Chemical Properties of Monosaccharides
3.6 Isomerism in Monosaccharides
3.7 Haworth Structures: Cyclic Structure of Monosaccharides
3.8 Monosaccharides as Reducing Agents
3.9 Biologically Important Disaccharides
3.10 Oligosaccharides
3.11 Polysaccharides
3.12 Glycoconjugates
3.13 Tests for Carbohydrates
3.14 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 4: Lipids
4.1 Introduction
4.2 Features of Lipids
4.3 General Structure and Nomenclature
4.4 Classification of Lipids
4.5 Physical Properties
4.6 Chemical Properties
4.7 Simple Lipids
4.8 Compound Lipids
4.9 Derived Lipids
4.10 Lipid Tests
4.11 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 5: Proteins-I
5.1 Introduction
5.2 Amino Acids: The Building Blocks of Proteins
5.3 Classification of Amino Acids
5.4 Physicochemical Properties of Alpha Amino Acids
5.5 General Chemical Reactions of Alpha Amino Acids
5.6 The Peptide Bond
5.7 Protein Sequencing
5.8 Protein Sequencing by Edman Degradation
5.9 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 6: Proteins-II
6.1 Introduction
6.2 Primary Structure
6.3 Nature of Peptide Bond
6.4 Secondary Structure
6.5 Tertiary Structure
6.6 Quaternary Structure
6.7 Classification of Proteins
6.8 Physical Properties
6.9 Chemical Properties
6.10 Denaturation and Renaturation of Protein
6.11 Protein Folding
6.12 Chaperones
6.13 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 7: Nucleic Acids
7.1 Introduction
7.2 Historical Background of Nucleic Acids
7.3 Nucleic Acid and Its Components
7.4 Nucleosides
7.5 Nucleotides
7.6 Types of Nucleic Acids
7.7 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 8: Enzymes
8.1 Introduction
8.2 Chemical Nature of Enzymes
8.3 Nomenclature and Classification
8.4 Mechanism of Action of Enzymes
8.5 Enzyme Kinetics
8.6 Regulation of Enzyme Action
8.7 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 9: Photosynthesis
9.1 Introduction
9.2 Photosynthetic Pigments
9.3 Organization of the Photosynthetic Apparatus
9.4 Photosynthesis
9.5 Light Dependent Reactions
9.6 Two-pigment Systems
9.7 Dark Reactions
9.8 Stoichiometry of Carbon Fixation
9.9 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 10: Carbohydrate Metabolism
10.1 Introduction
10.2 Role of Glucose in Metabolism
10.3 The Site of Carbohydrate Metabolism in Cells and Tissues
10.4 Glycolysis
10.5 Fates of Pyruvate
10.6 Fermentation
10.7 Gluconeogenesis
10.8 Pentose Phosphate Pathway of Glucose Oxidation
10.9 Conversion of Pyruvate into Acetyl-CoA
10.10 Citric Acid Cycle or Krebs Cycle
10.11 Glyoxylate Cycle
10.12 Overall Stoichiometry of Aerobic Oxidation of Glucose
10.13 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 11: Electron Transport and Oxidative Phosphorylation
11.1 Introduction
11.2 Mitochondria: The Site for ATP Synthesis
11.3 Components of the Electron Transport Chain
11.4 Standard Redox Potentials
11.5 The Respiratory Chain
11.6 Conservation of Energy in Form of ATP
11.7 The Chemiosmotic Theory of ATP Synthesis
11.8 Transport of ATP and ADP Across the Mitochondrial Membrane
11.9 Route of Reducing Equivalents from Cytosolic NADH into Mitochondria
11.10 Stoichiometry of Oxygen Consumption and ATP Synthesis
11.11 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 12: Biosynthesis of Lipids
12.1 Introduction
12.2 Biosynthesis of Saturated Fatty Acids
12.3 Regulation of Fatty Acid Synthesis
12.4 Biosynthesis of Unsaturated Fatty Acids
12.5 Biosynthesis of Eicosanoids
12.6 Biosynthesis of Triacylglycerols
12.7 Biosynthesis of Membrane Phospholipids
12.8 Biosynthesis of Cholesterol, Steroids, and Isoprenoids
12.9 Plasma Lipoproteins
12.10 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 13: Lipid Catabolism
13.1 Introduction
13.2 Digestion of Dietary Lipids
13.3 Lipolysis
13.4 Fatty Acid Oxidation
13.5 ATP Production in Fatty Acid ß- oxidation
13.6 Regulation of Fatty Acid Oxidation
13.7 Oxidation of Very Long Chain Fatty Acids in Peroxisomes
13.8 Oxidation of Unsaturated Fatty Acids
13.9 Ketogenesis
13.10 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 14: Biosynthesis of Amino Acids
14.1 Introduction
14.2 Nitrogen Fixation
14.3 Assimilation of Ammonia through Glutamate and Glutamine
14.4 Biosynthesis of Amino Acids
14.5 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 15: Catabolism of Amino Acids
15.1 Introduction
15.2 Digestion of Dietary Proteins in Gastrointestinal Tract
15.3 Degradation of Cellular Proteins
15.4 Breakdown of Amino Acids
15.5 Other Means of Ammonia Transport
15.6 The Urea Cycle
15.7 Degradation of C-skeletons of Amino Acids
15.8 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 16: Nucleotide Metabolism
16.1 Introduction
16.2 Nucleotide Biosynthesis
16.3 Role of PRPP
16.4 De novo Purine Synthesis
16.5 Catabolism of Purine Nucleotides
16.6 Salvage Pathways for Biosynthesis of Purine Nucleotides
16.7 De novo Pyrimidine Synthesis
16.8 Catabolism of Pyrimidine Nucleotides
16.9 Salvage of Pyrimidine Nucleotides
16.10 Biosynthesis of Thymidylate
16.11 Interconversion of Nucleotides
16.12 Formation of Deoxyribonucleotides
16.13 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Chapter 17: Biochemistry of Human Hormones
17.1 Introduction
17.2 Hormones and Receptors
17.3 Mechanisms of Hormone Action
17.4 Types of Hormones
17.5 Summary
Multiple-Choice Questions
Short Answer Type Questions
Long Answer Type Questions
Index
Backcover

Citation preview

Textbook of Biochemistry

Textbook of Biochemistry

Abhilasha Shourie Professor & Head Department of Biotechnology Faculty of Engineering & Technology Manav Rachna International Institute of Research & Studies Faridabad

Shilpa S. Chapadgaonkar Associate Professor Department of Biotechnology and Head of Molecular Biosciences Research Cluster Manav Rachna International Institute of Research and Studies Faridabad

Anamika Singh Assistant Professor Department of Botany Maitreyi College University of Delhi Delhi

©Copyright 2019 I.K. International Pvt. Ltd., New Delhi-110002. This book may not be duplicated in any way without the express written consent of the publisher, except in the form of brief excerpts or quotations for the purposes of review. The information contained herein is for the personal use of the reader and may not be incorporated in any commercial programs, other books, databases, or any kind of software without written consent of the publisher. Making copies of this book or any portion for any purpose other than your own is a violation of copyright laws. Limits of Liability/disclaimer of Warranty: The author and publisher have used their best efforts in preparing this book. The author make no representation or warranties with respect to the accuracy or completeness of the contents of this book, and specifically disclaim any implied warranties of merchantability or fitness of any particular purpose. There are no warranties which extend beyond the descriptions contained in this paragraph. No warranty may be created or extended by sales representatives or written sales materials. The accuracy and completeness of the information provided herein and the opinions stated herein are not guaranteed or warranted to produce any particulars results, and the advice and strategies contained herein may not be suitable for every individual. Neither Dreamtech Press nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Trademarks: All brand names and product names used in this book are trademarks, registered trademarks, or trade names of their respective holders. Dreamtech Press is not associated with any product or vendor mentioned in this book. ISBN: 978-81-947463-1-7 EISBN: 978-81-947463-0-0

Preface Biochemistry is a fundamental subject for students pursuing education in any domain related to life sciences. A number of subjects related to biological and life sciences find their roots in biochemistry. This book is a sincere endeavour to present the basic concepts of biochemistry in a comprehensive manner and has been designed to serve as both, a textbook and a reference book. Every effort has been made to retain the essential information about the chemistry and functions of all the basic biomolecules. The text covers the syllabi of various universities and institutions across India and abroad. The introductory chapter of the book gives an insight into the evolutionary aspects of biomolecules and orients the reader towards their increasing complexity. A chapter highlighting the role of water as a universal solvent and its biochemical aspects has been included, which lays down the foundation of all the metabolic processes. The first half of the book is about structure and functions of important biomolecules— carbohydrates, lipids, proteins and nucleic acids, while the latter half elaborates their metabolic pathways. The chapter elaborating chemical nature and functioning of enzymes has been placed just before the chapters describing metabolism, to maintain a continuum of concepts. Further, various concepts in different chapters have been integrated throughout the text to create a greater understanding of metabolic phenomena. Well illustrated diagrams and flowcharts are the highlight of this book which have been redrawn meticulously for better cognition and understanding. The chapter end exercises have been designed to help in preparing for the quizzes and exams. These exercises are meant to provoke the analytical thinking of the reader and provide a measure of self-assessment. The writing of this book has been a long journey as the task was not only extensive, but also very demanding because of the vast ocean of knowledge accumulated over the recent years in the field. There has been tremendous advancement in Biochemistry which made the organization of the book contents challenging. All the care has been taken to incorporate significant advances in the field and assimilate necessary facts and research findings. We wish that the book proves to be useful for a wide spectrum of readers including students, faculty and others who want to understand the subject. Constructive criticism and suggestions are invited for further improvement of the book. Abhilasha Shourie Shilpa S. Chapadgaonkar Anamika Singh

Contents Preface

v

1. Introduction to Biochemistry 1.1 Introduction 1.2 Characteristic Features of the Living Organisms 1.3 Molecules of Life 1.4 Diversity in the Living World 1.5 Basic Unit of Life: Cell 1.6 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

1 1 5 6 12 14 28 29 30 30

2. Water: The Unique Solvent 2.1 Introduction 2.2 Structure and Properties of Water 2.3 Weak Non-covalent Interactions in Aqueous Solutions 2.4 Ionization of Water 2.5 The pH Scale 2.6 Ionization of Acids and Bases 2.7 Henderson-Hasselbalch Equation 2.8 Acid-base Titrations 2.9 Buffers 2.10 Physiological Buffer Systems 2.11 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

31 31 31 32 36 37 38 39 39 41 43 46 47 48 48

3. Carbohydrates 3.1 Introduction 3.2 Monosaccharides and Disaccharides

49 49 50

viii Contents

3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14

Nomenclature and Classification of Monosaccharides Physical Properties of Monosaccharides Chemical Properties of Monosaccharides Isomerism in Monosaccharides Haworth Structures: Cyclic Structure of Monosaccharides Monosaccharides as Reducing Agents Biologically Important Disaccharides Oligosaccharides Polysaccharides Glycoconjugates Tests for Carbohydrates Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

51 55 56 56 58 60 60 62 64 71 73 74 75 76 76

4. Lipids 4.1 Introduction 4.2 Features of Lipids 4.3 General Structure and Nomenclature 4.4 Classification of Lipids 4.5 Physical Properties 4.6 Chemical Properties 4.7 Simple Lipids 4.8 Compound Lipids 4.9 Derived Lipids 4.10 Lipid Tests 4.11 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

77 77 77 77 79 80 81 83 86 90 92 92 93 94 94

5. Proteins-I 5.1 Introduction 5.2 Amino Acids: The Building Blocks of Proteins 5.3 Classification of Amino Acids 5.4 Physicochemical Properties of Alpha Amino Acids 5.5 General Chemical Reactions of Alpha Amino Acids

95 95 96 101 105 111

Contents ix

5.6 5.7 5.8 5.9

The Peptide Bond Protein Sequencing Protein Sequencing by Edman Degradation Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

112 115 121 122 123 124 124

6. Proteins-II 6.1 Introduction 6.2 Primary Structure 6.3 Nature of Peptide Bond 6.4 Secondary Structure 6.5 Tertiary Structure 6.6 Quaternary Structure 6.7 Classification of Proteins 6.8 Physical Properties 6.9 Chemical Properties 6.10 Denaturation and Renaturation of Protein 6.11 Protein Folding 6.12 Chaperones 6.13 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

125 125 126 126 129 131 132 133 136 137 141 142 143 145 146 147 147

7. Nucleic Acids 7.1 Introduction 7.2 Historical Background of Nucleic Acids 7.3 Nucleic Acid and Its Components 7.4 Nucleosides 7.5 Nucleotides 7.6 Types of Nucleic Acids 7.7 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

148 148 148 149 152 153 154 164 165 166 167

x Contents

8. Enzymes 8.1 Introduction 8.2 Chemical Nature of Enzymes 8.3 Nomenclature and Classification 8.4 Mechanism of Action of Enzymes 8.5 Enzyme Kinetics 8.6 Regulation of Enzyme Action 8.7 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

168 168 169 171 173 185 197 208 210 213 213

9. Photosynthesis 9.1 Introduction 9.2 Photosynthetic Pigments 9.3 Organization of the Photosynthetic Apparatus 9.4 Photosynthesis 9.5 Light Dependent Reactions 9.6 Two-pigment Systems 9.7 Dark Reactions 9.8 Stoichiometry of Carbon Fixation 9.9 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

215 215 215 218 219 219 220 223 228 230 231 232 232

10. Carbohydrate Metabolism 10.1 Introduction 10.2 Role of Glucose in Metabolism 10.3 The Site of Carbohydrate Metabolism in Cells and Tissues 10.4 Glycolysis 10.5 Fates of Pyruvate 10.6 Fermentation 10.7 Gluconeogenesis 10.8 Pentose Phosphate Pathway of Glucose Oxidation 10.9 Conversion of Pyruvate into Acetyl-CoA 10.10 Citric Acid Cycle or Krebs Cycle 10.11 Glyoxylate Cycle

233 233 234 234 236 256 257 258 266 270 274 286

Contents xi

10.12 Overall Stoichiometry of Aerobic Oxidation of Glucose 10.13 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

287 288 289 290 290

11. Electron Transport and Oxidative Phosphorylation 11.1 Introduction 11.2 Mitochondria: The Site for ATP Synthesis 11.3 Components of the Electron Transport Chain 11.4 Standard Redox Potentials 11.5 The Respiratory Chain 11.6 Conservation of Energy in Form of ATP 11.7 The Chemiosmotic Theory of ATP Synthesis 11.8 Transport of ATP and ADP Across the Mitochondrial Membrane 11.9 Route of Reducing Equivalents from Cytosolic NADH into Mitochondria 11.10 Stoichiometry of Oxygen Consumption and ATP Synthesis 11.11 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

291 291 291 294 298 298 301 301 304 305 305 308 308 310 310

12. Biosynthesis of Lipids 12.1 Introduction 12.2 Biosynthesis of Saturated Fatty Acids 12.3 Regulation of Fatty Acid Synthesis 12.4 Biosynthesis of Unsaturated Fatty Acids 12.5 Biosynthesis of Eicosanoids 12.6 Biosynthesis of Triacylglycerols 12.7 Biosynthesis of Membrane Phospholipids 12.8 Biosynthesis of Cholesterol, Steroids, and Isoprenoids 12.9 Plasma Lipoproteins 12.10 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

311 311 311 315 315 316 317 320 323 325 327 329 330 331

xii Contents

13. Lipid Catabolism 13.1 Introduction 13.2 Digestion of Dietary Lipids 13.3 Lipolysis 13.4 Fatty Acid Oxidation 13.5 ATP Production in Fatty Acid C-oxidation 13.6 Regulation of Fatty Acid Oxidation 13.7 Oxidation of Very Long Chain Fatty Acids in Peroxisomes 13.8 Oxidation of Unsaturated Fatty Acids 13.9 Ketogenesis 13.10 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

332 332 334 335 337 340 341 341 343 344 346 348 349 349

14. Biosynthesis of Amino Acids 14.1 Introduction 14.2 Nitrogen Fixation 14.3 Assimilation of Ammonia through Glutamate and Glutamine 14.4 Biosynthesis of Amino Acids 14.5 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

350 350 350 353 354 369 369 370 370

15. Catabolism of Amino Acids 15.1 Introduction 15.2 Digestion of Dietary Proteins in Gastrointestinal Tract 15.3 Degradation of Cellular Proteins 15.4 Breakdown of Amino Acids 15.5 Other Means of Ammonia Transport 15.6 The Urea Cycle 15.7 Degradation of C-skeletons of Amino Acids 15.8 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

372 372 373 373 376 380 381 386 402 403 403 404

Contents xiii

16. Nucleotide Metabolism 16.1 Introduction 16.2 Nucleotide Biosynthesis 16.3 Role of PRPP 16.4 De novo Purine Synthesis 16.5 Catabolism of Purine Nucleotides 16.6 Salvage Pathways for Biosynthesis of Purine Nucleotides 16.7 De novo Pyrimidine Synthesis 16.8 Catabolism of Pyrimidine Nucleotides 16.9 Salvage of Pyrimidine Nucleotides 16.10 Biosynthesis of Thymidylate 16.11 Interconversion of Nucleotides 16.12 Formation of Deoxyribonucleotides 16.13 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

405 405 406 408 409 415 417 418 421 421 423 424 426 428 430 431 431

17. Biochemistry of Human Hormones 17.1 Introduction 17.2 Hormones and Receptors 17.3 Mechanisms of Hormone Action 17.4 Types of Hormones 17.5 Summary Multiple-Choice Questions Short Answer Type Questions Long Answer Type Questions

432 432 433 434 439 449 450 450 451

Index

453

1 Introduction to Biochemistry 1.1

INTRODUCTION

Biochemistry is the study of the chemical processes occurring in the living matter. Though the definition appears simple, it embraces an immensely diverse field that encompasses the sciences of molecular biology, immunochemistry, and neurochemistry, as well as bioinorganic, bioorganic, and biophysical chemistry. The disease’s biology, pathology, inflammation rely on different natural biochemical processes and hence an understanding of biochemistry is essential for medical science. In order to understand the biochemical processes at the molecular level, early biochemists developed techniques for isolation and purification of biomolecules. They established the techniques for determination of structure and function which, in turn, accelerated the discoveries in other fields such as genetics and immunology.

Fig. 1.1

Facets of biochemistry

2 Textbook of Biochemistry

The genetic and immunological techniques are reciprocally boosting the research in biochemistry. In fact, the barriers between the different scientific domains are merging and biochemistry is becoming their universal language (Figure 1.1). The study of biochemistry naturally commences with the origination of our living world.

1.1.1

Origin of the Earth and Its Atmosphere, and Simple Organic Molecules

The world as we see today has a vast diversity of living organisms having a striking similarity at the molecular level. All organisms have similar molecular components and follow the universal genetic code and possess DNA or RNA as the genetic material. This uniformity is a strong evidence to the theory that all organisms on the earth evolved from a common ancestor and the diversity in the life forms is the result of evolutionary processes acting for millions of years. The natural selection or the survival of the fittest was the key driver for the evolution leading to formation of life forms that are suitably adapted to the environments. Many thinkers and philosophers have dedicated their entire life in discovering the origins of life. Several interesting theories have been proposed some of which are mentioned here: (i) ‘The theory of special creation by God’; (ii) ‘Panspermia’ a theory that considers that the first living cells on the earth (bacteria) came from the outer space and gradually evolution took place; (iii) ‘abiogenesis’ a theory that proposed that life generated spontaneously from the decaying organic substances, e.g., flies from the putrid organic matter. This theory was later proven wrong conclusively by experiments demonstrated by Louis Pasteur; (iv) ‘Origin in the clay’ in a recent theory scientists proposed that the clay might have adsorbed the early molecules and might have provided the requisite proximity of molecules to react. Clay can also form hydrogels which may form a protective layer over the primitive cells; (v) ‘The theory of biochemical evolution’ considers that the life evolved along with the origin and evolution of the earth and its atmosphere. Scientists O.I. Oparin (1923) and J.B.S. Haldane (1928) proposed the theory of biochemical origin of life separately. They hypothesized that the life did not originate abruptly but from a long series of physico-chemical changes brought about by ever-changing environmental conditions existing in the prehistoric era. Though the question of the most appropriate theory is debatable, we would focus on the modern biochemical theory in the present text. The earth evolved approximately 4.6 million years ago. In the beginning, the earth existed as a hot gaseous fireball having temperature between 5000-6000°C. Elements present in the molten mass of the earth got stratified according to their densities where lighter elements like hydrogen (H2), helium (He), carbon (C) and nitrogen (N2) remained at the outer strata forming the atmosphere whereas the heavy metals such as iron (Fe) and nickel (Ni) got concentrated in the core of the earth. Due to absence of oxygen, this was reducing in nature. Hydrogen being highly reactive in nature reacted with other elements to form methane (CH4), ammonia (NH3), hydrocyanic acid (HCN) and water (H2O). Further cooling of the earth resulted in condensation of water vapour and rain on the earth. The rain falling on the earth converted to vapour due to high temperature of the earth. This

Introduction to Biochemistry 3

water cycle continued for several years and after millions of years water bodies like lakes, ponds, rivers and oceans came into existence on the earth. Methane (CH4) and ammonia (NH3) in the atmosphere got dissolved in rainwater and ultimately mixed in the ocean. Further cooling resulted in the formation of saturated and unsaturated hydrocarbons which reacted with water vapours to create compounds like ketones, alcohols and organic acids. The atmosphere of the early earth had high UV radiation, frequent high energy thunderstorms and volcanic eruptions which catalyzed reactions that resulted in the formation of simple organic molecules like sugars, amino acids, purines and pyrimidines. The hot ocean containing the simple organic molecules was named primordial soup by J.B.S. Haldane. CH4 + H2O m
sugar, fatty acid, glycerol

...(1.1)

CH4 + H2O + NH3 m Amino acid

...(1.2)

CH4 + H2O + NH3 + HCN m
Purine + Pyrimidine

...(1.3)

1.1.2 The Miller-Urey Experiment The scientists, Stanley Miller and Harold C. Urey (1953) designed an apparatus to recreate the conditions that existed in the atmosphere on the primitive earth. The apparatus consisted of two glass chambers interconnected with tubes (Figure 1.2). Gaseous chamber contained gases like hydrogen (H2), methane (CH4), ammonia (NH3) and water vapours (H2O). This chamber had two electrodes for generating sparks that simulated the lightning’s happening on the primitive earth. The chambers were heated and the gases were allowed to condense and collect in the liquid chamber. The experiment was continued for a prolonged period of one week and the liquid collected was analyzed. The experiment yielded a non-random

Fig. 1.2

Apparatus for Miller-Urey experiment

4 Textbook of Biochemistry

mixture of amino acids such as glycine and alanine in about 2% concentration in the soup. Other more complicated amino acids such as glutamic acid and leucine were obtained in smaller proportions. The experiment proved that the primitive atmospheric conditions were conducive to the formation of complex organic molecules.

1.1.3

Formation of Complex Organic Compounds and Coacervates

It was further envisaged that simple organic compounds present in the primordial soup polymerized to form complex organic molecules such as polysaccharides, polypeptides, proteins, lipids, etc. The nucleotides were produced by combination of sugars, phosphates, purines and pyrimidines. Nucleotides got polymerized into the nucleic acids; the bearers of the genetic code of life. The complex organic compounds in the ocean aggregated spontaneously to form colloidal droplets named coacervates. The spontaneously aggregated coacervates had hydrophobic molecules hidden deep inside an outer layer of more hydrophilic Fig. 1.3 Coacervates in the surrounding compounds that were exposed towards the water aqueous surroundings (Figure 1.3). The formation of fatty acids gave the first fatty acid membrane bound protocells. The enclosed structure of the coacervates accelerated the chemical changes within the coacervates. The coacervates were able to absorb and assimilate the organic molecules from the primordial soup. Gradually, hydrolytic reactions occurring within the membrane bound coacervates provided energy for the synthetic reactions. Coacervates, therefore started growing and multiplying.

1.1.4

Biogeny: Formation of the First Cells and Their Evolution into Complex Organisms

Though coacervates had some properties of living systems such as absorption of nutrients, excretion, growth and multiplication, but they lacked the complex molecular organization, biocatalytic reactions and genetic control. Primitive nucleoprotein molecules had selfduplicating property and hence were capable of performing hereditary function. Small chains of nucleic acids might have aggregated into a large unit called protovirus. The process of nucleic acid replication and genetic control became precise in due course leading to formation of first cells or protobionts or eobionts. The first life forms thus originated about 3.7 billion years ago in the ocean. Some of the proteins had catalytic properties and they catalyzed the metabolic reactions. In this way, the first unicellular and heterotrophic

Introduction to Biochemistry 5

organisms came into existence. They were anaerobic due to the absence of oxygen on the earth and its atmosphere. The increase in the population of heterotrophs might have led to depletion of nutrients in the primordial soup leading to origination of newer methods of nutrition such as saprophytic, parasitic and chemosynthetic. Some of the protobionts synthesized chlorophyll from the magnesium porphyrin in the ocean. The chlorophyll pigment could absorb sunlight and provide energy for the synthesis of carbohydrates. The first prokaryotic, anaerobic and photoautotrophic organisms thus came into existence. The photoautotrophs were responsible for producting oxygen (O2) in large quantities. Ozone layer was formed from the free oxygen blocking the harmful UV rays of the sun and making the migration of the organisms from ocean to land possible. The abundance of oxygen in the atmosphere made aerobic respiration possible, presumably about 27 billion years ago. Some of the prokaryotes adapted the aerobic mode of respiration. Nucleus and other cell organelles developed causing evolution of eukaryotes from prokaryotes. The synergy between the cells and division of labour in the cell groups gradually caused the evolution of complex organisms around 1500 million years ago (Figure 1.4).

Fig. 1.4

1.2

Evolution of earth and life on it

CHARACTERISTIC FEATURES OF THE LIVING ORGANISMS

The living beings show several distinguishing features such as (i) chemical complexity and organizations; (ii) well-defined systems for extracting, transforming and utilizing energy from the environment. This provides energy to the organisms to carry out metabolic activities and maintain their functions; (iii) replication or reproduction is the most fundamental property of the living organisms in order to exist in the environment; (iv) mechanisms for sensing and responding to environment and constantly adapting to the surrounding

6 Textbook of Biochemistry

conditions; and (v) capability to change the inherited life processes or evolution which is at the root of diversity observed in the living world. The key driving force for evolutionary process is the competition amongst the organisms due to limitation of nutrition resources. Competition allows survival of the fittest providing selective pressure that gives advantage to the variants best suited for their survival and gradually an evolved and more efficient population comes into existence. The transfer of the acquired traits occurs by vertical gene transfer from parent to progeny. In contrast to this gradual process of evolution, recent evidence has established that transfer of genetic material between genetically distinct lineages can occur by horizontal gene transfer (HGT) or lateral gene transfer. The transfer of genetic material directly from one genetically distinct species to another leads to abrupt evolution of new species which shares some of the characteristics of genetically distant organisms. Different mechanisms such as transformation, transduction and conjugation are responsible for HGT. The genetic elements such as plasmids, transposons, integrons as well as genomic DNA can be transferred from one organism to another. HGT has been observed in a vast variety of organisms found in different ecological niches. HGT has made the phylogenetic representations more complicated. In a pioneering study, American microbiologist Sol Spiegelman experimentally demonstrated the evolution of replicating RNA molecules in vitro. This path breaking experiment demonstrating nucleic acid hybridization and evolution laid the foundations of modern-day recombinant DNA technology. In the Sol Spiegelman’s experiment, the genomic RNA isolated from bacteriophage QC; QC replicates protein complex for catalysing RNA replication and nucleic acid bases as the precursors for nucleic acid synthesis were mixed. Bacteriophage QC genome consists of a single strand of 3300 base pairs of RNA. When ample amount of precursors as well as time for hybridization was provided during the reaction, the newly synthesized RNA obtained was identical to the parent. Conversely, when a selective pressure such as decreasing the time or limiting the precursors was applied; the product molecules were found to be different from the parent molecules. When the time for the experiment was reduced from 20 minutes to 5 minutes for continuously over 75 generations; the product that was obtained was dominated by single species of shorter molecules having chain length of only 550 bases. This resultant population was capable of replicating 15 times more rapidly as compared to the parent RNA population and was popularly known as Spiegelman’s Monster. Similarly, evolution of new species of molecules could be observed by limiting the concentration of precursors or by adding inhibitors.

1.3

MOLECULES OF LIFE

Biomolecules are the basic units of life possessing special features in their structure that are translated to their specific functions in the living world. Therefore, before embarking on this momentous journey to understand biochemistry, let us first get introduced to the molecules of life. These macromolecules may be arranged into supramolecular complexes forming functional units, e.g., cell organelles such as ribosomes, biomembranes, chromosomes and

Introduction to Biochemistry 7

many more. Table 1.1 shows the major macromolecules found in E. Coli, the most popular model organism. Water is the most abundant molecule found as a constituent of all types of cells and the major macromolecules are proteins, nucleic acids (DNA and RNA) and polysaccharides. Table 1.1

Water Proteins Nucleic acids

DNA RNA

Approximate chemical composition of the cells % of total weight of cell

Types of molecular species (approx.)

70

1

15

3,000

6

1

3

> 3,000

Polysaccharides

3

5

Lipids

2

20

Inorganic ions

1

20

Most of the biomolecules are macromolecules which are high molecular weight polymers of simple precursors, compounds similar to those formed in the historic Miller-Urey experiment (Figure 1.5).

Fig. 1.5

Simple precursors that form complex macromolecules

8 Textbook of Biochemistry

Nucleic acids are polymers of nucleotides made from pentose sugar, nitrogenous base and a phosphate, proteins are the polymers of B carboxylic acids while the lipids are polymers of the fatty acids. In the RNA world theory, it has been proposed that the primitive organisms depended on RNA molecules for all the major life functions of heredity, storage and catalysis. The discovery of ribozymes by Tom Cech and Sidney Altman in 1980 proved the existence of catalytic RNA molecules. The molecular structure of the nucleic acids enables the process of self-replication where the single strands of nucleic acids act as templates for the directed synthesis of their complementary strand and the process can occur even with the synthetic replication machinery outside the cells. In the primitive RNA world, continuous RNA replication might have led to limitation in RNA precursor molecules thereby triggering the development of alternate mechanisms for their synthesis. Polypeptides may have been components of ribozymes to furnish specific reactivity. Polypeptides and proteins are polymers of amino acids—the B-amino carboxylic acids. The capacity of long-chain proteins to spontaneously fold into well-defined threedimensional geometries might have favoured the evolution of proteins as the biocatalysts of today and also as one of the most important structural building blocks. Sufficient evidence has been gathered to prove that DNA originated from RNA in an RNA protein world. DNA contains deoxyribose sugar which can be obtained by reducing the ribose sugar found in RNA. The simple base uracil of RNA is methylated to form thymidine in DNA. In the modern cells, DNA precursors can be produced by reducing ribonucleotide phosphates by action of ribonucleotide reductases. Gradually, DNA replaced RNA as the genetic material because it is more stable and can be repaired more easily. Replacement of RNA by DNA as genetic material paved the way for the evolution of larger genomes of complex organisms.

1.3.1

Nucleic Acids

Nucleic acids have been considered to be the first biomolecules that sustained life on the earth. They store and transfer cellular information and are the energy currency of the cells. Deoxyribonucleic acid (DNA) carries the hereditary information in segments known as genes whereas the ribonucleic acid (RNA) translates the genetic information from DNA to make proteins. Chemically, nucleic acids are the polymers of nucleotides that are composed of: a 5-carbon sugar, a phosphate group and a nitrogenous base. RNA has ribose sugar and adenine, uracil, guanine, and cytosine as nitrogenous bases while DNA has deoxyribose sugar and adenine, thymine, guanine, and cytosine as the nitrogenous bases (Figure 1.6). Though the linear sequence that constitutes the primary structure of nucleic acids is essentially similar; their conformations differ significantly. RNA usually exists as a single-chain molecule whereas DNA is double stranded with a typical helical structure. The structure and function of these molecules are discussed in Chapter 7: Nucleic Acids. The adenosine triphosphate (ATP) is the molecule that stores the chemical energy of the cell. ATP is a mononucleotide made up of one adenine, a ribose sugar, and three serially

Introduction to Biochemistry 9

Fig. 1.6

Structure of nucleic acids and nitrogenous bases

linked phosphate groups. Reactions that break the phosphate group leading to formation of adenosine diphosphate (ADP) release energy which is used for carrying out other cellular reactions. Dinucleotides such as nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP) and flavin adenine dinucleotide (FAD) function as

10 Textbook of Biochemistry

coenzymes that participate in electron transfer reactions. Their role in energy metabolism has been discussed in Chapters 9, 10 and 11.

1.3.2

Proteins

Proteins, polymers of amino acids, are the largest macromolecular fraction of cells. More than twenty different types of amino acids are found in nature. The greater diversity of amino acid structure suggests that proteins evolved later than the nucleic acids. Proteins function as biocatalyst enzymes, structural components, receptors or transport channels. Figure 1.7 shows the structures of different types of amino acids whereas formation of peptide bond has been shown in Figure 1.8. The amino acids based on their chemical structure can be distinguished into polar, nonpolar and aromatic amino acids. The structure and function of amino acids and their assembly into proteins has been detailed in Chapters 5 and 6.

Fig. Contd.

Introduction to Biochemistry 11 Fig. Contd.

Fig. 1.7 Types of amino acids

Fig. 1.8

Formation of a peptide bond

12 Textbook of Biochemistry

1.3.3

Polysaccharides and Lipids

Polysaccharides are polymers of simple sugars such as glucose. They are important structural elements and as the stores of energy. Oligosaccharides, the shorter polymers of sugars bind to proteins or lipids and play a significant role in cell signalling. The carbohydrates are dealt with in the Chapters 3 and 10. Lipids are hydrocarbon derivatives that function as the structural components, e.g., biomembranes and as energy-rich food reserve. The lipid synthesis and their metabolism have been dealt with in Chapters 12 and 13.

1.4

DIVERSITY IN THE LIVING WORLD

The living world comprises more than 1.6 million different species of organisms which are classified into three major domains, namely, eukarya, bacteria and archae. Eukarya consists of all organisms that have a well-defined nucleus within the cell. Macroscopic organisms and some unicellular organisms such as yeast belong to eukarya. Bacteria are unicellular and they lack a well-defined nucleus. Archaea were first grouped with bacteria but were put in a separate domain by American microbiologist, Carl Woese in 1977. It is believed that the archaea diverged from bacteria quite early during the evolution and they are closer to the eukaryotes as compared to the prokaryotes. Woese et al. classified the living world into three major domains, viz., eukarya, bacteria and archaea (Figure 1.9) based on following distinguishing characteristics: (i) differences in the nucleotide sequences in ribosomal RNAs (rRNA), (ii) cell membrane lipid structure and (iii) sensitivity of the cells to antibiotics (Table 1.2). The origination of all the species from a last common universal ancestor could thus be envisaged. A ‘superphylum’, of bacteria called PVC has organisms that share some characteristics with both archaea and eukaryotes, e.g.,

Fig. 1.9

Phylogenetic tree constructed on the basis of rRNA data showing three major domains —bacteria, archaea and eukaryota

Introduction to Biochemistry 13

phyla Planctomycetes, Verrucomicrobia and Chlamydiae. These bacteria display some of the characteristic features of eukaryotes such as cellular compartmentalization, some divide by budding or contain sterols in their cell membrane. A recent hypothesis proposes that the eukaryotic cells originated through a fusion between a bacterial and archaeal cell and the information processing genetic machinery has been inherited from the archea whereas the membrane phospholipid synthetic genes and energy metabolism genes from bacteria.

s 4HE ARCHAEA ARCHAEBACTERIA  !RCHAEA ARE MORE COMMON IN EXTREME ENVIRONments and include methanogens, halophiles and hyperthermophiles. The archaea are prokaryotes that possess membranes composed of branched hydrocarbon chains with ether linked glycerol moieties unlike the ester linked unbranched fatty acid chains found in bacteria and eukarya. The cell walls do not have peptidoglycan. Moreover, due to differences in protein synthetic mechanisms, archaea and eukarya are not sensitive to the antibiotics that inhibit protein synthesis, e.g., Chloramphenicol. The rRNA of archaea is different from that of bacteria and eukarya.

s 4HE BACTERIA EUBACTERIA  4HE BACTERIA ARE PROKARYOTES HAVING CELL MEMBRANES made of unbranched fatty acid chains bound to glycerol by ester linkages. The cell walls contain peptidoglycan and bacterial rRNA is different from eukarya and archaea.

s 4HEEUKARYOTAEUKARYA  4HEEUKARYOTAAREFURTHERSUBDIVIDEDINTOTHEFOLLOWING kingdoms: (i) Protista consisting of unicellular eukaryotic organisms including slime moulds, euglenoids and protozoans, (ii) Fungi that are unicellular or multicellular organisms, (iii) Plantae which are macroscopic multicellular complex organisms capable of photosynthesis including mosses, ferns, conifers and flowering plants and (iv) Animalia that are multicellular organisms having complex organization of tissues. They lack cell walls and photosynthetic capabilities. They obtain nutrients by ingestion. The examples include sponges, worms, insects and vertebrates. Table 1.2

Differences between bacteria, archaea and eukarya

Trait

Bacteria

Archaea

Eukarya

Lipid carbon linkage

Ester

Lipid phosphate backbone

Glycerol-3-phosphate

Glycerol-1-phosphate

Glycerol-3-phosphate

Well-defined organelles

No

No

No

Introns

No

No

Yes

Telomeres

No

No

Yes

DNA replication

Bacterial

Eukaryote-like

Eukaryotic

Transcription

Bacterial

Eukaryote-like

Eukaryotic

Translation

Bacterial

Eukaryote-like

Eukaryotic

Ether

Ester

14 Textbook of Biochemistry

1.5

BASIC UNIT OF LIFE: CELL

All organisms are made of cells that are capable of carrying out all the fundamental functions of life. Cells vary in size and shape depending upon the type of organism and also the tissue they belong to in case of multicellular organisms. Most of the bacteria are 0.2-10 Nm, animal cells are 10-30 Nm and plant cells range between 10-100 Nm in size. All prokaryotes are single-celled organisms enclosed in a cell wall that protects and maintains the cell shape. The intracellular cavity of each cell is filled with cytoplasm. Table 1.3

Relative sizes of different types of organisms, organelles and biomolecules Eukaryotic cell

10-100 μm

Prokaryotic cell

1-5 μm

Nucleus

10-20 μm

Chloroplast

2-10 μm

Mitochondria

0.5-5 μm

Large Virus

1-4 μm

Ribosome

25 nm

Cell membrane

7.5 nm (thickness)

DNA double helix

2 nm

Hydrogen bond

0.1 nm

Fig. 1.10

Comparison of prokarotic and eukaryotic cells

The internal organization of cells and central metabolic pathways is similar in all plants, animals, and unicellular eukaryotic organisms. Eukaryote cells have various membrane bound structures collectively known as the endomembrane system. It consists of endoplasmic reticulum, lysosomes, vacuoles and the Golgi apparatus suspended in the cytoplasm. These

Introduction to Biochemistry 15

organelles are responsible for performing different cellular activities and the division of labour allows greater work efficiency. The endoplasmic reticulum and the Golgi apparatus are organelles that are responsible for segregation and transport of proteins that are required at different destinations, for example, secretion outside the cells, integration into different cellular constituents and inclusion into lysosomes. In the following paragraphs, the cell organelles have been discussed briefly that would be essential to appreciate the intricately interwoven fabric that forms our living world.

1.5.1 The Cell Wall Bacterial cell walls The cell walls are rigid structures that determine the shape and protect the cell from bursting due to difference in osmotic pressure between the cell and its surroundings. Mycoplasma, some archaea and animal cells lack cell walls. The different structure of bacterial cell wall separates prokaryotic that can be distinguished into Gram-positive and Gram-negative using a very popular staining technique known as Gram staining, named after Danish bacteriologist Christian Gram. The principal component of the bacterial cell wall is peptidoglycan, a polymer of two sugar derivatives N-acetylglucosamine and N-acetyl muramic acid. A tetrapeptide of D- and L amino acids, D-glutamic acid, D-alanine and mesodiamino pimelic acid are attached to carboxyl group of N-acetyl muramic acid. Peptidoglycan subunits are crosslinked by the peptide bonds (Figure 1.11).

Fig. 1.11

Structure of cell walls of Gram-positive and Gram-negative bacteria

16 Textbook of Biochemistry

Gram-positive bacteria have 20-80 nm thick peptidoglycan cell walls and no outer phospholipid membrane whereas the Gram-negative bacteria have thinner 2-7 nm peptidoglycan layer and an outer phospholipid layer (7-8 nm). The Gram-positive cell walls also contain large amounts of teichoic acids. Penicillins, a class of antibiotics act by inhibiting the enzyme responsible for crosslinking of peptidoglycan strands and therefore interfere with cell wall synthesis. The space between the plasma membrane and the outer membrane is known as periplasmic space and it contains many proteins required for nutrition and transport such as hydrolytic enzymes for nutrient acquisition and binding proteins involved in transport of material into the cells.

Fig. 1.12 Peptidoglycan consists of alternating polysaccharide chains of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) residues joined by beta 1-4 glycosidic bonds. Parallel chains are crosslinked by tetrapeptides linked to NAM residues.

Eukarotic cell walls The cell walls of eukaryotes including fungi, algae and higher plants are made of different types of polysaccharides. Fungal cell walls are made of chitin polymer of N-acetyl

Introduction to Biochemistry 17

glucosamine residues while the cell walls of algae and higher plants are made of cellulose, a linear polymer of glucose with C (1m4) linkages as the main component (Figure 1.13).

Fig. 1.13 Polysaccharides of fungal and plant cell walls. Chitin, the principal component of fungal cell walls is a linear polymer of N-acetyl glucosamine residues, whereas cellulose is a linear polymer of glucose. The carbohydrate monomers are joined by C (1 m
4) linkages

18 Textbook of Biochemistry

Cellulose microfibrils in plant cell walls are enmeshed in a matrix made of proteins, hemicellulose and pectins. Hemicelluloses are highly branched polysaccharides that crosslink the cellulose microfibrils into a strong network. Pectins are polymers of negatively charged galactouronic acid residues. Additionally, plant cell walls also contain variety of glycoproteins incorporated in the matrix that strengthens the cell wall (Figure 1.14).

Fig. 1.14

Plant cell wall showing hemicellulose, pectin and cellulose

Growing plant cells have relatively thin flexible walls known as primary cell walls. These allow the plant cells to grow in size. After the cessation of growth, plants usually develop secondary cell walls between the plasma membrane and the primary cell walls. Primary and secondary cell walls differ in composition. The primary cell walls are composed of approximately equal amounts of cellulose, pectin and hemicellulose whereas secondary cell walls lack pectins. Secondary cell walls are more rigid than the primary and are responsible for conduction of water and structural rigidity of the plants. Most of the secondary walls are further strengthened by lignin, a complex polymer of phenolic residues (Figure 1.15).

Introduction to Biochemistry 19

Fig. 1.15

Primary and secondary cell walls in plant cells

Though the animal cells lack cell walls, the extracellular matrix (ECM) composed of proteins and polysaccharides provides the structural support, nutrition and is involved in cell signalling.

1.5.2

Cell Membrane

Cell membrane is the basic structure defining organelle of all cell types. All the known bio-membranes are made of two layers of amphipathic phospholipids that have hydrophobic fatty acid chains bound to hydrophilic head group. When placed in aqueous solutions, phospholipids have a property to aggregate spontaneously into bilayers organized in such a way that the hydrophilic head groups point towards the aqueous side and the hydrophobic tails are in contact with each other (Figure 1.16).

Fig. 1.16

Spontaneous assembly of lipid molecules into layers in aqueous solution

20 Textbook of Biochemistry

It can thus be imagined that the first primordial cell was formed due to such spontaneous aggregation of phospholipid molecules that enclosed RNA molecule in the primitive RNA world. Bacteria and Eukarya have phospholipids with fatty acid bound to D-glycerol with ester linkage whereas Archaea have branched isoprene units bound to L-glycerol with ether linkage (Figure 1.17). The isoprene units and the ether bonds confer greater heat and pH stability to archaea which are known to inhabit extreme environments.

Fig. 1.17 Types of lipids in bacteria and archaea

The cell envelope of Gram-negative bacteria is made of cell wall sandwiched between two different membranes, outer membrane (OM) and inner membrane (IM). OM is mostly composed of lipopolysaccharides (LPS) whereas IM is composed of phosphatidyl ethanolamine and phosphatidyl glycerol. LPS are the agents that cause endotoxin shock caused by infections with Gram-negative bacteria. It has majorly two types of proteins – lipoproteins and C-barrel proteins. In contrast to the bacterial inner membranes, the OM is highly porous due to presence of porins, the water filled open channels. aqueous channels. The inner membrane (IM) is composed of phosphatidyl ethanolamine and phosphatidyl glycerol. Since the prokaryotic cell lacks any well-defined membrane bound organelles, bacterial cell membrane has a responsibility to carry out other vital functions such as oxidative phosphorylation that produces most of the ATP during aerobic respiration, photosynthesis, synthesis of cell wall components, DNA replication, fixation of CO2 and oxidation of NH3. The metabolic enzymes required for these functions are located on the cell membrane. Eukaryotic plasma membrane consists of four major phospholipids: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin. The outer layer mainly consists of phosphatidylcholine and sphingomyelin and the inner layer predominantly consists of phosphatidylethanolamine and phosphatidylserine. Phosphatidylinositol is also present in the inner layer which plays a significant role in cell signalling. The head groups of both phosphatidylserine and phosphatidylinositol are negatively charged, so their

Introduction to Biochemistry 21

predominance in the inner leaflet results in a net negative charge on the cytosolic face of the plasma membrane. Glycolipids are minor outer layer components with their carbohydrate moieties exposed to the surface. Cholesterol is a major component of membranes which is inserted into the phospholipid bilayer with its polar head close to phospholipid head group. At high temperatures, cholesterol restricts the movement of phospholipid fatty acid chains making the membranes less fluid and reducing the permeability to the small molecules. Conversely, at low temperatures cholesterol prevents freezing of membranes due to interaction between fatty acid chains. Eukaryotic cell membranes show presence of sterols (cholesterol in animals, ergosterol in fungi, and phytosterols in plants) and sphingolipids which are absent in bacteria and archaea. Chemically, sphigolipids are fatty acids attached to amino acid serine. They are found in the outer layer of the eukaryotic membranes. The presence of sterols reduces the membrane fluidity and permeability. Sterols and sphingolipids together form lipid rafts in the membranes that function to organize proteins into cell signalling complexes. The fatty acid chain length also influences the membrane fluidity with longer chain lengths giving greater rigidity to the membrane. The naturally occurring fatty acids may have one or more double bonds with cis configuration. Cis unsaturated fatty acids have a bend in their shape which interferes with close packing with each other, hence increasing the membrane fluidity (Figure 1.18). According to the fluid mosaic model given by Jonathan Singer and Garth Nicolson in 1972, the membrane can be visualized as a mosaic of proteins embedded in phospholipid bilayer. The membrane bears two types of proteins—peripheral and integral. Peripheral proteins are associated with the membranes through protein-protein interactions. These interactions can be disrupted relatively easily by treatments such as high pH and salt concentration and the peripheral proteins can be separated from the membrane. In contrast, integral membrane proteins are transmembrane glycoproteins that span the membrane and they can be separated only on harsh treatments such as use of detergents that disrupt the hydrophobic interactions. The membrane spanning portions of the protein are usually helices made from 20-25 hydrophobic amino acids and have the oligosaccharide exposed on the surface. Some other proteins are anchored in the plasma membrane by covalently attached lipids or glycolipids, e.g., some of these proteins are attached to the outer layer of the membrane by glycosyl phosphatidyl inositol anchors. Some other proteins are anchored in the inner layer by covalently attached lipids. The carbohydrate portions of the glycolipids as well as glycoproteins are exposed to the outer surface of plasma membrane creating a carbohydrate coated surface of the cell which is known as glycocalyx. Glycocalyx protects the cell and serves as marker for a variety of cell-cell interactions.

1.5.3

Nucleus

The nucleus is the largest cellular organelle that contains the genetic information. Nucleus is enclosed in a double membrane known as the nuclear envelope which has nuclear pores

22 Textbook of Biochemistry

Fig. 1.18 Eukaryotic cell membrane showing a phospholipid bilayer composed of outer leaflet (layer) with carbohydrates (glycolipids and glycoprotein) and a cytoplasmic leaflet. Integral membrane proteins have some part of the protein embedded in the hydrophobic bilayer.

for transport of material. The outer nuclear membrane is mainly continuous with the rough endoplasmic reticulum (rough ER) and the space between the inner and outer membrane is continuous with the lumen of rough ER (Figure 1.19). The nucleus contains nucleoplasm with high DNA concentration. The nuclear DNA is packed into chromosomes that remain dispersed throughout the cell. Chromosomes can be visualized by light microscopy only during the cell division when they condense into thick, well-defined structures. Nucleus is the centre of RNA synthesis. A suborganelle of nucleus known as nucleolus is visible under light microscope. Nucleolus is the site of ribosomal RNA synthesis. The rRNA synthesised in the nucleolus is transported to cytoplasm through the nuclear pore.

Introduction to Biochemistry 23

Fig. 1.19

1.5.4

Structure of nucleus, endoplasmic reticulum, lysosome, and Golgi apparatus

Ribosomes

Ribosomes are the cellular machines for protein synthesis composed of protein and ribonucleic acid. The elucidation of ribosome structure has given an insight into the mechanism of protein synthesis in the cell. Ribosomes can be visualized bound to the rough endoplasmic reticulum (ER), free in the cytoplasm and in mitochondria and chloroplasts. Prokaryotes have ribosomes suspended in the cytoplasm. Bacterial ribosomes as well as eukaryotic ribosomes have two subunits, small and large (Figure 1.20). The size of ribosomes has been typically described in terms of Svedberg units (S) which is a measure of velocity of sedimentation. Prokaryotic ribosomes are 70S having 30S and 50S subunits – with the 30S subunit containing 16S RNA and 21 peptides. Eukaryotic ribosomes are 80S units with 40S and 60S subunits – with the 40S subunit containing 18S RNA and 33 polypeptides and the 60S containing 28S RNA, 5.8S RNA, 5S RNA and 49 polypeptides. During protein synthesis, these two subunits unite when they bind to messenger RNA (mRNA). They translate the specific sequence of amino acids as encoded by mRNA accurately. Amino acids are selected, collected and carried to the ribosome by transfer RNA (tRNA) molecules.

1.5.5

Plasmids

Many bacteria have extrachromosomal double stranded circular DNA molecules called plasmids. Plasmids are capable of existing and replicating independently which are passed

24 Textbook of Biochemistry

Fig. 1.20

Structure of a ribosome with growing amino acid strand

on to the progeny. They are not attached to the plasma membrane and may be lost during cell division, and are not essential for the host cells but confer selective genes that may give special advantage to the host cell, e.g., may bear antibiotic resistance genes or they may make the cells pathogenic. Plasmids can be easily spread within a bacterial population.

1.5.6

Mitochondria

Eukaryotic cells consist of one or more mitochondria depending on the energy requirements of the cells. Mitochondria are the site of aerobic respiration where oxidative phosphorylation takes place. Therefore, they are also known as powerhouse of the cell. Mitochondria are surrounded in phospholipid bilayer double membrane system separated by intermembrane space. The outer membrane is made up of half-lipid and half-protein making it permeable to high molecular weight compounds. The inner membrane is relatively impermeable and consists of 20% lipid and 80% protein. The inner mitochondrial membrane is invaginated to form structures known as cristae that extend into the matrix of the mitochondria.

1.5.7

Chloroplasts

Chloroplasts are present in blue green algae and green plants. Similar to the mitochondria, chloroplasts also possess double membrane and have interconnected membranous structures known as thylakoids. Thylakoids are grouped into stacks called grana and are surrounded by matrix called stroma. The membranes of thylakoids contain green chlorophyll pigments and enzymes for production of ATP by the process of photosynthesis (Figure 1.21). Molecular mechanisms involved in ATP generation in mitochondria and chloroplasts are strikingly similar. These are discussed in detail in Chapters 9 and 11.

Introduction to Biochemistry 25

Fig. 1.21

Structures of typical mitochondrion and chloroplast

Chloroplasts and mitochondria share several structural features– both have a double membrane and contain stacked membranous structures. Mitochondria as well as chloroplasts possess their own DNA and replicate autonomously. The DNA in these organelles code for key organelle specific proteins that are synthesized on the ribosomes located within these organelles. ‘The endosymbiotic origin’ proposed by Lynn Margulis of Boston University believes that an anaerobic cell engulfed aerobic bacteria further developed symbiotic relationship with the host and evolved into mitochondria. These cells further evolved into eukaryotic cells. The cells that engulfed aerobic as well as photosynthetic cyanobacteria developed into mitochondria and chloroplasts, respectively.

1.5.8

Lysosomes and Peroxisomes

Lysosomes and peroxisomes contain various digestive enzymes which are responsible for digesting complex molecules like cellular waste and compounds ingested by endocytosis. They are bound by single membranes. When cellular components are degraded by the lysosomes, it is known as autophagy (Figure 1.22). Lysosomes have enzymes such as nucleases, proteases and phosphatases which degrade the respective cellular and ingested materials. They have capability to degrade phospholipids and other polysaccharides. Tay-Sachs disease is caused by defect in a lysosomal enzyme that degrades glycolipids called gangliosides which are found in nerve cells. The affected children show dementia and blindness by age 2 and die soon after. The pH in the lysosomal cavity is maintained in the acidic range (around 4.8) by a hydrogen pump and a chloride channel. The enzymes present in the lysosomes are acid hydrolases and function efficiently. The acidic pH also denatures proteins and makes them more accessible for the hydrolytic enzymes.

26 Textbook of Biochemistry

Fig. 1.22

Structure of a typical lysosome showing phagocytosis and exocytosis

Most of the plant and animal cells except for the erythrocytes possess small (0.2-1 Nm), single membrane bound organelles called peroxisomes. Peroxisomes contain oxidases that use molecular oxygen for oxidation of organic substances and produce hydrogen peroxide (H2O2) which is harmful for the cell. However, the hydrogen peroxide produced is degraded by catalase enzyme that degrades hydrogen peroxide to produce water and oxygen. In the liver and kidney cells, peroxisomes are responsible for the degradation of many toxic substances that enter the blood stream. X-linked adrenoleukodystrophy (ADL) is a genetic disease where the peroxisomal degradation of long chain fatty acids is defective. The ADL gene product is a peroxisomal membrane protein that transports the enzyme responsible for the oxidation of fatty acids into the peroxisomes. The individuals suffering from ADL are unaffected till mid childhood after which neurological defects appear and die in few years. 2 H2O2 catalase 2H2O + O2 Glyoxisomes are present in plant seeds that oxidize the stored lipids as a source of carbon and energy. They possess enzymes similar to peroxisomes.

1.5.9 Vacuoles Vacuoles are single membrane bound organelles first named by de Vries. Vacuoles have enzymes for digestion of macromolecules and store waste products and nutrients for further transportation. Plant cells have large vacuoles; they provide strength to the cell.

1.5.10

Endoplasmic Reticulum

Endoplasmic reticulum (ER) is a network of membranous tubules. It consists of numerous sacs known as cisternae that extend from the nuclear membrane to cytoplasm and cell membrane. Endoplasmic reticulum is responsible for transport of material in and out of the cells and provides mechanical strength to the cell. Electron micrographs have revealed

Introduction to Biochemistry 27

presence of two different types of endoplasmic reticulum smooth endoplasmic reticulum and rough endoplasmic reticulum. Rough endoplasmic reticulum begins from the nuclear envelope and consists of tubules with ribosomes attached on the surface. The attached ribosomes are sites of protein synthesis and the endoplasmic reticulum is responsible for protein modification. The cells that specialize in the secretion of proteins particularly have a large part of cytosol filled with rough endoplasmic reticulum. The proteins synthesized in rough ER are transported in small vesicles that bud-off from the rough ER tubules and are carried to lumen of another cellular organelle called the Golgi complex where further modification and sorting takes place (Figure 1.23). Smooth endoplasmic reticulum is involved in the synthesis of lipids and detoxification. This organelle is particularly abundant in hepatocytes. The smooth endoplasmic reticulum liver cells have enzymes to degrade hydrophobic chemicals such as pesticides and carcinogens and derivatize them into more water-soluble conjugates so that these can be easily excreted.

1.5.11 The Golgi Complex The Golgi complex or apparatus is made of flattened membranous sacs and spherical membrane vesicles. It has three distinct parts: the cis, the medial, and the trans. The rough ER vesicles fuse with the cis region of the Golgi complex. Different enzymes are present in the

Fig. 1.23 The transport of material through the endomembrane system consisting of the endoplasmic reticulum, the Golgi apparatus and lysosomes

28 Textbook of Biochemistry

Golgi apparatus that modify the proteins depending on their structure and their intended final destinations. The proteins are tagged for transport to plasma membrane, lysosomes or other organelles. After modification, the proteins are transported using different transport vesicles that bud-off from the trans side of the Golgi apparatus.

1.5.12

Cytoskeleton

Structure of a cell is supported by a network of protein filaments that extend all through the cytoplasm. The key proteins filaments involved are: microfilaments composed of actin, intermediate filaments made of many different proteins, and microtubules that have tubulin as a basic subunit. Cytoskeleton filaments also help in cell movement and intracellular transportation, arrangement of organelles in the cell and movement of cellular components such as chromosomes during cell division.

1.6









SUMMARY s 4HE -ILLER 5REY EXPERIMENT PROVED THAT THE PRIMITIVE ATMOSPHERIC CONDITIONS WERE conducive to the formation of complex organic molecules. Simple organic compounds polymerized to form complex organic molecules such as polysaccharides, polypeptides, proteins, lipids, etc. The complex organic compounds in the ocean aggregated spontaneously to form colloidal droplets named coacervates. s 4HEFIRSTLIFEFORMSONTHEEARTHORIGINATEDABOUTBILLIONYEARSAGOINTHEOCEAN Some of the proteins had catalytic properties and they catalyzed the metabolic reactions. s 4HE PHOTOAUTOTROPHS WERE RESPONSIBLE FOR PRODUCING OXYGEN /2) in large quantities. The abundance of oxygen in the atmosphere made aerobic respiration possible, presumably about 27 billion years ago. s 4HE TRANSFER OF THE ACQUIRED TRAITS OCCURS BY vertical gene transfer from parent to progeny. In contrast to this gradual process of evolution, recent evidence has established that transfer of genetic material between genetically distinct lineages can occur by horizontal gene transfer (HGT) or lateral gene transfer. s .UCLEICACIDSARETHEPOLYMERSOFNUCLEOTIDESMADEFROMPENTOSESUGAR NITROGENOUS base and a phosphate. Proteins are the polymers of B carboxylic acids while the lipids are polymers of the fatty acids. s )N THE PRIMITIVE 2.! WORLD continuous RNA replication might have led to limitation in RNA precursor molecules thereby triggering the development of alternate mechanisms for their synthesis. s 4HE ADENOSINE TRIPHOSPHATE !40 IS THE MOLECULE THAT STORES THE CHEMICAL ENERGY of the cell. ATP is a mononucleotide made from one of adenine, a ribose sugar, and three serially linked phosphate groups. Reactions that break the phosphate group leading to formation of adenosine diphosphate (ADP) releases energy which is used for carrying out other cellular reactions.

Introduction to Biochemistry 29





s 0ROTEINS ARE THE POLYMERS OF AMINO ACIDS THE LARGEST MACROMOLECULAR FRACTION OF cells. More than twenty different types of amino acids are found in nature s 0OLYSACCHARIDES ARE THE POLYMERS OF SIMPLE SUGARS SUCH AS GLUCOSE s #ARL7OESEet al. classified the living world into three major domains, viz., eukarya, bacteria and archaea based on the following distinguishing characteristics: (i) differences in the nucleotide sequences in ribosomal RNAs (rRNA), (ii) cell membrane lipid structure, and (iii) sensitivity of the cells to antibiotics. s !LL ORGANISMS ARE MADE OF CELLS THAT ARE CAPABLE OF CARRYING OUT ALL FUNDAMENTAL functions of life. Cells vary in size and shape depending upon the type of organism and also the tissue they belong to in case of multicellular organisms. Scientists and their discoveries Cell Membrane

1665

Robert Hooke

Centriole and centrosome

1883

Edourd Van Benedon

Cytoskeleton

1903

Nikolai K Koltkov

Microtubules

1953

De Roberts and Franchi

Microfilament

1968

Edward David Kon

Intermediate filaments

1968

Howard Holtzer

Golgi Apparatus

1897

Camillo Golgi

Lysosomes

1947

Christian de Duve

Mitochondria

1984

Albert von Kolliker

Nucleus

1833

Robert Brown

Ribosomes

1955

George Palade

Endoplasmic reticulum

1945

Albert Claude

Vacuole

1976

Antony van Leeuwenhoek

Nucleolus

1774

Felice Fontana

Chromosomes

1842

Karl Wilhelm Nageli

MULTIPLE-CHOICE QUESTIONS 1. The earth originated .................... billion years ago. (a) 4.6 (b) 2.5 (c) 6.9 (d) 1.2 2. The first living cell originated .................... billion years ago. (a) 4.6 (b) 3.8 (c) 6.9 (d) 1.2 3. The first formed cells were: (a) Autotrophs (b) Saprotrophs (c) Chemoautotrophs (d) Heterotrophs 4. The first autotrophic organisms were: (a) Algae (b) Virus (c) Cyanobacteria (d) Plants 5. Scientists who tried to demonstrate the origin of life on the earth were: (a) Meselson and Stahl (b) Oparin and Haldane (c) Watson and Crick (d) Urey and Miller

30 Textbook of Biochemistry 6. Urey and Miller created the primitive environment in the spark chamber and used hydrogen, ammonia, methane and water vapours to simulate the chemical origin of life. The surprising molecules they obtained after the experiment was: (a) Proteins (b) Lipids (c) Amino acids (d) Polysaccharides 7 The functional unit of a life is called: (a) Cell (b) Egg (c) Nucleus (d) None of these 8. Which cell organelle connects the nucleus with the cell membrane? (a) Golgi apparatus (b) Ribosomes (c) Endoplasmic reticulum (d) Mitochondria 9. Protein synthesis occurs in: (a) Cytoplasm (b) Nucleus (c) Golgi bodies (d) Lysosome 10. The most important function of cell membrane is that it: (a) controls the entry and exit of materials from cells. (b) controls only the entry of materials into cells. (c) controls only the exit of materials from cells. (d) allows entry and exit of materials without any control.

Answers 1. (a) 9. (a)

2. (b) 10. (a)

3. (c)

4. (c)

5. (d)

6. (c)

7. (a)

Short Answer Type Questions 1. 2. 3. 4. 5.

Discuss some of the ancient theories of origin of life on the earth. How did formation of coacervates take place? Describe Sol Spiegelman’s experiment. Give some major differences between prokaryotic and eukaryotic cells. Explain the basis of Carl Woese’s classification.

Long Answer Type Questions 1. Describe Miller-Urey experiment? 2. Explain in detail the functions of cell membrane. 3. Describe in detail the structure and function of the following organelles: (a) Mitochondria (b) Golgi apparatus (c) Endoplasmic reticulum (d) Lysosome (e) Nucleus 4. Differentiate between archaea, eubacteria and eukarya.

8. (c)

2 Water: The Unique Solvent 2.1

INTRODUCTION

Life on the earth has been made possible due to the presence of water, the elixir of life. Water forms about 70% of all living organisms on the earth and life on the earth is believed to have originated in water. Water acts as a solvent, buffer, metabolite, thermo-regulator and much more. These important roles can be understood on the basis of unique structure and properties of water.

2.2

STRUCTURE AND PROPERTIES OF WATER

Water is a compound of oxygen with hydrogen with molecular formula H2O. Oxygen and hydrogen atoms are bound with the help of strong covalent bonds. The two hydrogen atoms and oxygen atom share one electron each and form two covalent bonds. The shape of the molecule largely resembles a tetrahedron with each of the hydrogen atom occupying the two corners and the unshared pair of electrons occupying the other two corners. The bond angle is 104.5° which is little less than the perfect tetrahedron because of crowding by the non-bonding orbitals of the oxygen atom. Oxygen atom has a greater electronegativity than the hydrogen, therefore the sharing of electrons between hydrogen and oxygen is unequal, i.e., the hydrogen atoms bear a partial positive charge (E+) and oxygen (2E-) atom bears a partial negative charge. This unequal sharing of electrons creates two dipole moments in the molecule, one each in the direction of OH bond (Figure 2.1). – O +

H

+

Hydrogen bond O

H

+

Fig. 2.1

H

H

–

H

+

O +

–

H

Covalent bond +

Structure of water and different types of bonds in water

32 Textbook of Biochemistry

Since water molecules have slightly positive and slightly negative ends, they can interact with each other extensively forming hydrogen bonds. One water molecule forms hydrogen bond with as many as four hydrogen bonds. Hydrogen bonds have bond energy of about 23 kJ/mol. This bond energy is very weak as compared to the 470 kJ/mol bond energy of the covalent OH bond in water. The unusual properties of water are due to the presence of hydrogen bonds. Apart from hydrogen bonds, weak interactions like van der Waals and hydrophobic interactions have significant impact on the three-dimensional structure of biological macromolecules like proteins, nucleic acids, polysaccharides and membrane lipids as would be discussed in the following chapters.

2.3 WEAK NON-COVALENT INTERACTIONS IN AQUEOUS SOLUTIONS 2.3.1

Hydrogen Bonding

Water has many unique properties that are result of hydrogen bonds in water. The higher melting point, boiling point and heat of vaporization are a result of extensive hydrogen bonding in water molecules. It is due to the higher boiling point of water that it exists in liquid state on the earth. Moreover, higher specific heat of water makes it a good temperature regulator in the living organisms. During melting or evaporation, the entropy (%S) of the system increases due to increase in randomness as in formation of liquid water from highly ordered ice or from water to highly disordered gaseous state as steam. This makes the overall free energy change to (%G = %H – T%S) negative, making the melting and evaporation spontaneous processes at room temperature even though %H is positive. H2O (solid) H2O (liquid)


H2O (liquid) %H = +5.9 kJ/mol
H2O (gas) %H = +44.0 kJ/mol

Cohesive forces of hydrogen bonds make the water liquid at room temperature and also give the highly ordered structure of ice. The melting point of water is 0°C. Ice has a lower density than water, hence hydrogen bonding is the reason behind this anomalous behaviour. Hydrogen bonds make water molecules line up less efficiently in ice as compared to liquid water, as a result the intermolecular distance in ice is greater than that in liquid water which results in lower density of ice as compared to water. The cohesive forces between water molecules are responsible for its high surface tension. The surface water molecules do not have other water molecules on their upper side, therefore they have stronger attraction towards other water molecules interacting with them creating high surface tension. The hydrogen bonds are dynamic and constant rearrangement allows for a small fraction of hydrogen bonds being formed with another substance. This is responsible for capillary action, i.e., the ability of a liquid to oppose gravity and flow in upward direction spontaneously. The adhesive and capillary actions play a significant role in transport of water in plants as well as transport of nutrients through blood in animals.

Water: The Unique Solvent 33

Polar molecules such as sugars, alcohols, aldehydes, ketones and compounds with NOH bonds can form hydrogen bonds with water molecules and solubilize in water (Figure 2.2). Hydrogen bonds can be formed between an electronegative atom (hydrogen acceptor) and an electronegative atom (hydrogen donor). The hydrogen atoms bound to carbon do not participate in hydrogen bonding as carbon is only slightly more electronegative than hydrogen.

Fig. 2.2 Hydrogen bonding including oxygen and nitrogen atoms

Polar molecules are easily soluble in water as they can replace the water-water interactions with energetically favourable water solute interactions. However, nonpolar molecules are unable to form energetically favourable interactions with water and hence they are poorly insoluble in water. The compounds that easily dissolve in water are known as hydrophilic whereas compounds that cannot dissolve in water are known as hydrophobic. Hydrophobic compounds dissolve in nonpolar solvents such as chloroform and benzene. Biologically important gases like oxygen, carbon dioxide and nitrogen are nonpolar. In oxygen and nitrogen, electrons are shared equally by the two atoms. Though CO2 is polar, the C=O bond is polar; the two dipoles cancel each other due to their opposite direction. Water soluble carrier proteins like haemoglobin, and myoglobin transport these gases in organisms. Carbon dioxide forms carbonic acid in water and is transported as bicarbonate (HCO3–).

2.3.2

Electrostatic or Ionic Interactions

Electrostatic interactions exist when electric charges are present in the atoms and molecules. The energy of an electrostatic interaction is given by Coulomb’s law: E = kq1q2 /Dr where, E q1 and q2 r D k The value of k

– Energy – charge on two atoms – the distance between two atoms – Dielectric constant (it accounts for the effect of medium) – Proportionality constant is 332 kCal/mol or 1389 kJ/mol.

...(2.1)

34 Textbook of Biochemistry

Ionic compounds like salts are easily solubilized by water since positive and negative ions are stabilized by the partial negative and positive charges present in water.

2.3.3

Hydrophobic Interactions

When nonpolar molecules are mixed with water molecules, they interfere with hydrogen bonding in water, however they are not able to form any hydrogen bonds with water. Water molecules surround the nonpolar molecules forming “cages” around the molecules. This highly ordered structure of water molecules surrounding the nonpolar molecules decreases the entropy and hence is thermodynamically unfavourable. In aqueous environments, the nonpolar solutes associate with each other to avoid interaction with water and attain thermodynamic stability (Figure 2.3).

Fig. 2.3 Hydrophobic interaction between nonpolar molecules (red) and water molecules (blue)

Amphipathic compounds have polar (charged) as well as nonpolar regions in their molecular structure. Amphipathic compounds when mixed with water, their hydrophilic regions are able to interact favourably with water whereas their hydrophobic regions try to cluster together to avoid interacting with water. The interactions among the nonpolar regions are known as hydrophobic interactions. Hydrophobic interactions are not due to the attractions between the nonpolar regions but due to the thermodynamic stability they offer in aqueous solvents. The nonpolar regions interact in such a way to minimize their interactions with the aqueous solvent whereas polar regions are presented to maximize their interactions with aqueous solvent. The result of these interactions is the stable structure of micelles (Figure 2.4). Micelles contain thou- Fig. 2.4 Micelles are amphipathic molecules sands of molecules.

Water: The Unique Solvent 35

Many biomolecules are amphipathic in nature. Proteins, pigments, phospholipids of membranes all have polar as well as nonpolar regions in their structures. The three-dimensional structures of these biomolecules are stabilized largely due to hydrophobic interactions in the nonpolar regions of these molecules. Biological membranes are stabilized by the hydrophobic interactions in phospholipids. The 3D structures of proteins are also supported by the hydrophobic interactions.

2.3.4

van der Waals Forces

van der Waals forces have been named after Dutch scientist, Johannes Diderik. van der Waals are weak interactions that come into being with proximity of the atoms. When two uncharged molecules come sufficiently closer, their surrounding electron clouds interact. Transient variations in position of electrons may create a transient electric dipole which induces a transient opposite electric dipole in the neighbouring atom. This creates an attractive force between the two atoms. As the two nuclei come closer, their electron clouds start repelling each other. The distance between the two nuclei at which the van der Waals forces exactly balance the repulsive forces is known as van der Waals diameter. Every atom has a characteristic van der Waals radius which is a measure of distance to which another atom would be permitted to approach (Figure 2.5).

Fig. 2.5 Energy of van der Waals interactions: The energy is minimum at the van der Waals contact distance. At distances lesser than the van der Waals contact distance, the energy rises due to the repulsive forces between electrons.

The three weak interactions—hydrogen bonding, hydrophobic interactions, ionic, and van der Waal’s interactions— are comparatively weaker than covalent bonds. At room temperature (25°C), the thermal energy available is equally favourable for interaction between the solute and solvent as well as that between the solute and solute. Therefore, these interactions

36 Textbook of Biochemistry

are continuously being made and broken in aqueous solutions. Though these interactions are weak individually, the collective effect of these interactions is significant and plays a vital role in the chemistry of life. These are responsible for precise replication of DNA, folding of proteins in their native active forms, and biological recognition.

2.4

IONIZATION OF WATER

Pure water conducts electricity and H+ migrates towards the cathode and OH_ towards the anode. The conductivity of water is because water undergoes slight reversible dissociation to yield a hydrogen ion (proton) and a hydroxide ion. H 2O

H+ + OH–

...(2.2)

The above reaction depicts that free protons do not exist in solution as hydrogen ions formed in water are instantaneously hydrated to the hydronium ion (H3O +).

The position of equilibrium of a chemical reaction is given by its equilibrium constant, Keq which is the ratio of products of concentration of products to the concentration of reactants. Equilibrium constant is characteristic of any chemical reaction at a particular temperature. It gives the composition of the reactants and products at the equilibrium regardless of the starting amounts of the reactants and products. The equilibrium constant of reversible ionization of water is given as equation 2.3. [H+][OH–] Keq = _________ [H2O]

...(2.3)

The concentration of pure water can be calculated as 55.5 M which essentially remains constant as compared to the smaller amounts of H+ and OH– ions. Grams H2O in 1L = 1000 g Gram molecular weight = 18.015 g/mol Concentration = (1000 g/L)/(18.015 g/mol) = 55.5M We can therefore substitute the value of concentration of water in equation 2.3 as [H+][OH–] Keq = _________ 55.5 M

...(2.4)

On rearranging, (55.5M)(Keq) = [H+][OH–] = Kw

...(2.5)

Water: The Unique Solvent 37

Kw is known as the ionic product of water. The value of Keq is measured by the electrical conductivity of water and it is measured to be 1.8 × 10 –16 at 25°C. Kw = [H+][OH–] = (55.5M)(1.8 × 10 –16 M) = 1.0 × 10 –14 M2

...(2.6)

Therefore, the product of hydrogen and hydroxide ions in the aqueous solutions is always equal to 1 × 10 –14. In neutral solution, the concentrations of H+ and OH– ions are exactly equal and can be calculated by the above equation as follows: Kw = [H+][OH–] = [H+] 2 Solving for [H+] gives ___

___________


w = •
1 × 10 –14 M2 [H+] = •K [H+] = [OH–] = 10 –7 M

...(2.7)

2.5 THE pH SCALE The ion product of water has been used to define pH scale which is a method to designate the concentration of H+ and (OH–) ions in aqueous solutions. pH is the negative log of hydrogen ion concentration given by the following equation: 1 pH = log ____ = – log[H+] [H+]

...(2.8)

Thus, neutral solution which has got 1 × 107 hydrogen ions has a pH of 7. 1 = log (1.0 × 107) pH = log _________ 1.0 × 10 –7 = log 1.0 + log 107 = 0 + 7 = 7 pH lower than 7 signifies acidic solution whereas pH greater than 7 signifies alkaline solution. Table 2.1 gives the pH scale with concentrations of H+ and OH– ions and respective pH/pOH. pOH is the negative logarithm of hydroxide ion concentration. The pH scale is convenient to use as it avoids complication of using exponential notation for the strength of acid or base. The value of pH indicates the value of the exponent of the hydrogen ion concentration and every unit change in the pH scale corresponds to a tenfold change in hydrogen ion concentration. pH can be measured by various indicator dyes such as litmus that changes colour at specific pH or it is frequently measured using pH electrode which is sensitive to H+ ions. The pH of the human blood is maintained at 7.4 by various mechanisms of homeostasis. When the pH of blood falls below 7.4, the condition is known as acidosis which is frequently encountered in people with diabetes whereas the condition in which blood pH rises above 7.4, is known as alkalosis.

38 Textbook of Biochemistry Table 2.1 The pH scale [H+](M) 100 –1

10

[OH–] (M)

pOH

0

10–14

14

1

–13

13

–12

10

–2

10

2

10

12

10–3

3

10–11

11

–4

10

4

–10

10

10–5

5

10–9

9

–6

10

6

10

–8

8

10–7

7

10–7

7

–8

10

8

10

–6

6

10–9

9

10–5

5

–10

10

10

–4

4

10–11

11

10–3

3

12

10

–2

2

10

–1

1

100

0

10

2.6

pH

10

–12

10

–13

13

10–14

14

10

IONIZATION OF ACIDS AND BASES

Acids are proton donors and bases are proton acceptors. Strong acids and bases are completely ionized in dilute aqueous solutions. Hydrochloric acid, sulphuric acid and nitric acid are examples of strong acids and sodium hydroxide and potassium hydroxide are strong bases. Weak acids or bases do not dissociate completely in water. The dissociation of weak acid can be represented by an equilibrium reaction, HA m H+ + A–

...(2.9)

where, HA is any generic acid, and A– is known as the conjugate base. The dissociation of any acid forms a conjugate base (A–) which readily accepts protons. Conversely, dissociation of a base results in the formation of a conjugate acid. The equilibrium constant for the dissociation of a weak acid is given by, [H+][A–] Ka = ________ [HA]

...(2.10)

Equilibrium constants are commonly known as dissociation constants. The dissociation constants (Ka) of common acids are given in Table 2.2. It can be observed that stronger acids, such as formic and lactic acids, have higher dissociation constants; weaker acids, such as dihydrogen phosphate (H2PO4), have lower dissociation constants.

Water: The Unique Solvent 39 Table 2.2

Dissociation constants and pKa of some common weak acids

Acid HCOOH CH3COOH

Name Formic acid Acetic acid

Ka

pKa

1.78 × 10–4

3.75

–5

4.76

–5

1.74 × 10

CH3CH2COOH

Propionic acid

1.35 × 10

4.87

CH3CH(OH)COOH

Lactic acid

1.38 × 10–4

3.86

–3

H3PO4

Phosphoric acid

7.25 × 10

2.14

H2PO4–

Dihydrogen phosphate

1.38 × 10–7

6.86

2–

–13

HPO

Monohydrogen phosphate

3.98 × 10

H2CO3

Carbonic acid

1.70 × 10–4 –11

HCO3–

Bicarbonate

6.31 × 10

NH4–

Ammonium

5.62 × 10–10

2.7

12.4 3.77 10.2 9.25

HENDERSON-HASSELBALCH EQUATION

The Henderson-Hasselbalch equation is derived from equation 2.10 by taking the logarithm of both sides and rearranging to get [A–] – log[H+] = – log Ka + log _____ [HA]

...(2.11)

By definition, pH is – log[H+] and – log Ka is defined as pKa. Rewriting equation 2.11, we obtain the famous Henderson-Hasselbalch equation. [A–] pH = pKa + log _____ [HA]

...(2.12)

We can think of the pKa as the pH at which the number of molecules of conjugate base [A–] and weak acid [HA] are equal. Strong acids have lower pKa and strong bases have higher pKa. The pKa can be determined experimentally by titrating against acid/base. pKa is the pH at the midpoint of the titration curve for the acid or base. The above equation explains the dependence of pH on the constant, pKa, as well as the ratio of conjugate base to weak acid, [A–]/[HA] existing in the solutions. HendersonHasselbalch equation is an extremely useful equation from which pH of the solutions of various ratios of concentrations of conjugate acid and conjugate base forms of a substance can be determined. Conversely, it can also be used to find out the required ratio of conjugate acid-base pair of known pKa to obtain a buffer of desired pH.

2.8

ACID-BASE TITRATIONS

The pKa values are determined by titrating the free acid form of any substance with an appropriate base. The pH of the solution is measured continuously during the titration. A titration curve is made with equivalents of base plotted on abscissa against pH as the

40 Textbook of Biochemistry

ordinate. Let us consider the titration of strong acid (HCl) with a strong base (NaOH) (Figure 2.6).

Fig. 2.6 Titration curve of strong acid (0.1M HCl) with strong base (0.1M NaOH)

As the base is added to the acid, pH rises slowly in the beginning. As the added base approaches near equivalence, pH rises sharply as shown in Figure 2.6. The point where the base added is enough to neutralize all acid is known as equivalence point. The equivalence point of a strong acid-base titration is achieved at pH 7. Beyond the point of equivalence, pH slowly increases. In contrast, the titration curve of weak acid shows that pH rises more slowly near the equivalence point. Weak acids and bases do not ionize completely in water. Hence, pH of a weak acid is higher than that of a strong acid at same concentration. The titration curve of weak acids shows that pH changes rapidly in the first part of the titration whereas the rise in pH is very slow near the equivalence point. Acetic acid (CH3COOH) dissociates in aqueous solutions as given in the equilibrium by equation 2.13: CH3COOH ==H+ + CH3COO –

...(2.13)

Acetic acid has pKa of 4.76; the equivalence point of weak acids is achieved at more than 7.0. Once equivalence point is reached (acid has been neutralized), the pH of the solution is dependent on the concentration of excess NaOH. Figure 2.7 shows the titration curve for acetic acid. The point of inflection indicates the pKa value.

Water: The Unique Solvent 41

Fig. 2.7 Titration curve of weak acid (acetic acid) with strong base (NaOH): The change in pH on each increment of NaOH added to the acetic acid solution is measured and plotted against the fraction of the total amount of NaOH required to neutralize the acetic acid. At pH below pKa the acids mostly exist as undissociated form as indicated in the figure. At midpoint of the titration, the concentrations of proton donor and proton acceptor are equal. The pH at this point is numerically equal to the pKa of acetic acid. The shaded zone is the useful region of buffering power.

Figure 2.8 shows the titration curve for phosphoric acid, a weak triprotic acid. Triprotic acids can dissociate up to three times, giving up a hydrogen ion each time as shown in the following reactions: H3PO4 H2PO4– HPO4–2

2.9

H+ + H2PO4– +

H + +

H +

HPO4–2 PO4–3

K1 = 7.5 × 10 –3

pK1 = 2.12

...(2.14)

K2 = 6.2 × 10

–8

pK2 = 7.21

...(2.15)

K3 = 4.8 × 10

–13

pK3 = 12.30

...(2.16)

BUFFERS

The titration curves of weak acids and bases show that weak acid and its anion—a conjugate acid-base pair can resist small changes in pH due to addition of acids and bases. This property to resist pH change is known as buffering. Buffers are the chemical compounds that resist pH change in systems. A buffer system consists of a weak acid (the proton donor)

42 Textbook of Biochemistry

Fig. 2.8 Titration curve of polyprotic acid (phosphoric acid) with strong base (NaOH)

and its conjugate base (the proton acceptor). Consider mixture of equal concentrations of acetic acid and acetate ion as an example of buffer system (Figure 2.7). In the titration curve we can see that there exists a relatively flat region extending about 0.5 pH units on either side of its pKa value of 4.76. In this zone, addition of OH– (or H+) brings about negligible change in the pH. This range is known as the buffering range of acetate buffer system. At the midpoint of the buffering region, the concentration of the proton donor (acetic acid) exactly equals that of the proton acceptor (acetate). The buffering power of the system is maximal at pH equal to pKa. Though pH changes a little on addition of small amount of H+ or OH– but this change is negligible as compared to pH change in absence of buffer in the system. Let us understand the buffering action of acetate buffer system using the dissociation equilibria that exist in the aqueous solutions containing acetate buffer. Acetate buffer consists of acetic acid and sodium acetate as an example. In solution, acetic acid will dissociate as follows: CH3COOH (aq)

CH3COO – (aq) + H+(aq)

...(2.17)

Since acetic acid is a weak acid, the undissociated form of acetic acid will predominate. However, the sodium acetate salt will completely dissociate into ions. Therefore according

Water: The Unique Solvent 43

to Le Chatelier’s principle, that will swing the position of the equilibrium even further to the left. When acid is added to this buffer system, the added hydrogen ions combine with the acetate ions to make acetic acid. Although the reaction is reversible, since acetic acid is a weak acid, most of the new hydrogen ions are removed in this way and pH remains comparatively constant. CH3COO –(aq) + H+(aq)

CH3COOH (aq)

...(2.18)

When alkali (OH) ions are added to the buffer solutions, the added OH– ions are removed by two different processes. Some of the hydroxide ions react with acetate acid molecules to form acetate ions and water. Ionization of acetic acids leads to formation of hydrogen ions. These hydrogen ions react with hydroxide ions to form water. CH3COOH (aq) + OH–(aq) CH3COOH (aq)

CH3COO –(aq) + H2O (l) CHCOO –(aq) + H+(aq)

...(2.19) ...(2.20)

The equilibrium shifts more towards right to replace them. This keeps on happening until most of the hydroxide ions are removed. In this way, the pH of the buffered solutions is maintained. Equilibrium moves to replace the removed hydrogen ions.

 

–

+

CH3COO (aq) + H (aq)

Hydroxide ions combine with these to make water.

2.10

...(2.21)

PHYSIOLOGICAL BUFFER SYSTEMS

Biological processes are highly dependent on the pH even when hydrogen ion (H+) is not a direct participant in the reactions. Enzyme activity which is essential for all the metabolic reactions is highly pH dependent. They have an optimum pH at which their activity is at maxima. Enzymes lose their activity at pH values greatly different from the optimum. Amino acids bear acidic as well as basic groups hence they can act as weak acid or base depending on the pH of the surroundings. Ionic interactions of proteins are important for their structural stability and the binding of ligands. Therefore, in most higher organisms, pH is maintained constant to maintain the biomolecules in their optimal ionic state. In higher animals, plasma proteins, phosphate and bicarbonate act as physiological buffers that control the pH of body fluids and intracellular fractions. The excretory organs like

44 Textbook of Biochemistry

the kidneys also help in control of pH by excreting excess of hydrogen ions and generation of bicarbonate to maintain the pH. Protein buffer systems are mostly responsible for maintenance of intracellular pH (Figure 2.9).

Fig. 2.9

Physiological buffer systems. Buffers are present in intracellular fluid (ICF) as well as in extracellular fluid (ECF). They maintain the pH in the body.

Proteins are made up of amino acids which possess positively charged amino groups and negatively charged carboxyl groups. These charged groups can bind with hydrogen and hydroxyl ions, and thus function as buffers. Haemoglobin efficiently acts as buffer. It is the principal protein found in the red blood. During the conversion of CO2 into bicarbonate, hydrogen ions liberated in the reaction are buffered by haemoglobin, which is reduced by the dissociation of oxygen. Conversely, this process is reversed in the lungs where CO2 is released to be exhaled to the atmosphere. Phosphates are another prominent physiological buffer. Phosphate buffer system is made of sodium dihydrogen phosphate (Na2H2PO4−), a weak acid, and sodium monohydrogen phosphate (Na2HPO42–), which is a weak base. When Na2HPO42– reacts with a strong acid, such as HCl, weak acid Na2H2PO4− and sodium chloride, NaCl are formed. HCl + Na2HPO4 m NaH2PO4 + NaCl (strong acid) + (weak base) m (weak acid) + (salt)

...(2.22)

When Na2HPO42– encounters a strong base, such as sodium hydroxide (NaOH), it produces salt and water. Thus, phosphate buffer system is capable of resisting the pH change.

Water: The Unique Solvent 45

NaOH + NaH2PO4 m Na2HPO4 + H2O (strong base) + (weak acid) m (weak base) + (water)

...(2.23)

Mechanism of buffering of bicarbonate-carbonic acid buffer system is similar to phosphate buffers. The concentration of bicarbonate and phosphate ions in blood are regulated by sodium ions. The ratio of concentration of bicarbonate to carbonic acid in blood is about 20:1. This is because most of the metabolic wastes such as lactic acid and ketones are acidic in nature. Carbonic acid levels can be controlled by exhalation of CO2 through lungs. In contrast, the concentration of bicarbonate ions is controlled through the kidneys where the bicarbonate is retained and released back to blood. The respiratory system plays an important role in maintaining the acid-base balance in body by regulating the concentration of carbonic acid in blood (Figure 2.10). CO2 in the blood dissolves in water to form carbonic acid and equilibrium between CO2 and carbonic acid is established. If the concentration of CO2 increases, the excess CO2 makes carbonic acid and tends to lower the pH of blood. Increasing the exhalation rate can help to remove more of CO2 hence mitigate acidosis. This process can be reversed in case of alkalosis. Minor adjustments in rate of breathing are very effective in adjusting the pH of blood to normal levels. This situation is commonly encountered during strenuous exercise. The higher energy demand in muscles leads to formation of lactic acid as well as higher CO2 production. In order to balance the excess acid, the rate of respiration increases to remove the CO2 and maintain the pH. Chemoreceptors are present in the brain (medulla oblongata) as well as in the arteries that sense the level of CO2 in cerebrospinal fluid and in blood,

Fig. 2.10 Control of pH imbalance in body

46 Textbook of Biochemistry

respectively and they initiate the regulation of respiratory rate to control the levels of CO2. Hypercapnia or abnormally high levels of CO2 may result in impairment of respiratory systems like pneumonia and congestive heart failure. Moreover, drugs like morphine, barbiturates, or ethanol may also result in hypercapnia. Hypocapnia, or abnormally low blood levels of CO2, might be the result of salicylate toxicity or hyperventilation. Metabolic component of buffering is accomplished by the renal regulation. The renal system controls the blood levels of bicarbonate. Inhibition of carbonic anhydrase by certain diuretics or loss of bicarbonates due to extreme diarrhoea may result in decrease in bicarbonate levels. Bicarbonate levels are increased in some other conditions like renal damage and Addison’s disease wherein aldosterone levels are reduced and also due to ketosis which are common in unmanaged diabetes mellitus.

2.11









SUMMARY

s 7ATER MOLECULE CONSISTS OF TWO COVALENT BONDS FORMED BY SHARING OF ONE ELECTRON each from hydrogen and oxygen. The greater electronegativity of oxygen as compared to hydrogen creates two dipole moments in the direction of –OH bond. The sharing of electrons between hydrogen and oxygen is unequal, i.e., the hydrogen atoms bear a partial positive charge (E+) and oxygen atom bears a partial negative charge (2E–). s 7ATER MOLECULES HAVE SLIGHTLY POSITIVE AND SLIGHTLY NEGATIVE ENDS THEY CAN INTERACT with each other extensively forming hydrogen bonds. s 4HE UNUSUAL PROPERTIES OF WATER ARE DUE TO THE PRESENCE OF HYDROGEN BONDS !PART from hydrogen bonds, weak interactions like van der Waals and hydrophobic interactions have significant impact on the three-dimensional structure of biological macromolecules like proteins, nucleic acids, polysaccharides and membrane lipids. s 0OLARMOLECULESAREEASILYSOLUBLEINWATERASTHEYCANREPLACETHEWATER WATERINTERactions with energetically favourable water-solute interactions. However, nonpolar molecules are unable to form energetically favourable interactions with water and hence they are poorly insoluble in water. s )ONICCOMPOUNDSLIKESALTSAREEASILYSOLUBILIZEDBYWATERSINCEPOSITIVEANDNEGATIVE ions are stabilized by the partial negative and positive charges present in water. s 4HE COMPOUNDS THAT EASILY DISSOLVE IN WATER ARE KNOWN AS HYDROPHILIC WHEREAS compounds that cannot dissolve in water are known as hydrophobic. s P( IS THE NEGATIVE LOG OF HYDROGEN ION CONCENTRATION 4HUS NEUTRAL SOLUTION WHICH has got 1 × 107 hydrogen ions has a pH of 7. pH lower than 7 signifies acidic solution whereas pH greater than 7 signifies alkaline solution. s 4HE (ENDERSON (ASSELBALCH EQUATION IS [A–] pH = pKa + log_____ [HA] s PKa is the pH at which the number of molecules of conjugate base [A–] and weak acid [HA] are equal. Strong acids have lower pKa and strong bases have higher pKa.

Water: The Unique Solvent 47



The pKa can be determined experimentally by titrating against acid/base. pKa is the pH at the midpoint of the titration curve for the acid or base. s 4HETITRATIONCURVESOFWEAKACIDSANDBASESSHOWTHATWEAKACIDANDITSANIONˆA conjugate acid-base pair can resist small changes in pH due to addition of acids and bases. This property to resist pH change is known as buffering. Buffers are the chemical compounds that resist pH change in systems. A buffer system consists of a weak acid (the proton donor) and its conjugate base (the proton acceptor). s "IOLOGICAL PROCESSES ARE HIGHLY DEPENDENT ON THE P( EVEN WHEN HYDROGEN ION (+) is not the direct participant in the reactions. s 4HEREFORE IN MOST HIGHER ORGANISMS P( IS MAINTAINED CONSTANT TO MAINTAIN THE biomolecules in their optimal ionic state. In higher animals, plasma proteins, phosphate and bicarbonate act as physiological buffers that control the pH of body fluids and intracellular fractions.

MULTIPLE-CHOICE QUESTIONS 1. Water is a: (a) Polar solvent (b) Nonpolar solvent (c) Amphiphilic solvent (d) Nonpolar uncharged solvent 2. Polar molecules can easily dissolve in water because: (a) Polar molecules can easily form hydrogen bonds with water (b) Polar molecules can easily replace water-water interaction with more energetically favourable water-solute interactions (c) Water can replace the charge interactions in solutes (d) Water reacts with polar molecules 3. Anamolous behaviour of water is due to: (a) Covalent bonds in water (b) Ionization of water (c) Hydrogen bonding in water (d) Covalent bond angle in water 4. Which of the following statements is true? (a) Hydrogen is more electronegative as compared to oxygen. (b) Oxygen is more electronegative as compared to hydrogen. (c) Hydrogen and oxygen have equivalent electronegativity. (d) Hydrogen and oxygen both are not electronegative. 5. The bond energy of hydrogen bonds is: (a) 30 kJ/mol (b) 23 kJ/mol (c) 100 kJ/mol (d) 10 kJ/mol 6. Why is oil not soluble in water? (a) Water and oil are hydrophilic (b) Oil and water are hydrophobic (c) Oil is hydrophobic (d) Oil is hydrophilic 7. The aggregation of nonpolar molecules or groups in water is thermodynamically due to: (a) Increase in entropy of the nonpolar molecules when they associate (b) Decrease in enthalpy of the system

48 Textbook of Biochemistry (c) Increase in entropy of the water molecules (d) van der Waals forces among the nonpolar molecules or groups 8. What is the concentration of hydrogen ions in water? (a) 1 × 107 M (b) 1 × 10 –7 M (c) 1 × 1014 M (d) 1 × 10 –14 M 9. The equilibrium constant for dissociation of pure water is: (a) 1.0 × 1014 (b) 1.0 × 10 –14 (c) 1.0 × 10 –16 (d) 1.0 × 107 10. Why is water neutral? (a) pH of pure water is 7 (b) In pure water, the concentration of H+ and OH– ions is equal. (c) Water does not ionize. (d) Water does not contain any free H+ and OH– ions.

Answers 1. (a) 9. (b)

2. (b) 10. (b)

3. (c)

4. (b)

5. (b)

6. (c)

7. (c)

8. (b)

Short Answer Type Questions 1. 2. 3. 4. 5.

Why is water known as unique solvent? What causes unequal sharing of electrons between hydrogen and oxygen atoms in water? List some unique properties of water. Compare hydrogen bond and van der Waals forces. How do buffers maintain the pH?

Long Answer Type Questions 1. What is the pH when 20 ml of 0.1M NaOH are added to 100 ml of 0.1M lactic acid? The pKa of lactic acid is 3.85. 2. Aspirin (acetylsalicylic acid) has a pKa of 2.97. (a) Draw the structure and give the name of the conjugate base of aspirin. (b) Calculate the percentage of aspirin available for absorption in the stomach at (pH = 2.0) and in the duodenum at (pH = 4.5).

3. Find the pH of a 2 L solution containing 80 g of lactic acid (MW = 90.8 g/mol) and 120 g of sodium lactate (MW = 112.06 g/mol) and the Ka of lactic acid = 1.38 × 10 –4. Calculate the pH of a buffer system consisting of 0.25 M benzoic acid and 0.75 M benzoate (pKa = 4.2).

3 Carbohydrates 3.1

INTRODUCTION

Carbohydrates are commonly known as saccharides, defined as “staff of life”. They are the most abundant biomolecules having a series of compounds of carbon, hydrogen, and oxygen in which the atoms of the latter two elements are in the ratio of 2:1 (as in water). Its empirical formula is Cm (H2O) n (where m could be different from n). Carbohydrates are actually hydrates of carbon. They act as energy source, fuel, storage, metabolic product and intermediate, also as genetic material in the form of ribose and deoxyribose sugars (DNA and RNA). Plants utilize carbon dioxide and water (CO2 and H2O) and convert them into glucose, cellulose and other derivatives. Carbohydrates possess aldehyde or ketone groups with multiple hydroxyl groups. There are three major classes of carbohydrates: monosaccharides, oligosaccharides and polysaccharides. The word “saccharide” derives from the Greek sakcharon, meaning “sugar”. Monosaccharides are sugars, with single polyhydroxy aldehyde or ketone units. Six-carbon D-glucose sugar (dextrose) is the most abundant monosaccharide in nature. Monosaccharides of more than four carbons tend to have cyclic structures. Carbohydrates are actually monosaccharides, and contain three to nine carbon atoms. These monosaccharides may be linked together to form a large variety of oligosaccharide structures. Disaccharides are sugars having two monosaccharide units. Most abundant disaccharide is sucrose (cane sugar), consisting of the six-carbon sugars, D-glucose and D-fructose. All monosaccharides and disaccharides have names ending with the suffix “-ose.” Oligosaccharides are short chains of monosaccharide units, joined together by glycosidic bonds. The polysaccharides are sugar polymers containing more than 20 or so monosaccharide units, and some have hundreds or thousands of units. Some polysaccharides, such as cellulose are linear chains while glycogens are branched structures. Both glycogen and cellulose consist of repeating units of D-glucose, but they differ from each other by glycosidic bonds. Polysaccharides are the structural elements in the cell walls of bacteria and plants. Cellulose, the main constituent of plant cell walls, is one of the most abundant organic compounds in the biosphere. Glycoconjugates are carbohydrates linked to many proteins and lipids that help in cell-cell interaction. Simple classification of carbohydrates is explained below (Figure 3.1).

50 Textbook of Biochemistry

Fig. 3.1

3.2

Classification of carbohydrates

MONOSACCHARIDES AND DISACCHARIDES

Monosaccharides are simplest sugars having three to ten carbons. They may have aldehydes or ketones groups with two or more hydroxyl groups. Glucose and fructose are the six-carbon monosaccharides having five hydroxyl groups. Most of the hydroxyl groups are attached to the carbons having chiral centres (four different groups attached to single carbon) and it gives a specific characteristic to all monosaccharides, known as stereoisomers. The monosaccharide molecules are generally unbranched carbon chains and all the carbon atoms are linked together by single bonds. One of the carbon atoms is double-bonded to an oxygen atom to form a carbonyl group while other carbon atom has a hydroxyl group. If the aldehyde (carbonyl) group is at an end of the carbon chain, the monosaccharide is known as aldose and if the ketone (carbonyl) group is at any other position the monosaccharide is a ketose. Simplest monosaccharides are the three-carbon trioses: glyceraldehyde, an aldotriose, and dihydroxyacetone, a ketotriose (Figure 3.2(a)). Monosaccharides with four, five, six and seven carbon atoms in their backbones are respectively, tetroses, pentoses, hexoses, and heptoses (Figure 3.2(a) and (b)). There are aldoses and ketoses of each of these chain lengths: aldotetroses and ketotetroses, aldopentoses and ketopentoses, and so on (Figure 3.3(a)). The hexoses, which include the aldohexose D-glucose and the ketohexose D-fructose (Figure 3.3(b)), are the most common monosaccharides in nature. Aldopentoses D-ribose and 2-deoxy-D-ribose (Figure 3.3(c)) are the main components of nucleotides and nucleic acids.

Carbohydrates 51

3.3

NOMENCLATURE AND CLASSIFICATION OF MONOSACCHARIDES

Monosaccharides are the simplest carbohydrates and cannot be hydrolyzed into smaller carBOHYDRATES 4HE GENERAL CHEMICAL FORMULA OF AN UNMODIFIED MONOSACCHARIDE IS #s(2O) n. Monosaccharides are important biological fuel molecules and are building blocks for nucleic acids. The smallest monosaccharides, for which n=3, are dihydroxyacetone and D- and L-glyceraldehydes.

3.3.1

How to Name Acyclic Monosaccharides

Acyclic monosaccharides have three different characteristics: 1. Number of carbon atoms it contains 2. Its D or L configuration 3. Placement of its carbonyl group (aldehyde or ketone). Three carbon monosaccharides are called trioses, four carbons are called tetroses, five carbons are called pentoses, six carbons are hexoses and so on. The number of carbon atoms in a molecule is used in the suffix of carbohydrate naming (Figure 3.4(a)).

Fig. Contd.

52 Textbook of Biochemistry Fig. Contd.

Fig. 3.2 Aldoses and ketoses. The series of (a) D-aldoses and (b) D-ketoses have three to six carbon atoms. The carbon atoms in pink are chiral centres. In all these D isomers, the chiral carbon most distant from the carbonyl carbon has the same configuration as the chiral carbon in D-glyceraldehyde.

Depending upon the position of carbonyl group (aldehyde or ketone) monosaccharides may be aldose or ketose (Figure 3.4(b)).

Carbohydrates

53

Fig. 3.3 Basic monosaccharides: (a) Two trioses, an aldose and a ketose. The carbonyl group in each is with pink colour, (b) Two common hexoses, (c) The pentose components of nucleic acids D-Ribose is a component of ribonucleic acid (RNA) and 2-deoxy-D-ribose is a component of deoxyribonucleic acid (DNA).

Fig. 3.4 (a)

Number of carbon atoms in a chain

54 Textbook of Biochemistry

Fig. 3.4 (b) Type of group (aldehyde or ketone) present in the chain

D or L letters are usually put in the beginning of a carbohydrate when naming the molecule. A monosaccharide is given D configuration if the hydroxyl group is to the right side of the last stereocentre carbon, whereas L configuration is given if the OH is to the left of the last stereocentre carbon (Figure 3.4 (c)). By putting all the concepts together, the final name of the carbohydrate is obtained (Figure 3.4 (d) and (e)).

Fig. 3.4 (c) Left- and right-handed configuration

Fig. 3.4 (d)

3.3.2

Final carbohydrate by combining all the rules

How to Name Cyclic Monosaccharide

Cyclic monosaccharides form a ring-like structure and are mostly 5 or 6 carbons (Figure 3.5). Five-carbon rings have the suffix fruscose and six carbon rings have the suffix

Carbohydrates 55

Fig. 3.4 (e)

Example showing method of naming chain carbohydrates

pyranose. Also, when acyclic molecules are turned into cyclic molecules, it creates a chiral centre at the anomeric carbon (chiral carbon) and this gives rise to B and C anomers. B means that the anomeric OH and CH2OH groups are trans whereas B means anomeric OH and CH2OH groups are cis. These anomers are placed in the beginning in naming cyclic carbohydrates.

Fig. 3.5

3.4

Nomenclature of cyclic carbohydrates

PHYSICAL PROPERTIES OF MONOSACCHARIDES

All monosaccharides are crystalline and sweet in taste, easily dissolve in water but are insoluble in nonpolar solvents. They can cross the plasma membrane. The water solutions of monosaccharides can rotate the angles of polarized light so, monosaccharides are optically active. Angle of rotation depends upon the type of chemicals, solvents, concentration of the chemical in water and temperature. If a solution rotates the plane of light clockwise, it is known as dextrorotatory. Alphabet, “D”, or (+) would be put before its name (Figure

56 Textbook of Biochemistry

3.6(a)) and if it rotates anticlockwise it is levorotatory, and alphabet “L” or (–) would be there before its name (Figure 3.6(b)). All carbohydrates that can be assimilated by living things are dextrorotatory. All amino acids utilized by living things are levorotatory.

Fig. 3.6 Forms of glyceraldehydes showing left- (a) and right-handed (b) conformations

3.5

CHEMICAL PROPERTIES OF MONOSACCHARIDES

Asymmetric Carbon: All monosaccharides have asymmetric carbon, i.e., the valencey of carbon is satisfied by four different groups attached at four sides (Figure 3.7(a)) and this carbon is known as chiral carbon. If any two groups are identical, it is not chiral. All monosaccharides have one or more asymmetric carbons except dihydroxyacetone, due to this all monosaccharides show optical activity and stereoisomerism (enantiomers). Compounds can have more Fig. 3.7 (a) A chiral carbon than one chiral carbon (Figure 3.7(b)).

Fig. 3.7 (b) Compound with more than one chiral carbon

3.6

ISOMERISM IN MONOSACCHARIDES

Isomers are compounds with the same molecular formulae but different structural formulae. There are two types of isomerism: structural isomerism and stereoisomerism. Structural isomerism is defined as isomers having identical molecular formulas but the order of connection of atoms varies. In stereoisomerism, the isomers have same molecular and structural formulas but differ in the spatial arrangement of the atoms in the molecule. The number of stereoisomers depends upon the number of chiral carbons present in any compound. The maximum number of stereoisomers is 2n, where n = number of chiral carbon atoms. Therefore, this compound with two chiral carbon atoms has 22 or 4 stereoisomers. The compound on the previous slide with four chiral carbon atoms has 24 or 16 stereoisomers.

Carbohydrates 57

For example, molecular model of CHClBrI molecule can be prepared by two methods. The first model attaches four different atoms (H, Cl, Br, and I) to the central carbon. The second model will be the “mirror image” of the first model. The two models are nonsuperimposable, that is, all atoms cannot be aligned at the same time (Figure 3.8). These two models are related to each other in a way similar to your right hand and left hand. A right hand is the nonsuperimposable mirror image of a left hand. A molecule that is nonsuperimposable on its mirror image is said to be chiral (pronounced “ki-ral”, from Greek Fig. 3.8 Nonsuperimposable word cheir, meaning “hand”). mirror image of CHClBrI The Nobel Prize winner German chemist, Emil Fischer devised a simple method for representating of three-dimensional molecule by using two-dimensional drawing and this representation is known as Fischer projection (Figure 3.9(a)). When drawing Fischer projections, the molecule is drawn in the form of a cross with the chiral carbon centred at the point of intersection. The horizontal lines represent bonds directed outward towards the observer and the vertical lines are bonds pointing away. We can draw Fischer projection for CHClBrI molecule as:

Fig. 3.9 (a)

Glyceraldehyde is the smallest monosaccharide that contains a chiral carbon

The two stereoisomers that are nonsuperimposable mirror images of one another are called a pair of enantiomers (Greek: enantios + meros, opposite + part). In detail, our right and left hands are considered a pair of enantiomers (Figure 3.9(b)). Pairs of stereoisomers that are not mirror images of each other are called diastereomers (Figure 3.9(b)). The two isomers are identified using Fischer projections and differentiated using the prefixes D and L. By convention, the carbon chain is written vertically with the carbonyl group (most oxidized carbon) at the top. Identify the carbon containing four different groups (chiral carbon). It is the middle carbon in this case, if the –OH group is on the left, the

58 Textbook of Biochemistry

Fig. 3.9 (b)

Enantiomers of glyceraldehyde

isomer is assigned “L-” (remember L for left), if on the right, it is assigned “D-”. L- is from the Latin, levo meaning left and D- is from the Latin, dextro meaning right. The D and L symbols are typically typeset in small caps (D- and L-). If there are two or more chiral carbons in the molecule, the chiral carbon most distant from the carbonyl group determines the D- or L- isomers. This will always be the second to the last carbon in the Fischer projection. Therefore, when assigning D- or L- to more complex isomers, focus only on the second to the last carbon.

3.7

HAWORTH STRUCTURES: CYCLIC STRUCTURE OF MONOSACCHARIDES

English chemist, Sir Walter N. Haworth has given a method of representation of cyclic structure of monosaccharides and it is known as Haworth projections. Open-chain form of D-glucose can be converted into cyclic form through a ring closure reaction which involves the OH group on carbon five and the C-1 carbonyl carbon (Figure 3.10). The carbonyl group at C-1 reacts with the hydroxyl group at C-5 to give a six-membered ring structure containing oxygen. The newly formed ring is a rigid structure that has two planes, one above the ring, the other below. When the ring closes, the C-5 OH loses the hydrogen and the oxygen is incorporated into the ring. The C-1 carbonyl carbon loses its double bond to oxygen and becomes a single bond to OH. The newly formed OH at C-1 (formerly the carbonyl carbon) can be drawn above or below the ring producing two different isomers. These two isomers are called anomers, namely, B (alpha) and C (beta) anomers. In the B anomer, the C-1 hydroxyl group is below the ring, in the C anomer, the C-1 hydroxyl group is above the ring. Isomeric forms of monosaccharides that differ only in their configuration about the hemiacetal or hemiketal carbon atom are called anomers. The hemiacetal (or

Carbohydrates 59

carbonyl) carbon atom is called the anomeric carbon. The B and C anomers of D-glucose interconvert in aqueous solution by a process called mutarotation. The B and C anomers are diasteriomers of each other and usually have different specific rotation.

Fig. 3.10

Howarth structures and anomers of glucose

Diastereomerism occurs when two or more stereoisomers of a compound have different configurations at one or more (but not all) of the equivalent (related) stereocentres and are not mirror images of each other. When two diastereoisomers differ from each other at only one stereocentre, they are epimers. Two sugars that differ only in the configuration around one carbon atom are called epimers; D-glucose and D-mannose, which differ only in the stereochemistry at C-2, are epimers, as are D-glucose and D-galactose (which differ at C-4) (Figure. 3.11).

Fig. 3.11

Epimers: D-glucose and its two epimers, differ from each other in the configuration of one chiral carbon (shaded)

60 Textbook of Biochemistry

3.8

MONOSACCHARIDES AS REDUCING AGENTS

Monosaccharides such as glucose and fructose are crystalline solids at room temperature, but they are quite soluble in water, each molecule having several OH groups that readily engage in hydrogen bonding. The chemical behaviour of these monosaccharides is likewise determined by their functional groups. An important reaction of monosaccharides is that the aldehydes group can be oxidized to carboxylic acids by using mild oxidizing reagents like Tollens’ or Benedict’s reagents, while ketoses do not. Tollens’ and Benedict’s reagents are basic solutions, while some ketones rearrange to aldoses which do oxidize. For example, fructose (ketose) will rearrange into an aldose when treated with a basic Benedict’s solution. Any carbohydrate (sugar) that undergoes reactions without first undergoing hydrolysis that can reduce the Benedict’s reagent is called the reducing sugar. In a positive Benedict’s test, the blue colour of the solutions turns greenish, and a dark red, orange, brown, or yellow precipitate forms. Benedict’s test requires heating the test solutions in the boiling water for several minutes (Figure 3.12). General reaction: Reducing sugar + copper(II) oxidized compound + copper(I) oxide (blue solution) (red-orange precipitate). All monosaccharides and all common disaccharides (except sucrose) are reducing sugars.

Fig. 3.12

3.9

Reaction showing how carbohydrate interacts with Tollen’s and Benedict’s reagents and showing carbohydrates as reducing agent

BIOLOGICALLY IMPORTANT DISACCHARIDES

Two monosaccharides react to form a disaccharide molecule and loss of water molecule occurs, this reaction is dehydration reaction (loss of water) (Figure 3.13(a)). Disaccharides are joined together through an “oxygen bridge”. The carbon-oxygen bonds are called glycosidic bonds. The most common disaccharides are sucrose, lactose and maltose (Figure 3.13(b)).

Carbohydrates 61

Fig. 3.13 (a)

3.9.1

Disaccharide formation dehydration reaction (b) Monosaccharide unit in common disaccharides

Sucrose

Sucrose, commonly known as table sugar, is the most abundant disaccharide in the biological system. Sucrose contains an B-D-glucose unit and a C-D-fructose unit joined by B, C(1 m 2) glycosidic linkage. Sucrose is a nonreducing sugar because its glycosidic bond involves both anomeric carbons, therefore there is no free aldehyde group (Figure 3.14 (a)).

62 Textbook of Biochemistry

3.9.2

Lactose

Lactose (milk sugar) is made up of a C-D-galactose unit and a D-glucose unit joined by a C(1 m 4) glycosidic linkage (Figure 3.14(b)). Lactose is reducing sugar (glucose ring on the right has a free anomeric carbon that can open to give an aldehyde).

3.9.3

Maltose

Maltose (malt sugar) is made up of two D-glucose units. The glycosidic linkage between two glucose units is an (1 m 4) linkage (Figure 3.14(c)). Maltose is a reducing sugar (glucose ring on the right can open to give an aldehyde).

Fig. 3.14

3.10

Disaccharide formation and types of bonds in disaccharides: (a) Sucrose (b) Lactose (c) Maltose

OLIGOSACCHARIDES

The word oligosaccharide is derived from Greek olígos, “a few”, and sácchar, “sugar” is a saccharide polymer containing a small number typically two to ten of simple sugars (monosaccharides). Oligosaccharides can have many functions including cell recognition and cell binding and in immune response. Generally, they are attached to either N- or O-linked to compatible amino acid side chains in proteins or to lipid moieties. N-linked oligosaccharides are found attached to asparagine through beta linkage to the nitrogen of amine. Alternately, O-linked oligosaccharides are generally attached to threonine or serine on the alcohol group of the side chain.

Carbohydrates 63

3.10.1

N-linked Oligosaccharides

Fig. 3.15 (a) N-linked oligosaccharide, with GlcNAc

The process of N-linked glycosylation occurs cotranslationally, or concurrently while the protein is being translated. Since it is added cotranslationally, its N-linked glycosylation helps to determine the folding of polypeptides due to the hydrophilic nature of sugars. They are small carbohydrates which are formed by condensation of 2-9 monosacchrides. This asparagine has increased nucleophilicity in the amide group (Figure 3.15(a)). The unique arrangement of N-linked oligosaccharides usually has the oligosaccharide linked to the amide nitrogen of the Asn residue, in the sequence Asn-X-Ser/Thr. X can be any amino acid except for proline (though it is rare to see Asp, Glu, Leu, or Trp).

3.10.2

O-linked Oligosaccharides

Fig. 3.15 (b) O-linked oligosaccharide, with GlcNAc

64 Textbook of Biochemistry

Oligosaccharides that participate in O-linked glycosylation are attached to threonine or serine on the alcohol group of the side chain. O-linked glycosylation occurs in the Golgi apparatus, in which monosaccharide units are added to a complete polypeptide chain. Cell surface proteins and extracellular proteins are O-glycosylated. Glycosylation sites in O-linked oligosaccharides are specified only in the secondary and tertiary structures of the polypeptide, which will dictate where glycosyltransferases will add sugars (Figure 3.15(b)). Both glycoproteins and glycolipids have a covalently attached carbohydrate attached to their respective molecules. They are abundant on the surface of the cell, and their interactions contribute to the overall stability of the cell.

Oligosaccharide and its role in blood group determination Carbohydrate is very important as it is used for the classification of blood group. The blood groups are classified on the basis of antigen found at surface of red blood cells (RBC), these antigens are simple chains of sugars (oligosaccharides). Types of oligosaccharide attached at the surface of RBC determines the person’s blood group (Table below). Table Types of blood groups due to different oligosachrides attached at RBC. S. No.

3.11

Antigen

Blood group

Can receive blood

1.

O type

O

O

2.

A type

A

A, O

3.

B type

or B

B, O

4.

AB type

AB

A, B, O

POLYSACCHARIDES

A polymer is a large molecule composed of many small, repeating structural units that are identical. The repeating structural units are called monomers. Most of the carbohydrates in nature are found as polysaccharides. Polysaccharides are carbohydrates formed by more than nine monosaccharide units linked by glycosidic bonds. They are polymers of high molecular weight. Polysaccharides, also called glycans, differ from each other by means of monosaccharide units, length of their chains, types of bonds linking the units, and in the branching pattern. For the synthesis of polysaccharides, there is no template required (different from protein synthesis) but it depends upon enzymatic action that catalyzes the polymerization of monomeric units. For each type of monosaccharide to be added to the growing polymer there is a separate enzyme, and each enzyme acts only when the enzyme that inserts the preceding subunit has acted. The alternating action of several enzymes produces a polymer with a precisely repeating sequence, but the exact length varies from molecule to molecule, within a general size class. Depending upon the monomeric units, polysaccharide is homoor heteropolysaccharide. Homopolysaccharides have single type of monomeric unit while heteropolysaccharides contain two or more kinds of monomeric units (Figure 3.16 (a), (b),

Carbohydrates 65

(c), (d)). Bacterial cell envelope (the peptidoglycan) is a heteropolysaccharide. The rigid layer built from two alternating monosaccharide units. While in animal tissues, extracellular space is occupied by different types of heteropolysaccharides.

Fig. 3.16

3.11.1

Different types of polymerization (a, b) Homopolymers unbranched and branched (c, d); Heterpolymers branched and unbranched

Homopolysaccharides

In animal and plant kingdom, homopolysaccharides play a vital role. They may act as storage molecules as well as structural molecules. Storage forms of monosaccharides are used as fuels like starch and glycogen. Cellulose and chitin are structural homopolysaccharides and are found in plant cell walls and animal exoskeletons, respectively. Three very important polysaccharides are starch, glycogen, and cellulose and all contain only glucose units. They are differentiated on the basis of type of glycosidic bonds and the degree of branching in the molecule. 1. Starch: It is the primary storage polysaccharide in the plants and the most common forms of starch is amylose and amylopectin. Starch molecules are heavily hydrated because they have many exposed hydroxyl groups easily available to form hydrogen bonds with water. Starch is a tightly coiled helical structure stabilized by hydrogen bonds. Starch contains two types of glucose polymers, amylose and amylopectin. The former consists of long, unbranched chains of D-glucose units connected by B(1 m 4) linkages (Figure 3.17(a)). Most of the starch contains 20 to 25% amylose and 75 to 80% amylopectins. It completely hydrolyses into amylose and amylopectins and produces only D-glucose. The glycosidic

66 Textbook of Biochemistry

linkages joining successive glucose residues in amylopectin chains are B(1 m 4), but the branch points, occurring every 24 to 30 residues, are B(1 m 6) linkages. An amylose molecule is a continuous chains of D-glucose units joined by B-1,4-glycosidic linkage. There are no branch points in the molecule which may contain as many as 4000 D-glucose units. While amylopectin is a branched-chain polysaccharide larger than amylose. Amylopectin molecules, on the average, consist of several thousand -D-glucose units joined by -1,4glycosidic linkage (Figure 3.17(b)). The molecular masses routinely approach 1 million or more. Wheat, rice, corn and potatoes are the important sources of starch.

Fig. 3.17

(a) Amylase and (b) Amylopectin

Starch and glycogen are nonreducing Carbon which contains a free aldehyde or ketone group is known as reducing and which does not have free aldehyde or ketone group is nonreducing in nature. For example, glycogen has B 1-4 linkage and B
1-6 linkage in branches. It is not having any terminal free aldehyde or ketone group, thus it is nonreducing. Similarly, starch is not having free aldehyde or ketone terminal groups, thus it is also nonreduicng in nature. Even sucrose is a disaccharide of glucose and fructose and it does not have any free group (aldehyde, ketone) carbon, therefore it is nonreducing in nature.

Carbohydrates 67

2. Glycogen: Glycogen is the main glucose storage molecule in animal cells. Structurally, glycogen is very similar to amylopectin (-D-glucose units joined by -1,4-glycosidic linkages), but glycogen is a highly branched structure. Similar to starch, it is highly hydrated as large number of exposed hydroxyl groups are present to form hydrogen bonding. Glycogen occurs intracellularly as large clusters or granules. Glycogen is a polymer of B(1 m
4)-linked subunits of glucose, with B(1 m
6)-linked branches and it is a compact structure. Like starch, glycogen is a tightly bound structure stabilized by hydrogen bonding. Glycogen is abundant in the liver and also found in skeletal muscle. In liver cells glycogen is found in the form of large granules, which are themselves clusters of smaller granules composed of single, highly branched glycogen molecules with an average molecular weight of several million.

Why not store glucose in its monomeric form Glucose is highly water soluble and it easily mixes in the cell and increases the osmotic potential of the cell. Glycogen is not water soluble and it will not upset the osmotic potential of cells if stored. Glucose increases hypertonicity of the cell and which causes cell lysis. Glycogen has B(1 m
4) and B(1 m
6) bonds, which are not easily degraded while glucose is a simple monomer and highly unstable structure. So the liver and skeletal muscle contain glycogen as stored food, it is essentially in soluble form and contributes very little to the osmotic strength of the cytosol.

3. Cellulose: Cellulose is the most abundant organic molecule found in nature. Cellulose is a fibrous, tough, water-insoluble substance, found in the cell walls of plants. Structurally, it is a long, unbranched, D-glucose polymer in which the glucose units are linked by C (1 m 4) glycosidic bonds (Figure 3.18). Because cellulose is a linear, unbranched homopolysaccharide of 10,000 to 15,000 D-glucose units, linked by C(1 m
4) glycosidic bonds. In cellulose, the glucose residues have the C configuration (Figure 3.18), Due to this, cellulose has very different three-dimensional structures and physical properties.

Fig. 3.18

Structure of cellulose

68 Textbook of Biochemistry

Why cellulose is not digested by humans Cellulose is considered a fibre molecule in our diet and it is not digested in contrast to glycogen, amylose, and amylopectin. This is because our digestive system does not have the enzymes to catalyze the hydrolysis of C-glycosidic bonds. Glycogen and starch ingested in the diet are hydrolyzed by B-amylases, enzymes in saliva and intestinal juice that break C(1 m
4) glycosidic bonds between glucose units. Cellulose cannot be used by most animals as a source of stored fuel, because the B(1 m
4) linkages of cellulose are not hydrolyzed by B-amylases. Termites readily digest cellulose (and therefore wood), but only because their intestinal tract harbours a symbiotic microorganism, Trichonympha, which secretes cellulase, an enzyme that hydrolyzes C(1 m
4) linkages between glucose units. Wood-rot fungi and bacteria also produce cellulase. The only vertebrates able to use cellulose as food are cattle and other ruminant animals (sheep, goats, camels, giraffes). The extra stomachs (rumens) of these animals team with bacteria and protists that secrete cellulase.

4. Chitin: It is the second most abundant polysaccharide. It is a linear homopolysaccharide composed of N-acetyl-D-glucosamine residues in C linkage (Figure 3.19). It is almost similar to cellulose but the only difference from cellulose is the replacement of a hydroxyl group at C-2 with an acetylated amino group. Chitin forms extended fibres and found commonly in vertebrate animals. Chitin is the principal component of the hard exoskeletons of nearly a million species of arthropods–insects, lobsters, and crabs, for example and is probably the second most abundant polysaccharide, next to cellulose, in nature.

Fig. 3.19

3.11.2

Structure of chitin

Heteropolysacchrides: Bacterial Cell Wall Contains a Heteropolysaccharide

The bacterial cell wall has an important component which gives rigidity to the cell and it is a heteropolymer of alternating C(1 m
4)-linked N-acetylglucosamine and N-acetylmuramic acid units linked by glycosidic linkage. These linear polymers lie side by side in the cell wall in many numbers and are crosslinked by short peptides. Its number depends on the bacterial species. Lysozyme is the enzyme which degrades these cross linked polymers and it hydrolyzes the glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid and it kills bacterial cells. Lysozyme is present in tears, presumably a defense against bacterial infections of the eyes. It is also produced by certain bacterial viruses to ensure their release from the host bacteria, an essential step of the viral infection cycle.

Carbohydrates 69

1. Glycosaminoglycans: It is a heteropolysaccharide and a linear polymer composed of repeating disaccharide units. One of the two monosaccharides is always either N-acetylglucosamine or N-acetylgalactosamine, the other is in most cases a uronic acid, usually glucuronic acid. When glycosaminoglycan chains are attached to a protein molecule, the compound is known as “proteoglycans”. Some of the examples are hyaluronic acid, chondroitin sulphate and heparin. (a) Hyaluronic acid: Hyaluronic acid is a disaccharide containing repeating units of N-acetylglucosamine and D-glucuronic acid linked together by C-1,3 linkage (Figure 3.20(a)). These heteropolysacchrides are clear, highly viscous solutions and serve as lubricants in the synovial fluids of the joints and provide a cushioning effect.

Hyaluronidase Hyaluronidase is an enzyme secreted by some pathogenic bacteria that hydrolyzes the glycosidic linkages of hyaluronate, and it causes easy invasion by the bacteria into cells. A similar enzyme found in sperm can hydrolyze an outer glycosaminoglycan coat found around the ovum, allowing sperm penetration.

(b) Chondroitin sulphate: It is a polysaccharide found in the cartilage and contains alternating units of D-glucuronic acid and N-acetyl-D-galactosamine linked by C-1,3 linkage (Figure 3.20(b)). The negatively charged groups at Carbon-6 of

70 Textbook of Biochemistry

D-glucuronic acid (carboxyl group) and the sulphate ester group at Carbon-4 of N-acetylgalactosamine is found. Due to presence of these groups, it repels each other and causes expansion of polysaccharides molecule. Similar to starch molecule, they are highly hydrated and form a gelatinous matrix, when placed in water it acts as a lubricant. (c) Heparin: It is made up of D-glucuronate sulphate/L-idurunote sulphate and N-sulphoglucosamine –6-sulphate linked by B (1 m 4) glycosidic bonds (Figure 3.20(c)). Almost 90% of uronic acids are iduronic acids. It is present in the liver, lungs, spleen, monocytes, etc. Commercially, heparin is prepared from lung tissues. It act as an anti-coagulant agent widely used for clinical studies. It is also used in vivo to prevent intravascular coagulation.

(d) Dermatan sulphate: It is alternating unit of L-iduronic acid and N-acetyl galactosamine–4–sulphate linked by C-1,3 linkage and repeating disaccharide by C-1,4 linkage (Figure 3.20(d)). It is present in the skin, blood vessels and heart valves.

(e) Keratan sulphate: It is a heterogeneous GAG with variable sulphate content. It is made up of alternating units of D-galactosamine and N-acetylglucosamine linked by C-1,4 linkage (Figure 3.20(e)).

Carbohydrates 71

(e) Fig. 3.20

Different heteropolysaccharides: (a) Hyaluronic acid (b) Chondroitin sulphate (c) Heparin (d) Dermatan sulphate and (e) Keratin sulphate

Glycosaminoglycans and proteoglycans are components of the extracellular matrix Extracellular space in animal tissues are filled with a gelly-like material known as ground substance. It holds the cells of a tissue together and provides a porous pathway for the diffusion of nutrients and oxygen to individual cells. The extracellular matrix is composed of an interlocking meshwork of heteropolysaccharides and fibrous proteins. Glycosaminoglycans and proteoglycans are the main components of the extracellular matrix.

3.12

GLYCOCONJUGATES

Membrane proteins and few other classes of membrane lipids are complex and are covalently attached to oligosaccharides, these are glycoproteins and glycolipids.

3.12.1

Glycoproteins

Eukaryotic cells secrete glycoproteins. Glycoproteins have specific oligosaccharides, which are responsible for the particular functions of glycoproteins like antigenicity, solubility and resistance to proteases. Most inportanat contribution of glycoproteins in cell is cellsurface receptors, cell-cell adhesion molecules, immunoglobulins, and tumour antigens. Particularly attached oligosaccharides to newly synthesized protein determines the target of intracellular cell organelles, and its receptor molecules are present at the other surface (attachment point).

Biological Function of Glycoprotein The carbohydrate chains covalently attached to glycoproteins are generally oligosaccharides. The carbohydrate portion commonly constitutes from 1% to about 70% of a glycoprotein by

72 Textbook of Biochemistry

weight, while it is 99% in the case of proteoglycans. Glycoproteins may have only one or a few carbohydrate groups. These oligosaccharide side chains may be linear or branched. Most of the plasma membrane proteins are glycoproteins, with their oligosaccharide moieties located on the exposed surface of the membrane.

Glycophorins These are membrane glycoproteins of the erythrocyte membrane. It contains 16 oligosaccharide chains which is 60% of carbohydrate by weight and it attaches covalently to residues near the amino terminus of the polypeptide chain. It is a membrane-spanning protein and carries sugar molecules. Glycophorins are rich in sialic acid, which gives the red blood cells a very hydrophilic-charged coat. This enables them to circulate without adhering to other cells or vessel walls.

3.12.2

Glycolpids

Glycolipids are lipid molecules bound to oligosaccharides and found in lipid bilayer. It helps in cell recognition, and act as receptors for membrane proteins. Additionally, they can serve as receptors for cellular recognition and cell signalling. The head of the oligosaccharide acts as a binding partner in receptor activity. Composition of the oligosaccharide sugars exposed at the surface actually help in the binding mechanisms of receptors. There is great biodiversity in the binding mechanisms of glycolipids, which is what makes them such a target for pathogens as a site for interaction and entrance.

Glycolipids and lipopolysaccharides are membrane components A few lipid molecules are also attached to oligosaccharides through covalent bonds. Lipopolysaccharides are the major components of the outer membrane of Gram-negative bacteria such as E. coli and Salmonella typhimurium and it is one of the most important features of Gram-negative bacteria; they are prime targets of the antibodies produced by the immune system in response to bacterial infection. The lipid portion of the lipopolysaccharide of some bacteria is toxic to humans and other animals.

Carbohydrates 73

Cell Recognition Glycoproteins or glycolipids are found in almost all cell surfaces and they decide the type of cell. Most importantly, blood group differentiation is only because of the type of glycan attached at the surface of blood cells. These can be visualized using mass spectrometry.

Cell Adhesion Lectins are specific carbohydrate-binding ligands, which mediate cell adhesion with oligosaccharides. Selectins is a family of lectins which helps to mediate certain cell-cell adhesions, including those of leukocytes to endothelial cells. In an immune response, endothelial cells can express certain selectins transiently in response to damage or injury to the cells. In response, a reciprocal selectin-oligosaccharide interaction will occur between the two molecules which allow the white blood cell to help eliminate the infection or damage. Protein-carbohydrate bonding is often mediated by hydrogen bonding and van der Waals forces.

3.13 TESTS FOR CARBOHYDRATES 1. Molisch’s test: Molisch’s reagent is 10% alcoholic solution of B-naphthol. This is a common chemical test to detect the presence of carbohydrates. Carbohydrates undergo dehydration by sulphuric acid to form furfural (furfuraldehyde) that reacts with B-naphthol to form a violet coloured product. 2. Fehling’s test: This is an important test to detect the presence of reducing sugars. Fehling’s solution-A is copper sulphate solution and Fehling’s solution-B is potassium sodium tartrate. On heating, carbohydrate reduces deep blue solution of copper (II) ions to red precipitate of insoluble copper oxide.

3. Benedict’s test: Benedict’s test distinguishes reducing sugar from nonreducing sugar. Benedict’s reagent contains blue copper (II) ions (Cu2+, cupric ions) that are reduced to copper (I) ions (Cu+, cuprous ions) by carbohydrates. These ions form precipitate as red colour cuprous copper (I) oxide. CuSO4 m Cu2+SO42– Cu2++ Reducing sugar m Cu+ Cu+ m Cu2O

74 Textbook of Biochemistry

4. Tollen’s test: Tollen’s reagent is ammoniacal silver nitrate solution. On reacting with carbohydrate elemental silver precipitates out of the solution, occasionally onto the inner surface of the reaction vessel. This produces silver mirror on the inner wall of the reaction vessel. AgNO3 + NH4OH m NH4NO3 + AgO 2AgOH m Ag2O + H2O Ag2O + 2NH4OH m 2[Ag (NH3)2]OH + 3H2O (Soluble)

5. Iodine test: Iodine test is used to detect the presence of starch. Iodine is not much soluble in water so iodine solution is prepared by dissolving iodine in water in the presence of potassium iodide. Iodine dissolved in an aqueous solution of potassium iodide reacts with starch to form a starch/iodine complex which gives characteristic blue-black colour to the reaction mixture (Figure 3.21).

Fig. 3.21

3.14

Starch molecule and its reaction with iodine.

SUMMARY

s 3UGARS ALSO KNOWN AS SACCHARIDES CONTAIN ALDEHYDE OR KETONE GROUPS WITH TWO OR more hydroxyl groups. s -ONOSACCHARIDESSHOWSTEREOCHEMICALPROPERTIESDUETOTHEPRESENCEOFONEORMORE chiral carbons.

Carbohydrates 75







s -ONOSACCHARIDES ARE COMMONLY FOUND AS UNBRANCHED OR CYLIC STRUCTURES #YCLIC structures are represented as a Haworth perspective formula. s 3TEREOISOMERISM MUTAROTATION AND EPIMER FORMATION ARE THE IMPORTANT FEATURES OF monosaccharides. s $IFFERENT MONOMERS JOIN TOGETHER THROUGH GLYCOSIDIC BONDS FORMING DISACCHARIDES s /LIGOSACCHARIDESARESHORTPOLYMERSOFSEVERALMONOSACCHARIDESJOINEDBYGLYCOSIDIC bonds. s -ONOMERS JOIN TOGETHER TO FORM POLYMERS AND ARE HOMOPOLYSACCHARIDES OR heteropolysacchrides. s 3TARCH AND GLYCOGEN ARE IMPORTANT RESERVE FOOD AND ARE HOMOPOLYSACCHRIDE s 4HE HOMOPOLYSACCHARIDES CELLULOSE AND CHITIN ARE STRUCTURAL UNITS s #ELLULOSE IS THE MOST ABUNDANT POLYSACCHARIDE IN THE BIOSPHERE

MULTIPLE-CHOICE QUESTIONS 1. Fructose is metabolized by: (a) Fructose 1-phosphate pathway (b) Fructose 6-phosphate pathway (c) Glyceraldehyde 3-phosphate pathway (d) Both (a) and (b) 2 Cells capture the energy released during the breakdown of large molecules and it is accepted by small molecules and this reaction is known as: (a) Biosynthesis (b) Metabolism (c) Reduction (d) Catalysis 3 How many ATP equivalents per mole of glucose input are required for gluconeogenesis? (a) 2 (b) 6 (c) 8 (d) 4 4. The main site for gluconeogenesis is the: (a) Kidney (b) Liver (c) Brain (d) Muscle 5. Cellulose fibres resemble the protein structure in the form of: (a) C-sheets (b) C-helices (c) B-turns (d) None of these 6. B-amylose is similar to: (a) C-sheets (b) C-turned coils (c) B-helices (d) Hydrophobic core 7. The glycosidic bond: (a) in maltose is not hydrolyzed in lactose intolerant humans (b) in sucrose is hydrolyzed by bees (c) joins glucose and fructose to form sucrose (d) both (b) and (c) 8. The sugar which forms major component of nucleic acids is: (a) Ribose (b) Galactose (c) Mannose (d) Maltose 9. Which of the following is not a disaccharide? (a) Amylose (b) Cellobiose (c) Lactose (d) None of these

76 Textbook of Biochemistry 10. Insulin: (a) stimulates gluconeogenesis and glycolysis. (b) stimulates gluconeogenesis and inhibits glycolysis. (c) inhibits gluconeogenesis and glycolysis. (d) inhibits gluconeogenesis and stimulates glycolysis.

Answers 1. (d) 9. (a)

2. (c) 10. (d)

3. (b)

4. (b)

5. (a)

6. (c)

Short Answer Type Questions 1. 2. 3. 4. 5.

Discuss classification of carbohydrates. What are D and L forms of carbohydrates? Describe the important physical properties of carbohydrates? What is isomerism in carbohydrates? What is nonreducing sugar? Discuss with examples.

Long Answer Type Questions 1. 2. 3. 4. 5.

What are structural polysaccharides? Discuss storage polysaccharides in detail. Give a brief account of dissacharides of biological importance. What are sugar alchohols? How are they produced in the body? Explain monosaccharides in detail.

7. (d)

8. (a)

4 Lipids 4.1

INTRODUCTION

Lipids are important for diet as they are high sources of energy. In body, fats are stored in the adipose tissue that acts as an insulating layer. Lipids are natural molecules that include fats, waxes, sterols, vitamins, etc. Lipids are very important as they play a role in energy storage, signalling, and as a structural component. They have the ability to combine with other biomolecules and form diverse group of combinations like lipoproteins, lipopolysacchrides, etc. Lipids are natural organic compounds commonly known as oils and fats. They are found in plants, animals, microorganisms, and in every cell type. Lipids are hydrophobic in nature. They are heterogeneous in nature.

4.2

FEATURES OF LIPIDS

1. Lipids are insoluble in water. 2. They are soluble in chloroform, ether and methanol, i.e., they are soluble in nonpolar solvents. 3. They have higher energy content and their breaking down releases energy. 4. Lipids insulate nerve axons and act as electrical insulators. 5. Lipids are generally fats and are saturated or unsaturated, saturated fats are solid at room temperatures like animal fats. Unsaturated fats are plant fats and are liquid at room temperature. 6. Pure fats are colourless and sparingly soluble in water (hydrophobic). 7. Fats are soluble in organic solvents like ether, acetone and benzene. 8. The melting point of fats depends on the length of the chain of the fatty acid and the degree of unsaturation. 9. Fats have insulating capacity; they are bad conductors of heat. 10. Generally, fats break or hydrolyse into fatty acids and glycerol.

4.3

GENERAL STRUCTURE AND NOMENCLATURE

Fatty acids are hydrocarbon chains of various lengths and degrees of unsaturation that terminate with carboxylic acid groups. The nomenclature of fatty acid is derived from the

78 Textbook of Biochemistry

name of its parent hydrocarbon by the substitution of oic. For example, the C18 saturated fatty acid is called octadecanoic acid because its parent hydrocarbon is octadecane. A C18 fatty acid with one double bond is called octadecenoic acid and if it has two double bonds it is octadecadienoic acid and with three double bonds, octadecatrienoic acid. The notation 18:0 denotes a C18 fatty acid with no double bonds. While 18:2 is that there are two double bonds. Fatty acid carbon atoms are numbered starting at the carboxyl terminus. Fatty acids are generated by hydrolysis or breaking down of fats. In natural fats, fatty acids contain even number of carbons, as they synthesize from two carbon units and are straight-chain derivatives. Fatty acids may be saturated or unsaturated depending upon double bonds. Unsaturated fatty acids contain one or more double bonds while saturated ones contain no double bonds. Carboxylic group carbon is always numbered as carbon one (c1) while adjacent to it is carbon two (c2) known as the B-carbon. Carbon 3 is the C-carbon and the end methyl carbon is known as the H-carbon (Figure 4.1). Different conventions are used to indicate the number and position of carbon. The position of a double bond is represented by the symbol % % followed by a superscript number atom like %9 indicates, double bond between carbon nine (c9) and ten (c10) of the fatty acid. trans- % %2 means that there is a trans double bond between carbon atoms 2 and 3. Alternatively, the position of a double bond can be denoted by counting from the distal end, with the %-carbon atom (the methyl carbon) as number 1.





Fig. 4.1

CH3–(CH2) n–CH2–CH2–COOH X C B Nomenclature of fatty acids, carbon close to –COOH group is alpha carbon, then beta and omega

Fatty acids found in biological systems usually have even number of carbon atoms, typically between 14 and 24 among them the 16- and 18-carbon fatty acids are the most common. Animal fatty acids hydrocarbon chains are invariably unbranched. The alkyl chain may be saturated or it may contain one or more double bonds. In general, configuration of the double bonds in most unsaturated fatty acids is cis. The double bonds in polyunsaturated fatty acids are separated by at least one methylene group. The properties of fatty acids mainly depend on chain length and degree of saturation. Unsaturated fatty acids have lower melting points than saturated fatty acids of the same length. The melting points of polyunsaturated fatty acids are even lower. So chain length and bonds decide important features of fatty acids. Fatty acids with short chains and unsaturated hydrocarbons are more fluid. For example, the melting point of stearic acid is 69.6°C, whereas that of oleic acid (which contains one cis double bond) is 13.4°C as illustrated by the fact that the melting temperature of palmitic acid (C16) is 6.5 degrees lower than that of stearic acid (C18). Lipids may be classified on the basis of their physical properties at room temperature, i.e.,

Lipids 79

solid or liquid. Chain length also affects the melting points. Most important classification of a lipid is based upon its structure (Figure 4.2).

Fig. 4.2

4.4

Classification of lipids

CLASSIFICATION OF LIPIDS

Lipids are generally classified as simple, complex, and derived lipids. Each category and its subtypes are described below.

4.4.1

Simple Lipids

They are esters of fatty acids with different alcohols. These lipids belong to a heterogeneous class of nonpolar compounds and are insoluble in water, but soluble in nonpolar organic solvents such as chloroform and benzene. (a) Fats: They are esters of fatty acids with glycerol. Oils are fats and are liquid at room temperature. Fats are triglycerides as all the three hydroxyl groups of glycerol are esterified. (b) Waxes: They are esters of fatty acids having higher molecular weight with monohydric alcohols. They are solid at room temperature and are esters of long-chain fatty acids such as palmitic acid with aliphatic or alicyclic. They show weak polarity and that’s why they are water-insoluble.

4.4.2

Complex Lipids

They are esters of fatty acids containing additional groups along with fatty acid. (a) Phospholipids: Lipids that contain phosphoric acid residue, in addition to fatty acids and an alcohol. They frequently have nitrogen containing bases and other

80 Textbook of Biochemistry

substituents, e.g., in glycerophospholipids the alcohol is glycerol and in sphingophospholipids, the alcohol is sphingosine. (b) Glycolipids (glycosphingolipids): Lipids containing a fatty acid, carbohydrate and sphingosine. (c) Complex lipids: Lipids such as sulfolipids and aminolipids. Lipoproteins may also be placed in this category.

4.4.3

Derived Lipids

These include fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, and ketone bodies, hydrocarbons, lipid soluble vitamins, and hormones. Because they are uncharged, acylglycerols (glycerides), cholesterol, and cholesteryl esters are termed neutral lipids.

4.5

PHYSICAL PROPERTIES

Pure form of fatty acids forms crystals and that contains stacked layers of molecules. Thickness of the layer is of two extended molecules. The molecular arrangements occur in such a way, that the water-fearing (hydrophobic) hydrocarbon chains form interior of the layer while the water-loving (hydrophilic) carboxylic acid groups form the outer two faces. Fatty acids have specificity and their molecular packing may vary, giving rise to different crystal forms known as polymorphs. Melting temperatures of saturated fatty acids vary from 27°C (81°F) or above and it rises with increasing length of the hydrocarbon chain. Monounsaturated and polyunsaturated molecules melt at lower temperatures as compared to saturated ones, with the lowest melting temperatures occurring when the carbon-carbon double bonds are too close to the centre of the hydrocarbon chain, as it is in most biological molecules. The result is that molecules form viscous liquids at room temperature. The hydrophobicity of hydrocarbon chain of most biological fatty acids exceeds the hydrophilic nature of the carboxylic acid group, making the water solubility of these molecules very low. So water solubility decreases exponentially with the addition of each carbon atom to the hydrocarbon chain. This relationship reflects the energy required to transfer the molecule from a pure hydrocarbon solvent to water. With each CH2 group, for instance, more energy is required to order water molecules around the hydrocarbon chain of the fatty acid, which results in the hydrophobic effect. In pure water, the carboxylate group dissociates into a positively charged hydrogen ion R−COOH m RCOO − + H+. Here R represents the hydrocarbon chain. The carboxylate ion having negative charge, is more polar than the undissociated acid. RCOOH can be converted completely to the ion RCOO − by adding an equal number of molecules of a base such as sodium hydroxide (NaOH). This effectively replaces the H+ with Na+ to give the salt of the fatty acid.

Lipids 81

4.6 4.6.1

CHEMICAL PROPERTIES Saponification Number

Fatty acid breaks into glycerol in the presence of alkali; this is known as saponification. Saponification number is defined as the mg of KOH required to saponify 1g of fat.

4.6.2

Acid Number

It is defined as the mg of KOH utilized to neutralize the free fatty acids present in 1g of fat or oil. N short acid number is the quantity of free fatty acid found in a fat. Acid value can be high or low, high acid number indicates a state oil or fat stored under improper conditions. Acid value measures hydrolytic rancidity and it is very important.

4.6.3

Iodine Number

It is the measure of degree of unsaturation. It is the number of carbon-carbon double bonds in relation to the amount of fat or oil. It is defined as the grams of iodine absorbed per 100 g of sample.

4.6.4

Peroxide Value

Peroxide value is the measure of the degree of lipid oxidation in fats and oils. It is defined as the number of milli equivalents of peroxide per kg fat. It is a measure of the formation of peroxide or hydroxide groups that are initial products of the lipid oxidation.

4.6.5

Riechert Messel Number

The Riechert Messel Number is a measure of the amount of H2O soluble volatile fatty acids. It is defined as the number of millilitres of 0.1 N alkali necessary to neutralize the volatile H2O soluble fatty acids in a 5 g sample of fat.

4.6.6

Polenske Number

Polenske number is the measure of the amount of volatile insoluble fatty acids. It can be defined as the number of millilitres of 0.1N alkali necessary to neutralize the volatile H2O insoluble fatty acids which are present in 5 g sample.

4.6.7

Hydrolysis

It is the reaction of fats with the water. Due to this, fats and oils split into fatty acids and give free fatty acids like monoglycerides and diglycerides. This reaction is accelerated by high temperatures and pressures in the presence of an excessive amount of water. This reaction occurs at the junction of the fatty acids and glycerol.

4.6.8

Hydrogenation

It is the reaction used to optimize the properties of fats and oils prepared for specific uses. Hydrogenation depends on a few facts like nature of substance to be hydrogenated

82 Textbook of Biochemistry

and concentration of the catalyst, temperature and pressure. Trans fatty acids, present in certain foods, arise as a byproduct of the saturation of fatty acids during hydrogenation, or “hardening,” of natural oils in the manufacture of margarine.

4.6.9

Isomerization

Isomers are two or more substances that are composed of same elements combined in the same proportions, hence having the same molecular formula. The two important types of isomerism are: geometric and positional isomerism. (a) Geometrical Isomerism: A double bond can have two configurations cis or trans. If the two atoms are on the same side of the carbon chain, this arrangement is called cis and if the two atoms are on opposite sides of the carbon chain, the arrangement is called trans. Natural fats and oils are in cis form. (b) Positional Isomerism: Unsaturated fatty acids can be isomerised in acids or alkaline conditions by applying high temperatures. This reaction causes movement of double bond from one position to another. Hydrogenation process mainly causes shifting in the location of double bonds in the fatty acid chains and may forms cistrans isomerisation. cis isomers occur in food fats, oils while trans isomers occur in fats from remnants. Most trans isomers result from the partial hydrogenation of fats and oils.

4.6.10

Esterification

It is reverse of hydrolysis and in this, triglycerides are formed from free fatty acids and glycerol. Mainly mono- and diglycerides are produced by esterification. Monoglycerides are important emulsifying agents in food products. Emulsifying agents are made by alcoholysis or by esterification.

4.6.11

Oxidation

It is the reaction of an oil or fat with O2 in the air, and with the food. Double bonds or points of unsaturation are the places where reaction occurs. This reaction is not desirable as it will adversely affect the flavour of the fat. Oxidation rate depends upon temperature, oxygen and light. By increasing temperature, rate of oxidation increases. In general, oxidation by air at room temperature is referred to as autoxidation.

4.6.12

Interesterification

This reaction is given by fats and oils and it is simply that fatty acid esters react with the other esters of a fatty acid and forms new esters by interchanging fatty acid groups. It is also referred to as randomization, rearrangement or modification. It is used to process edible fats, oils to produce confectionery fats, margarine oils and cooking fats, etc.

4.6.13

Halogenation

The halogens include chlorine, bromine and iodine can readily add to the double bonds of unsaturated fatty acids. So, degree of unsaturation in oils and fats can be measured by

Lipids 83

measuring iodine monosaccharide added to the quantities of fats or oils This is known as iodine number and is an important analytical measurement.

4.6.14

Polymerization

When oxidation increases to some limit or excessive oxidation is accompanied by polymerization. This reaction occurs at the points of unsaturation of fatty acid chains or at the juncture of the fatty acid and glycerol. Deep frying of foods at high temperature (325°F -375°F) leads to polymerization. Heat stress, oxidation presence of the radical and polar catalyst leads to polymerization of unsaturated fatty acid in lipids. Rate of polymerization increases with the amount of unsaturation and viscosity of fat or oil.

4.7 4.7.1

SIMPLE LIPIDS Fatty Acids

They are carboxylic acids with long hydrocarbon chains. Fatty acids (FA) differ from one another in: (a) length of the hydrocarbon tails, (b) position of the double bonds in the chain and (c) degree of unsaturation (double bond). A fatty acid is a molecule characterized by the presence of a carboxyl group attached to a long hydrocarbon chain. Therefore, these are molecules with a formula R–COOH where R is a hydrocarbon chain. Fatty acids can be said to be carboxylic acids, and come in two major varieties. Saturated fatty acids do not have any double bonds. A fatty acid is saturated and every carbon atom in the hydrocarbon chain is bonded to hydrogen atoms as possible (the carbon atoms are saturated with hydrogen). Fatty acids play an important role in metabolism. They act as major metabolic fuel, as storage and transport of energy, as essential components of all membranes, and as gene regulators. In addition, dietary lipids provide polyunsaturated fatty acids (PUFAs) that are precursors of powerful metabolites, i.e., the eicosanoids. Fatty acids are also important as work as thermal, electrical insulators and as mechanical protection. The triglycerides are the most abundant of all lipids that constitute about 98% of total dietary lipids, 2% phospholipids and cholesterol and its esters. They are nonpolar, hydrophobic molecules as they do not contain any electrically charged or highly polar functional groups. In animals, the fat cells or adipocytes contain large quantities of triglycerides in the form of fat droplets, which cover almost the entire volume of cell. Triglycerides can be stored and can supply the energy needs of the body for many months. Triglycerides are much better adapted than glycogen to serve as storage form of energy. They are not only stored in large amounts but also yield over twice as much energy as carbohydrates. Since fats tend to remain in the stomach longer than carbohydrates and are digested slowly. Whales, seals, walruses and penguins are amply padded with triglycerides that serve as energy storage and as insulation against low temperature. Most fats and oils, upon hydrolysis, yield several fatty acids as well as glycerol. In a human body approximately 10-20% of the body weight is lipid, mainly triacylglycerol (TAG). TAG is found in all organs of the human body, particularly in adipose tissue, in which droplets of triacylglycerols may represent more than 90% of the cytoplasm of the cells. Body lipid is a reservoir of

84 Textbook of Biochemistry

potential chemical energy. About 100 times more energy is stored as mobilizable lipid than as mobilizable carbohydrate in a normal human being. TAG is stored in a relatively water-free state in the tissue, in comparison to carbohydrate, which is heavily hydrated. Chemically, triglycerides are esters of glycerol with 3 fatty acid molecules. Their generic formula is shown in Figure 4.3. A simple fat molecule contains three moles of similar or dis- Fig. 4.3 General formula of triglyceraldehyde similar fatty acids. Those containing a single kind of fatty acid in all three positions (B, C, Ba) are called simple (or symmetrical) triglycerides. It is found infrequently in natural fats while it is synthesized in the laboratory, e.g., tristearin and triolein. Natural triglycerides are generally mixed (or asymmetrical) triglycerides, i.e., they contain 2 or 3 different fatty acid units in the molecule (Figure 4.4). Fatty acids are classified on the basis of chain length. Fig. 4.4 Two optical isomers of triglyceraldehyde 1. Short chain fatty acids (SCFA): They are fatty acids having aliphatic tails of not more then six carbon, e.g., butyric acid. 2. Medium-chain fatty acids (MCFA): They are fatty acids with six to twelve carbon long aliphatic chains. 3. Long-chain fatty acids (LCFA): They are fatty acids with aliphatic tails, thirteen to twenty-one carbons. 4. Very long chain fatty acids (VLCFA): Fatty acids with aliphatic tails longer than twenty-two carbons. (a) Saturated fatty acids have mainly monoglycerides and fatty acids. They are actually long carbon-chain compounds and there are single or double bonds between carbon atoms. Double bonds can react with hydrogen atoms and form single bonds that are known as saturated. Animal fats are saturated, while plant fats are generally unsaturated. Saturated fatty acids are straight chains of hydrocarbons having even carbon atoms (Figure 4.5). The simplest fatty acids are unbranched, linear chains of CH2 groups linked together by carbon-carbon single bonds, terminated with carboxylic acid group. The systematic names are based on numbering the carbon atoms, beginning with the acidic carbon. Most common fatty acids contain 12–22 carbon atoms and saturated fats have higher melting points as compared to the corresponding unsaturated fats. This is the reason that saturated fats tend to be solid at room temperature, while unsaturated fats are liquid at room temperature. The degree of viscosity varies and vegetable products have higher saturated fat content like coconut oil and palm oil. Shorter-chain fatty acids are biochemically important like butyric acid (C4) and caproic acid (C6) are lipids found in milk. Palm kernel oil, an important dietary source of fat in certain areas of the world, is rich in fatty acids. It contain 8 and 10 carbons (C8 and C10).

Lipids 85

–CH=CH–CH=CH Unsaturated fatty acid chain –CH–CH–CH Saturated fatty acid chain Fig. 4.5

Saturated and unsaturated fatty acids

(b) Unsaturated fatty acids are monounsaturated fatty acids having at least one carbon– carbon double bond, which can occur in different positions. Commonly, they have a chain length of 16-22 carbons. Plants are the main source of unsaturated fatty acids. Isomers are produced due to the variations in the locations of the double bonds in unsaturated fatty acid. Geometric isomerism mainly depends on the orientation of radicals around the axis of double bonds. In unsaturated fats, the orientation of hydrogen atoms may be cis or trans. Hydrogen bonds in same directions are in cis configuration and if hydrogen atoms on either side of the double bond are oriented in the different directions, they are known as trans configuration. cis configuration of fatty acids is thermodynamically less stable as compared to trans forms. The cis fatty acids have lower melting points than the trans fatty acids. The cis conformation is rigid as its double bond freezes and stabilises the isomer, that’s why the chain bends and it restricts the conformational freedom of the fatty acid. As in trans configuration, two adjacent hydrogen atoms at opposite sides of the chain cause not much bending of carbon chain, their shape is more similar to straight chain saturated fatty acids (Figure 4.6). There are more geometric isomers in the case of acids having higher degree of unsaturation. The unsaturated long chain fatty acids found in nature are almost the cis form and the molecules are “bent” at the position of the double bond. So they form a U-shaped structure of a molecule like in arachidonic acid. The differences in geometry of fatty acids, due to unsaturation and saturation play an important role in biology.

Fig. 4.6

cis and trans isomers of fatty acids

Fatty acids Energy – high per gram (37 kJ 21 g fat) Transportable form of energy – blood lipids (e.g., triacylglycerol in lipoproteins) Storage of energy, e.g., in adipose tissues and skeletal muscles Component of cell membranes (phospholipids) Insulation – thermal, electrical and mechanical Signals – eicosanoids, gene regulation (transcription)

86 Textbook of Biochemistry

Essential and Nonessential fatty acids When a fatty acid can only be obtained from the diet (for humans) then the fatty acid is an essential fatty acid. There are two types of essential fatty acids, the first one has a double bond three carbon atoms away from the methyl end, while other has a double bond six carbon atoms away from the methyl end. Human body does not synthesize fatty acids having double bonds beyond carbons 9 and 10 as counted from the carboxylic acid side. Linoleic acid (LA) and alpha-linolenic acid (ALA) are two essential fatty acids distributed in plant oils. These two unsaturated fatty acids are not synthesized in the human body and are essential. Nonessential fatty acids can be made by the human body.

4.7.2 Waxes The term ‘wax’ originated from weax meaning “the material of the honeycomb”. They are esters of long-chain saturated and unsaturated fatty acids with long-chain monohydroxy alcohols. Fatty acid chains range between C14 and C36 and the alcohols range from C16 to C36. Vertebrates have cutaneous glands and secrete waxes to protect and keep the skin pliable, lubricated and water-proof. Hair, wool and fur are also coated with wax. The leaves of Rhododendron, Calotropis are shiny because of the deposition of protective waxy coating. In plankton, waxes serve as the chief storage form of fuel. Since marine organisms (whale, herring, salmon) consume plankton in large quantities, waxes act as major food and storage lipids in them. Refined oil: It is prepared by removal of free fatty acids by alkali treatment in the first step. In the second step, colour is removed by using activated carbon whereas specific odour is removed by superheating. Hydrogenated oils: The refined oils are hydrogenated under optimum temperature and pressure with hydrogen in the presence of nickel catalyst. By this method, unsaturated fatty acids are converted into saturated fatty acids. The liquid oil becomes solid fat like Vanaspati is hydrogenated refined groundnut oil. Hydrogenation decreases the degree of unsaturation as in: Oleic acid

4.8 4.8.1

Hydrogenation

Stearic acid.

COMPOUND LIPIDS Phospholipids (Phosphatides)

They are the main structural entities of membranes, matrix of cell wall, myelin sheath, microsomes and mitochondria. They are esters of fatty acids with glycerol with esterified phosphoric acid and a nitrogen base. Mainly found in the nerve tissues brain, liver, kidneys, pancreas and heart, they increase the rate of fatty acid oxidation, act as prosthetic group to certain enzymes, help in blood clotting and also act as carriers of inorganic ions across the membranes.

Lipids 87 Comparison between animal and plant fats S. No.

Animal Fat

Plant Fat

1.

Relatively rich in saturated fatty acids.

Relatively rich in unsaturated fatty acids, C16 and C18 acids and polyunsaturated acids.

2.

Solid at ordinary room temperature.

Liquid at ordinary room temperaure.

3.

Low iodine number.

High iodine number.

4.

These have usually high Reichert-Meissel number.

These have usually low Reichert-Miessel number.

5.

Stored mainly in liver and bone marrow.

Stored mainly in seeds and fruits.

6.

Oxidative rancidity is observed more fre- Oxidative rancidity is observed less frequently. quently. Examples, butterfat, beef fat, tallow Examples, olive oil, castor oil, soybean oil, corn oil.

It is based on the type of alcohol present in the phospholipid. There are three types: 1. Glycerophosphatides: In this, glycerol is the alcohol group, e.g., Phosphatidyl ethanolamine (cephalin), phosphatidyl choline (lecithin), phosphatidyl serinem plasmalogens and phosphatidic acid. 2. Phosphoinositides: In this, inositol is the alcohol, e.g., phosphatidyl inositol (lipositol). 3. Phosphosphingoside: In this, sphingosine is an amino alcohol, e.g., sphingomyelin. The phospholipids include the following groups: 1. Phosphatidic acid: Phosphatidic acid is important as it is an intermediate in the synthesis of triacylglycerols and phospholipids. 2. Phosphatidyl glycerol, cardiolipin: It is formed from phosphatidyl glycerol. Chemically, it is di-phosphatidyl glycerol. It is found in inner membrane of mitochondria and bacterial wall. 3. Lecithin (Phosphatidylcholine): The lecithin contains glycerol and fatty acids, phosphoric acid and choline (nitrogenous base). Lecithin has saturated fatty acid at B position and an unsaturated fatty acid at C position. They can be in two forms, B or C. It is waxy, white substance but becomes brown when exposed to air. When aqueous solution of lecithin shaken with H2SO4, choline is split, forming phosphatidic acid. H2SO4 Phosphatidic acid + choline Lecithin Phosphatidic acid m Glycerophosphoric acid + fatty acids (2 mol) 4. Cephalitis (Phosphatidyl ethanolamine): They are found in tissues and always associated with lecithin due to similar properties. The only difference is the nitrogenous base.

88 Textbook of Biochemistry

5. Phosphatidyl inositol (Lipositol or Phosphoinositides): It acts as second messenger in Ca++ dependent hormone action and are more acidic than the other phospholipids. It provides communication between the hormone receptor on the plasma membrane and intracellular Ca++ reservoirs. 6. Lysophospholipids: These are phosphoacylglycerols containing only one acyl radical in a position, e.g., lysolecithin. It is formed by the action of phospholipase or by interaction of lecithin and cholesterol in the presence of the enzyme, lecithin cholesterol acyl transferase. Due to this, lysolecithin and cholesterol esters are formed. Lecithin + cholesterol n LCAT Lysolecithin + cholesterol ester 7. Plasmalogens: These are the contents of brain and muscle. Structurally, these resemble lecithin and cephalins but give a positive reaction when tested for aldehydes with Schiff’s reagent (fuchsin-sulphurous acid) after pretreatment of the phospholipid with mercuric chloride. They have an ether link in a position instead of ester link. The alkyl radical is an unsaturated alcohol. 8. Sphingomyelins: These are found in large quantities in brain and nerve tissues. Its concentration increase in liver and spleen in Niemann-Pick disease. They contain 18 carbons containing sphingosine fatty acid, phosphoric acid and choline without any glycerol. In sphingosine molecule, –NH2 group binds a fatty acid by an amide linkage to produce ceramide. When phosphate group is attached to ceramide, it is called ceramide phosphate. When choline is split from sphingomyelin, ceramide phosphate is left.

Niemann-Pick Disease It is due to amount of sphingomycin deposited in the brain, liver and spleen. It is a lipid storage disease and hereditary, mainly caused due to the deficiency of sphingomyelinase. Its symptoms are metal retardation, affected nervous system, enlarged liver and spleen, anaemia and leucocytosis.

4.8.2

Glycolipids

Commonly known as cerebrosides, they are the compounds of fatty acids with carbohydrates that contain nitrogen but no phosphoric acid. The glycolipids also include certain gangliosides, sulfolipids and sulfatids. These are further classified into: (i) Cerebrosides (ii) Gangliosides

Lipids 89

(i) Cerebrosides: Cerebrosides are sphingolipids and contains galactose, a high molecular weight fatty acid and sphingosine. They are the chief constituent of myelin sheath. They may be differentiated by the type of fatty acid in the molecule. The cerebrosides are in much higher concentration in medullated than in non-medullated nerve fibres.

Gaucher disease The reticuloendothelial cells (spleen) contains high amount of cerebroside. This disease is caused by deficiency of glucocerebrosidase. Enlarged spleen and liver, leucopenia and yellow brown wedgeshaped elevation appears in eyes.

(ii) Gangliosides: These are glycolipids found in the brain. Gangliosides contain ceramide (sphingosine + fatty acids), glucose, galactose, N-acetylgalactosamine and sialic acid. Some gangliosides also contain di-hydro-sphingosine or gangliosine in place of sphingosine. Most of the gangliosides contain a glucose, two molecules of galactose, one N-acetylgalactosamine and up to three molecules of sialic acid.

Taysachs disease Characterized by increased accumulation of GM2 ganglioside in brain and spleen. It is caused due to deficiency of hexosaminidase A enzyme. Its symptoms are mental retardation, blindness, muscular weakness.

4.8.3

Other Compound Lipids

1. Lipoproteins: Hydrophilic lipoprotein complex is formed by the combination of triacylglycerol (45%), phospholipids (35%), cholesterol and cholesteryl esters (15%), free fatty acids (less than 5%) and a few proteins. The density of lipoproteins increases as the protein content increases, and the lipid content falls and the size of the particle becomes smaller. Lipoproteins may be separated by its special feature, i.e., electrophoretic properties and immuno-electrophoresis. Physiologically, four major groups of lipoproteins have been identified which are important and clinical diagnosis in fat metabolism as some metabolic disorders. These are chylomicrons, very low density lipoproteins (VLDL or pre-C-lipoproteins), low density lipoproteins (LDL or C-lipoproteins) and high density lipoproteins (HDL or B-lipoproteins). Chylomicrons and VLDL are mainly triacylglycerol (50%) and cholesterol (23%). The concentrations of these are increased in atherosclerosis and coronary thrombosis, etc. Low density lipoproteins have main lipid as cholesterol (46%) and phospholipids (23%). Increase in atherosclerosis and coronary thrombosis, etc. High density lipoproteins predominant lipid is phospholipids (27%) and proteins (45%). The protein moiety lipoprotein is known as an apoprotein which constitutes nearly

90 Textbook of Biochemistry

60% of some HDL and 1% of chylomicrons. Many lipoproteins contain more than one type of apoprotein polypeptide. The larger lipoproteins (such as chylomicrons and VLDL) consist of a lipid core of nonpolar triacylglycerol and cholesteryl ester surrounded by more polar phospholipid, cholesterol and apoproteins. To transport and deliver the lipids to tissues. To maintain structural integrity of cell surface and subcellular particles like mitochondria and microsomes. The C-lipoprotein fraction increases in severe diabetes mellitus, atherosclerosis, etc. Hence, determination of the relative concentrations of B- and C-lipoproteins and pre-C-lipoproteins are of diagnostic importance. 2. Amino lipids: Phosphatidyl ethanolamine and serines are amino lipids and sphingomyelins and gangliosides contain substituted amino groups. 3. Sulpholipids (Sulphatides): These have been isolated from brain and other animal tissues. These are sulphate derivatives of the galactosyl residue in cerebrosides.

4.9

DERIVED LIPIDS

These are the lipid derivatives derived from simple and compound lipids by hydrolysis. These include fatty acids, alcohols, mono- and diglycerides, steroids, terpenes and carotenoids.

4.9.1

Alcohols

Alcohols found in lipid molecules include glycerol, cholesterol and higher alcohols (acetyl alcohol), usually found in the waxes. Usually, unsaturated alcohols are important pigments.

4.9.2

Steroids

The steroids are often found in association with fat. They have a cyclic nucleus similar to phenanthrene (rings A, B, C) to which a cyclopentane ring (D) is attached. The parent substance is better designated as cyclopentanoperhydrophenanthrene. The position on the steroid nucleus is numbered as shown in Figure 4.7. Methyl side chains occur at positions 10 and 13 (constituting C atoms 19 and 18). A side chain at position 17 is usual (as in cholesterol). If the compound has one or more hydroxyl groups and no carbonyl or carboxyl groups, it is a sterol, and the name terminates in -OL. The steroids are the most studied classes in biological compounds and are often found in association with fat. As they do not have any fatty acids, so Fig. 4.7 Structure of cyclopentanoperhydrophenanthrene they are nonsaponifiable, i.e., cannot be hydrolyzed by heating with alkali to yield soaps of their fatty acid components. All steroids may be treated as derivatives of a fused and fully saturated ring system called cyclopentanoperhydrophenanthrene or sterane. This system consists of 3

Lipids 91

cyclohexane rings (A, B and C) fused in nonlinear or phenanthrene manner and a terminal cyclopentane ring (D).

Classification of steroides Sterols: cholesterol, ergosterol, coprosterol. Bile acids: Glycocholic acid and taurocholic acid. Sex hormone: Testosterone, estradiol. Vitamin D: Vitamins D2 and D3. Adrenocortical hormones: Corticosterone. Cardiac glycosides: Stropanthin. Saponins: Digitonin.

(a) Cholesterol: It is widely distributed in all the cells of the body. It occurs in animal fats but not in plants. Cholesterol (Figure 4.8) has a molecular formula, C27H45OH. There is an OH group at C3 and a double bond at C5. The hydroxyl group constitutes its polar head, the rest of the molecule is hydrophobic. It is a white crystalline solid and shows optical activity. Cholesterol is the most publicized lipid in nature as it is correlated Fig. 4.8 Structure of cholesterol with health issues. Higher levels of cholesterol in the blood causes cardiovascular diseases in humans. It is an important component of cell membranes, plasma lipoproteins and also precursor of biologically important steroids, like bile acids and various steroid hormones. It is the principal sterol of higher animals and is especially abundant in nerve tissues and in gallstones. It occurs as either free or fatty esters in all animal cells. (b) Ergoesterol: It occurs in ergot (hence its nomenclature) and yeast. It is a precursor of vitamin D and also acquires anti-rachitic properties with the opening of ring B when irradiated with ultraviolet light. Ergosterol (Figure 4.9) has a molecular formula, C28H43OH with one OH group at C3 and 3 double bonds at C5, C7 and C22. It is also optically active. Rupture of the ring B by UV radiation produces vitamin D2 which is chemically known as ergocalciferol. Fig. 4.9 Structure of erogosterol Terpenes: Among the nonsaponifiable lipids found in plants are many hydrocarbons known as terpenes (from turpentine). These hydrocarbons and their oxygenated derivatives have less than 40 carbon atoms. The simplest terpenes are called monoterpenes and conform to the formula C10H16, those with the formula

92 Textbook of Biochemistry

C15H24 are called sesquiterpenes, with C20H32 diterpenes and with C30H48 triterpenes. Terpenes with 40 carbon atoms (or tetraterpenes) include compounds called carotenoids.

4.10

LIPID TESTS

1. Grease spot test: A drop of oil placed over a piece of ordinary paper. A translucent spot is visible and it indicates the presence of fat. 2. Emulsification test: 2 ml water is taken in one test tube and 2 ml of diluted bile salt solution in another test tube. Add 3 drops of the given oil to each test tube and shake vigorously. Note the stability of the emulsification formed. 3. Saponification test: Take 10 drops of coconut oil in a test tube. Add 20 drops of 40% NaOH and 2 ml of glycerol to it. Gently boil for about 3 minutes until complete saponification occurs. If oil globules are visible, boiling must be continued. Divide the solution into three parts to carry the following experiments in test tubes 1, 2, 3. Test tube No. 1

Test tube No. 2

Test tube No. 3

Add saturated solution of NaCl. Note Add a few drops of conc. HCl. An Add a few drops of CaCl2 soluthat the soap separates out and floats oily layer of the fatty acids rises tion. The insoluble calcium soap to the surface (salting out process). to the surface. is precipitated.

4. Unsaturation test: Add 10 drops of Hubble’s iodine reagent to 10 ml of chloroform. The chloroform assumes a pink colour due to the free iodine. The solution is divided equally into three test tubes as (a), (b) and (c) and three types of oil are added. Add oil No. 1 to test tube (a) drop by drop shaking the tube vigorously after each addition till the pink colour of the solution just disappears. The number of oil drops required is noted. The experiment is repeated by adding oils 2 and 3 to test tubes (b) and (c), respectively. The more the number of drops required to discharge the pink colour, the less is the unsaturation.

4.11







SUMMARY

s Lipids are main storage compounds and triglycerides serve as reserve energy of the body. s Lipids are important component of cell membranes in eukaryotic cells. s Lipids regulate membrane permeability. s They serve as source for fat soluble vitamins like A, D, E, K. s They act as electrical insulators to the nerve fibres, where the myelin sheath contains lipids. s Lipids are components of some enzyme systems. s Some lipids like prostaglandins and steroid hormones act as cellular metabolic regulators. s Cholesterol is found in cell membranes, blood, and bile of many organisms. s As lipids are small molecules and are insoluble in water, they act as signalling molecules.

Lipids 93









s Layers of fat in the subcutaneous layer, provides insulation and protection from cold. Body temperature maintenance is done by brown fat. s Polyunsaturated phospholipids are important constituents of phospholipids, they provide fluidity and flexibility to the cell membranes. s Lipoproteins that are complexes of lipids and proteins, occur in blood as plasma lipoprotein, they enable transport of lipids in aqueous environment, and their transport throughout the body. s Cholesterol maintains fluidity of membranes by interacting with lipid complexes. s Cholesterol is the precursor of bile acids, vitamin D and steroids. s Emulsification is the process by which a lipid mass is converted to a number of small lipid droplets. The process of emulsification happens before the fats can be absorbed by the intestinal walls. s The fats are hydrolyzed by the enzyme lipases to yield fatty acids and glycerol. s The hydrolysis of fats by alkali is called saponification. This reaction results in the formation of glycerol and salts of fatty acids called soaps. s Hydrolytic rancidity is caused by the growth of microorganisms which secrete enzymes like lipases. These split fats into glycerol and free fatty acids.

MULTIPLE-CHOICE QUESTIONS 1. Which is a characteristic of sphingolipids? (a) They all contain a fatty acid joined to glycerol. (b) They all contain a long-chain alcohol joined to isoprene. (c) They all contain ceramide joined to a polar group. (d) They all contain a carbohydrate joined to a phosphate group. 2. Which is a property of eicosanoids? (a) All eicosanoids contain three conjugated double bonds. (b) All eicosanoids contain arachidonic acid and sphingosine. (c) Prostaglandins and leukotrienes both contain a ring structure. (d) Thromboxanes and prostaglandins both contain a carboxyl group. 3. Which is a characteristic of all the fatty acid components in this lipid? (a) They all contain an unbranched carbon chain. (b) They all contain unconjugated cis double bonds. (c) They all are joined to glycerol through an ester bond. (d) They all are hydrophilic because they contain oxygen. 4. What is the proper designation for the unsaturated fatty acids in this lipid? (a) 18:2 (%9,12) (b) 18:2 (%6,9) (c) 17:2 (%9,12) (d) 17:2 (%5,8) 5. Which property does the lipid share with a typical triacylglycerol? (a) Both contain an ether bond. (b) Both contain a long-chain alcohol. (c) Both are amphipathic. (d) Both are saponifiable.

94 Textbook of Biochemistry 6. Which membrane lipid contains an amide bond? (a) Cholesterol (b) Phosphatidylserine (c) Phosphatidic acid (d) Sphingomyelin 7. Which type of membrane lipid contains an acidic oligosaccharide? (a) Phosphatidylinositol (b) Cerebroside (c) Ganglioside (d) Globoside 8. HDLs are synthesized in: (a) Liver (b) Blood (c) Intestine (d) Pancreas 9. Triacylglycerols are: (a) Soluble in water (b) Insoluble in water (c) Soluble in water at elevated temperature (d) Partially soluble in water 10. The key enzyme in the regulation of fatty acid synthesis is: (a) Acetyl-CoA carboxylase (b) AMP activated protein kinase (c) Protein phosphatase (d) None of these

Answers 1. (c) 9. (b)

2. (d) 10. (a)

3. (a)

4. (b)

5. (d)

6. (d)

7. (c)

8. (a)

Short Answer Type Questions 1. 2. 3. 4. 5.

What is saponification number? Discuss Polenske Number in short. What are essential and nonessential fatty acids? What are refined oils and hydrogenated oils? Differentiate between animal fat and plant fat.

Long Answer Type Questions 1. 2. 3. 4.

Define lipids with suitable examples. Define phospholipids. Classify them with suitable examples and state their functions. Classify different types of fatty acids with suitable examples. Write short notes on any three of the following: (a) Prostaglandins (b) Lipoproteins (c) Chemistry and functions of cholesterol (d) Sphingolipids (e) Biological wax 5. What are essential and nonessential fatty acids? Explain with examples. 6. Explain briefly: (a) Stereoisomers (b) Fat soluble vitamins (c) Prostaglandins (d) Thromboxanes

5 Proteins-I 5.1

INTRODUCTION

Proteins are most abundant biomolecules that occur in all living beings. These molecules serve as essential structural and functional components of a cell and drive all the metabolic activities of living organisms. The huge diversity of proteins is evident by the gamut of their molecular size ranging from very small peptides of just a few hundred daltons to very large proteins of molecular weight of about several million daltons. Glutathione is an example of one of the smallest proteins which is made up of only three amino acids and has the molecular mass of approximately 307 Da, while human titin is the largest protein known that has molecular mass of about 3800 kDa. Structural proteins are the most abundant class of proteins in nature, of which collagen is recognized as the most abundant mammalian protein. Collagen is a structural unit of connective tissues. Other common structural proteins are keratin, fibrin, actin, etc. Figure 5.1 depicts some structures made of protein such as skeletal muscle fibres, feathers of birds, spider web, human eye lens, hair, nails and claws of animals.

Lens a Fig. 5.1

b

c

d

Proteins as structural components: (a) muscle fibres, (b) bird feathers, (c) spider web, (d) human eye lens

Besides the structural role, proteins also play a vital role in metabolism. All biological catalysts or enzymes are proteinaceous in nature and serve to enhance the rate of

96 Textbook of Biochemistry

biochemical reactions. All the antibodies are composed of proteins. Many hormones that regulate the growth in animals and humans are also composed of proteins. The nucleoproteins carry genes that inherit the traits from one generation to another. Some important proteins along with their respective molecular weights are enlisted in Table 5.1. Table 5.1

Molecular weights (kDa) of some important proteins

Name of Protein Insulin (human)

Molecular Weight (kDa) 5.8

B-amylase

51-54

Keratin

40-68

Hemoglobin

64.5

Elastin

66

Hexokinase

100

Lactic Dehydrogenase (LDH)

140

Glyceraldehyde-3-phosphate dehydrogenase (GAPD)

144

Immunoglobulin G (IgG)

150

5.2

AMINO ACIDS: THE BUILDING BLOCKS OF PROTEINS

Biochemically, all proteins are polymers of simple organic compounds amino acids, which contain both a carboxyl (—COOH) and an amino (—NH2) group (Figure 5.2). There are more than 500 naturally occurring amino acids that exist in nature, but only 20 amino acids are encoded by the genetic code and used for the synthesis of numerous proteins. These 20 amino acids are ubiquitous and are common to all forms of life from bacteria to higher animals and plants. The history of amino acids dates back to 1806, when the first amino acid was discovered by French chemists Louis-Nicolas Vauquelin and Pierre Jean Robiquet from asparagus and was named asparagine. Other amino acids discovered after that were cysteine in 1810, leucine and glycine in 1820 and last to be discovered among 20 standard amino acids was threonine (1938). Some of the amino acids derived their Fig. 5.2 General names from their sources like glutamate from wheat gluten and asparstructure of an agine from asparagus, valine from valeric acid which is present in the amino acid plant Valerian and glycine was named so because of its sweet taste. The amino acids serving as the monomers or building blocks of a protein join in different numbers and different linear sequences to produce the huge diversity of proteins. Each amino acid residue is joined to another by a specific type of covalent bond called peptide bond, through which long chains are formed to constitute the polymeric form of protein. Conversely, proteins can be broken down to their constituent amino acids by hydrolysis. The reversibility of this process is the key to obtain nitrogenous biomolecules from food.

Proteins-I

97

The dietary protein is hydrolyzed by the special digestive enzymes in the stomach into their component amino acids. These amino acids are absorbed in the small intestine and distributed throughout the body through the bloodstream. All the cells use these amino acids to build specific proteins under the guidance of genetic information inherent in the DNA. Though the proteinogenic role of amino acids has been highlighted, several amino acids play many other important roles in metabolism. Glycine is a precursor of heme porphyrins. Tyrosine and phenylalanine are precursors of neurotransmitters dopamine, epinephrine and norepinephrine, while tryptophan is a precursor of the neurotransmitter serotonin. Glycine, glutamine and aspartate are precursors of nucleotides. Thus, all the 20 amino acids are indispensable. The vitality of amino acids can be exemplified by the fatal disease, sickle cell anaemia which is caused due to substitution of a single amino acid, glutamic acid with valine in an amino acid sequence of the protein chain in the haemoglobin molecule. This small defect takes away the reoxygenation capacity of sickle cell haemoglobin molecules and makes them extremely sensitive to oxygen deficiencies.

Fig. 5.3

Mutation causing sickle cell anaemia

98 Textbook of Biochemistry

Sickle cell anaemia It is a genetic disorder inherited in an autosomal recessive pattern. The mutation of a single nucleotide from a GAG to GTG codon on the coding strand of the C-globin gene results in substitution of glutamic acid by valine at the sixth position in the C-globin subunit of the haemoglobin protein. This causes sickling of red blood cells and their premature breakdown leading to severe anaemia (Figure 5.3).

5.2.1

General Structure of Amino Acids

As stated above, amino acids are the organic compounds containing carbon, hydrogen, oxygen and nitrogen. The 20 standard amino acids are all alpha amino acids and their empirical formula is RCH(NH2)COOH with an exception of proline. The proline is a cyclic amino acid having secondary amine group, therefore is known as imino acid. In general, amino acids have a tetrahedral structure due to the presence of an asymmetric carbon atom (B-C) in the centre (Figure 5.5). Other carbon atoms are designated as C, H, E
etc., according to their specific position around the asymmetric carbon atom, as shown in lysine (Figure 5.4). This central carbon atom is known as chiral carbon because it is attached with four different Fig. 5.4 Numbering of carbon atoms groups in amino acids— a carboxyl group, an in lysine amino group, a hydrogen atom and an R group. The R group in glycine is another hydrogen atom. Amino acids mainly differ from each other by their R groups which vary in structure, size, polarity, electric charge and hydrophobicity. The characteristics of R groups influence many properties of the amino acids that are the key to the structure and function of a protein.

(a)

(b)

Fig. 5.5 Structural representation of an amino acid: (a) General structure showing an asymmetric carbon atom (B-C) and (b) three-dimensional tetrahedral structure of an amino acid

5.2.2

Standard Amino Acids

The 20 standard amino acids which are used in biosynthesis of peptides and proteins are assigned a three-letter abbreviation and a one-letter symbol. The symbols and molecular

Proteins-I 99

mass of each amino acid is given in Table 5.2. Their molecular formulae are also presented in Figure 5.6. Table 5.2 S.No.

Symbols, systemic names and molecular masses of standard amino acids.

Amino Acid

Three-letter One-letter symbol symbol

Systemic Name

Molecular Mass (Da)

1

Alanine

Ala

A

2-Aminopropanoic acid

2

Arginine

Arg

R

2-Amino-5-guanidinopentanoic acid

174.203

3

Asparagine

Asn

N

2-Amino-3-carbamoylpropanoic acid

132.119

4

Aspartic acid

Asp

D

2-Aminobutanedioic acid

133.104

5

Cysteine

Cys

C

2-Amino-3-mercaptopropanoic acid

121.154

6

Glutamine

Gln

Q

2-Aminopentanedioic acid

147.131

7

Glutamic acid

Glu

E

2-Amino-4-carbamoylbutanoic acid

146.146

8

Glycine

Gly

G

Aminoethanoic acid

9

Histidine

His

H

2-Amino-3-(1H-imidazol-4-yl)-propanoic acid

155.156

10

Isoleucine

Ile

I

2-Amino-3-methylpentanoic acid

131.175

11

Leucine

Leu

L

2-Amino-4-methylpentanoic acid

131.175

12

Lysine

Lys

K

2,6-Diaminohexanoic acid

146.189

13

Methionine

Met

M

2-Amino-4-(methylthio)butanoic acid

149.208

14

Phenylalanine

Phe

F

2-Amino-3-phenylpropanoic acid

165.192

15

Proline

Pro

P

Pyrrolidine-2-carboxylic acid

115.132

16

Serine

Ser

S

2-Amino-3-hydroxypropanoic acid

105.093

17

Threonine

Thr

T

2-Amino-3-hydroxybutanoic acid

119.119

18

Tryptophan

Trp

W

2-Amino-3-(lH-indol-3-yl)-propanoic acid

204.228

19

Tyrosine

Tyr

Y

2-Amino-3-(4-hydroxyphenyl)-propanoic acid

181.191

20

Valine

Val

V

2-Amino-3-methylbutanoic acid

117.148

5.2.3

89.094

75.067

Non-Standard Amino Acids

As mentioned above, all the proteins are made up of only 20 amino acids. Besides these 20 standard amino acids, some other amino acids also occur in nature, which play important physiological roles in metabolic system of organisms, these are also known as non-standard amino acids. Some of these are not proteinogenic or do not contribute in the synthesis of protein structure, for example, GABA – gamma amino butyric acid, ornithine and citrulline.

100 Textbook of Biochemistry Fig. Contd.

Fig. Contd. Fig. Contd.

Proteins-I 101 Fig. Contd.

Fig. 5.6

Molecular formulae of amino acids

GABA is a neurotransmitter that has an inhibitory effect on mammalian central nervous system and regulates the activity of nerve cells. Ornithine and citrulline occur as intermediates in metabolic pathways of standard amino acids. Three amino acids are recognized as non-standard proteinogenic amino acids. These are N-formylmethionine, selenocysteine and pyrrolysine. N-formylmethionine is the first amino acid to be incorporated in polypeptide chain during translation in prokaryotes, chloroplasts and mitochondria. Selenocysteine is encoded by a stop codon UGA in both prokaryotes and eukaryotes. Pyrrolysine is encoded by another stop codon, UAG only in prokaryotes.

5.3

CLASSIFICATION OF AMINO ACIDS

The side chains of amino acids vary in size, shape, charge, hydrogen-bonding capacity, hydrophobic character, and chemical reactivity. Thus, the 20 standard amino acids can be categorized into different classes based on different criteria.

102 Textbook of Biochemistry

5.3.1

Classification based on the Properties of Their R Groups

(1) Aliphatic monoamino monocarboxylic acids This category comprises glycine, alanine, valine, leucine, isoleucine. These are nonpolar and hydrophobic and their side chains help to stabilize protein structure through hydrophobic interactions. (2) Hydroxy amino acids Serine and threonine are the two hydroxy amino acids. Their hydroxyl groups make them polar and their R groups are quite hydrophilic as they can form hydrogen bonds with water. (3) Amino acids with amide group The aliphatic amino acids, asparagine and glutamine are polar due to the presence of amide groups. They are hydrophilic as their side chains are involved in hydrogen bond interactions with the peptide backbone. (4) Acidic and dicarboxylic acids The two amino acids, aspartic acid and glutamic acid are acidic due to the presence of two carboxylic groups. The presence of carboxylic groups in their side chains makes them hydrophilic due to which they tend to orient themselves towards the outer side of a protein molecule. (5) Basic amino acids The amino acids with positively charged R groups are lysine, arginine and histidine. Their charge is due to the presence of amino group at the F position in lysine, guanidinium group in arginine, and an imidazole group in histidine. These groups make them quite hydrophilic and tend to orient them on the outside of the proteins. The F-amino group of lysine often participates in hydrogen bonding. The guanidinium group of arginine remains positively charged in neutral, acidic and even basic environments. It has the ability to form multiple H-bonds due to delocalization of positive charge.

Fig. 5.7

Ionization of histidine at physiological pH

Proteins-I 103

The ionisable R group of histidine has nearly neutral pKa value; thus its unprotonated imidazole is nucleophilic and can serve as a general base, while its protonated form can serve as a general acid (Figure 5.7). Due to its ability to switch between protonated and unprotonated states, histidine can participate in acid-base catalysis and is often a part of catalytic sites of enzymes. (6) Sulphur-containing amino acids Methionine is a sulphur-containing aliphatic amino acid that has a nonpolar thioether group in its side chain. It is quite hydrophobic and is found buried in the hydrophobic environment inside proteins. Methionine generally does not participate in covalent interactions, therefore are not generally involved in catalytic activities of enzymes. The sulphur of both methionine and cysteine is prone to oxidation. Two cysteine residues readily oxidize to yield a dimeric amino acid (cystine) linked by a covalent disulphide bond (Figure 5.8). This remarkable property of formation of disulphide linkages is useful for formation and stabilization of secondary and tertiary conformations of proteins by strong covalent linkages between different polypeptide chains.

Fig. 5.8

Dimerization of cysteine residues into cystine through disulphide bridges

(7) Secondary amino acids Proline is nonpolar and hydrophobic due to the presence of an aliphatic side chain that is bonded to both the nitrogen and the B-carbon atom. Its ring structure makes it conformationally more rigid and restricts the structural flexibility of the polypeptide chain. (8) Aromatic amino acids Phenylalanine, tyrosine, and tryptophan have aromatic side chains due to which these are nonpolar and hydrophobic. The aromatic ring of tyrosine contains a hydroxyl group that is quite reactive and can form hydrogen bonds. Tryptophan consists of an indole ring joined to a methylene (-CH2-) group. Tyrosine and tryptophan are more polar than phenylalanine, because of the tyrosine hydroxyl group and the nitrogen of the tryptophan indole ring. The aromatic rings of tryptophan and tyrosine contain delocalized Q-electrons that strongly

104 Textbook of Biochemistry

absorb ultraviolet light; this accounts for the characteristic strong absorbance of most proteins at 280 nm (Figure 5.9).

Fig. 5.9

5.3.2

Absorption spectra of tryptophan and tyrosine

Classification Based on Human Nutritional Requirement

Amino acids are classified on the basis of nutritional requirement by humans as: (i) Essential or indispensable amino acids: These are 9 in number which cannot be formed inside the human body but have to be acquired through the diet. Lack of these amino acids causes disease such as Kwashiorkar. These are histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. (ii) Non-essential amino acids: They include 11 amino acids which can be synthesized inside the body and humans are not dependent on diet to obtain them. These are asparagine, alanine, arginine, aspartic acid, glutamic acid, cysteine, glutamine, proline, glycine, tyrosine and serine.

5.3.3

Classification Based on Metabolism of Amino Acids

Metabolically, amino acids are classified into three groups i.e., (i) Glucogenic amino acids: They are those amino acids which break down to finally yield glucose. Example, all amino acids except leucine and lysine are glucogenic such as glycine, alanine, serine, asparagine, etc. (ii) Ketogenic amino acids: They are those which are directly degraded into acetyl-CoA, which act as a precursor of ketone bodies, i.e., they break down into end products that are converted further into ketone bodies. Example, leucine and lysine.

Proteins-I 105

(iii) Both keto and glucogenic amino acids: Those amino acids which form both ketone and glucose. They include isoleucine, phenylalanine and tyrosine.

5.4

PHYSICOCHEMICAL PROPERTIES OF ALPHA AMINO ACIDS

Amino acids are colourless crystalline solids and have very high melting point (>200°C). They may be either tasteless (tyrosine), sweet (glycine and alanine) or bitter (arginine). They are completely soluble in water but insoluble in organic solvents like benzene and ether. They have high dipole moments (N) in their aqueous solutions as compared to the amines and organic acids. For example, dipole moment of glycine is high (14D) as compared to propylamine (1.4D) and propionic acid (1.7D).

5.4.1

Stereoisomerism

All amino acids, except glycine, are stereo-specific in nature and have the ability to rotate the plane polarised light either to left or right, i.e., they are optically active constituents of proteins. This phenomenon of stereoisomerism is called chirality, and it occurs in all compounds having an asymmetric carbon atom with four different substituents. The four bonds of the chiral carbon (B-C) of an amino acid are directed towards the four corners of a tetrahedron and there are two possible spatial arrangements of the H and R group with respect to the carboxyl (–COOH) and amino (–NH2) groups. These arrangements are mirror images of each other and are not superimposable, therefore, the two mirror image compounds are also called enantiomers. Just like other chiral compounds, stereoisomers of amino acids are also designated as D or L, analogous to the D & L stereoisomers of glyceraldehyde (Figure 5.10). The classification is based only on the Emil Fischer’s convention in which L and D refer only to the absolute configuration of the four substituents around the chiral carbon. It does not refer to the specific optical properties of the amino acid. The stereoisomerism in amino acids is discussed in terms of the absolute configuration of the four different substituents in the tetrahedron around the asymmetric carbon atom, in reference to a three C sugar glyceraldehyde. The two possible stereoisomers of glyceraldehyde, designated as L and D are given as follows:

Fig. 5.10

Illustration of the structural analogy between glyceraldehyde and amino acid

106 Textbook of Biochemistry

The amino group on the asymmetric carbon atom of alanine can be stereochemically compared with the substituent hydroxyl group on the asymmetric carbon atom of glyceraldehyde, the carboxyl group of alanine can be compared with the aldehyde group of glyceraldehyde, and the R group of alanine can be compared with the –CH2OH group of glyceraldehyde. Thus, isomers stereochemically related in configuration to L-glyceraldehyde are designated L, and those related to D-glyceraldehyde are designated D, irrespective of the direction of rotation of plane polarized light. The amino acids with more than one chiral centre such as threonine (2-amino-3hydroxybutanoic acid) and isoleucine (2-amino-3-methylpentanoic acid) have four different stereoisomers; as depicted in Figure 5.11. The other two stereoisomers are called diastereoisomers or alloisomers because they differ in the configurations at one or more chiral centres and are not mirror images of each other. Diastereoisomers exhibit substantially different chemical and physical properties, while enantiomers have identical physical properties and differ only in optical rotations.

Fig. 5.11

Stereoisomers and alloisomers of (a) threonine and (b) isoleucine

The equal amount of both forms of stereoisomers, i.e., D & L, form an optically inactive mixture known as racemic mixture. Racemization of L-amino acids residues to their D-isomers in food proteins often occurs due to processing of food and affects the nutritional quality of food. It is noteworthy that all naturally occurring proteins consist exclusively of the L forms of amino acids. The presence of only L forms of amino acids in living systems probably ensures the steady polymerization of protein subunits into functional proteins. The specific chirality of active sites of enzymes might also contribute to the selectivity and specificity of enzyme action.

Proteins-I 107

D-amino acids rarely occur naturally in organisms except for some bacteria where they are found in the cell wall peptides and are produced by non-ribosomal mechanism of synthesis. In higher organisms, D-amino acids may be introduced into polypeptide chains through post-translational epimerization.

5.4.2

Acid-Base Properties of Amino Acids

In solution, the amino acids exist in their ionic form. The carboxyl group (–COOH) loses a proton giving a carboxylate ion, and the amino group (–NH2) is protonated to an ammonium ion. This structure is called a dipolar ion or a zwitterion (Figure 5.12).   

 

 





–



  

Fig. 5.12

    

Formation of a zwitterion

Zwitterion is an ampholyte as it acts both as a proton donor (an acid) or proton acceptor (a base). The acidic part of the amino acid molecule is the –NH3+ group instead of –COOH group, while the basic part is the –COO– group instead of –NH2 group. Amino acids are less acidic than most of the carboxylic acids and less basic than most of the amines (Figure 5.13).

Fig. 5.13

Acidic and basic properties of a zwitterion

The amino acid species that predominates in the solution depends upon the pH of the solution. In an acidic solution, the carboxylic group remains protonated to a free –COOH group, and the molecule has an overall positive charge. When the pH is raised at about pH 2.2, it loses its proton. This point is called the first acid-dissociation constant (pKa1). When the pH is raised further, the –NH3+ group loses its proton at about pH 9 or 10; this point is called the second acid-dissociation constant (pKa2). Beyond this pH, the amino acid has an overall negative charge (Figure 5.14).

Fig. 5.14

Change in ionic property of an amino acid with pH

108 Textbook of Biochemistry

The acid-base property of amino acids can be used to prepare the buffer of required pH through Henderson-Hasselbalch equation. [HA] pKa = pH + log _____ ...(5.1) [A–] Here, [HA] is a weak acid and [A–] is a conjugate base. Through Henderson-Hasselbalch equation, the nature of ionic species of amino acids that are present in aqueous solutions at different pH can be determined. As seen in the titration curves, when strong base is added, it removes protons from the solution, more and more acid is in the conjugate base form, and the pH increases. When the moles of base added are equal to the half of total moles of acid, the weak acid and its conjugate base are in equal amounts. The ratio of conjugate base and weak acid is equal to 1, i.e., according to the Henderson-Hasselbalch equation pH = pKa,

...(5.2)

–

when [HA] = [A ] On further addition of base, the conjugate base form becomes more till the equivalance point is reached when all of the acid is in the conjugate base form.

5.4.3 Titration Curve of Amino Acids The deprotonation pattern of amino acids can be studied by the acid-base titration method. In solution, acid dissociation constants of the two equilibrium reactions are given as: H3N+ CH2COOH(aq) R H3N+ CH2COO – (aq) + H+(aq) Ka1

+ – + [H 3N CH2COO ][H ] __________________ = [H3N+ CH2COOH]

H3N+ CH2COO – (aq) R H2NCH2COO – (aq) + H+(aq) Ka2

– + [H 2NCH2COO ][H ] __________________ = [H3N+ CH2COO –]

...(5.3 and 5.4)

Use the Henderson-Hasselbalch equation to calculate the pI. At isoelectric point, pH = pI [H3N+ CH2COO –] pI = pKCOOH + log ________________ [H3N+ CH2COOH] pI = pK Adding up: 2pI = pKCOOH + pK

+ NH3

+ NH3

[H2NCH2COO –] _______________ + log [H3N+ CH2COO –]

[H2NCH2COO –] ________________ + log [H3N+ CH2COOH]

when pH = pI, [H2NCH2COO –] = [H3N+ CH2COOH] 2pI = pKCOOH + pKNH+3 or pI = {pKCOOH + pKNH+3 }/2

...(5.5-5.8)

Proteins-I 109

Figure 5.15 shows a titration curve for glycine. At highly acidic pH, the predominant + species of glycine is entirely cationic ( H3N–CH2–COOH). On titrating it with the base, the first proton is lost by the –COOH group and at the midpoint of this titration equimolar +

+

concentrations of the proton-donor ( H3N–CH2–COOH) and proton-acceptor ( H3N–CH2– COO–) species are present. At this point the pH is equal to the pKa of the –COOH group i.e., 2.34. On further titration, another point of inflection is achieved at which the first proton has been dissociated completely and second proton is yet to be dissociated. At this +

pH the dipolar species of glycine exists H3N–CH2–COO– predominantly, which is known as a ‘zwitter ion’ and this point of pH is known as ‘isoelectric point’, designated as pI. It is defined as the characteristic pH at which the net electric charge of a molecule is zero. The pI of glycine occurs at pH 5.9. + Further titration yields another proton from the –NH3 group of glycine. The midpoint of pH of this titration that corresponds to the pKa2 is 9.60. Complete removal of protons is observed at about pH 12, where glycine exists as H2N–CH2–COO-.

Fig. 5.15 Titration curve for glycine

One useful application of the acid-base property of amino acids is that by varying the pH of the solution, the charge on the amino acids can be manipulated and utilized for

110 Textbook of Biochemistry

separating and identifying amino acids and proteins by chromatography or electrophoresis. The titration curve of glycine also defines two buffering regions, one near pKa1 of 2.34 that ranges widely upto 1 pH unit, and other near pKa2 of 9.6. Similarly, Figure 5.16 shows the titration curve of alanine

Fig. 5.16 Titration curve of alanine

Separation of proteins and peptides is largely based upon its amino acid composition, as they act as weak acids or bases. Isoelectric point is therefore a critical feature of most protein separation and analytical techniques such as isoelectric focusing and 2-D gel electrophoresis. The pKa values and the isoelectric points of the 20 standard amino acids are listed in Table 5.3. Table 5.3 The pKa values and the isoelectric points of the 20 standard amino acids Acid

pI

pKa1

pKa2

(B–COOH)

(B–+NH3)

Alanine

6.01

2.35

9.87

Arginine

10.76

1.82

8.99

Asparagine

5.41

2.14

8.72

Aspartic acid

2.85

1.99

9.9

Cysteine

5.05

1.92

10.7

Glutamic acid

3.15

2.1

9.47

Glutamine

5.65

2.17

9.13

Glycine

6.06

2.35

9.78

Histidine

7.6

1.8

9.33

Isoleucine

6.05

2.32

9.76 Contd.

Proteins-I 111 Table Contd...

5.5

Leucine

6.01

2.33

9.74

Lysine

9.6

2.16

9.06

Methionine

5.74

2.13

9.28

Phenylalanine

5.49

2.2

9.31

Proline

6.3

1.95

10.64

Serine

5.68

2.19

9.21

Threonine

5.6

2.09

9.1

Tryptophan

5.89

2.46

9.41

Tyrosine

5.64

2.2

9.21

Valine

6

2.39

9.74

GENERAL CHEMICAL REACTIONS OF ALPHA AMINO ACIDS

Amino acids perform most of the chemical reactions characteristic of both amines and carboxylic acids. However, the reactions require specific conditions of pH, so that the carboxyl group does not interfere with an amino group reaction, and vice versa. The most significant reactions are esterification of the carboxyl group and acylation of the amino group, which are used to protect one of the respective groups while the other group is undergoing a chemical reaction. Some key reactions are described ahead.

5.5.1

Carboxylic Acid Esterification

Under acidic conditions, carboxylic acid group undergoes esterification while amino group is converted to its protonated form (NH3+) so that it does not interfere with the process. Usually, it requires an acidic catalyst such as gaseous HCl. Esterification of amino acids, most commonly with methyl, ethyl, and benzyl esters, is often used to prevent the carboxyl group from reacting in an unwanted manner. A typical Fischer esterification of an amino acid with an alcohol that results into a stable ester is illustrated in the following example.

...(5.9)

5.5.2

Amine Acylation

At high pH, the amino group of an amino acid can be converted to an amide using an acylating reagent. Acylation of the amino group is used to protect it from undesirable nucleophilic reactions. The carboxylic groups exist as carboxylate anions at high pH, therefore do not interfere with the reaction. Many acid chlorides and anhydrides are used as acylating agents. A general reaction illustrating an amine acylation is given below.

112 Textbook of Biochemistry

...(5.10)

5.5.3 The Ninhydrin Reaction The alpha-amino acids, except proline, undergo a unique reaction with ninhydrin (2,2-dihydroxyindane-1,3-dione), to produce a purple coloured imino derivative called Ruhemann’s purple, which is a resonance-stabilized anion derived from the original amino acid. In this reaction, CO2 is released and the side chain of the amino acid is lost as an aldehyde. Ninhydrin reaction is commonly used for determining presence of amino acids, for quantitative estimation of amino acids by spectrophotometry and for visualizing spots or bands of amino acids that have been separated by chromatography.

...(5.11)

5.6 THE PEPTIDE BOND Amino acids polymerize to form peptides and proteins by forming a bond called peptide bond which is a covalent linkage between two neighbouring amino acids. Each unit of amino acid in the polymer is called a residue. Peptide bond formation between two amino acids yields a dipeptide such as glycylalanine. Three amino acids joined by two peptide bonds form a tripeptide. In the same manner four, five and six amino acids may link to form tetra, penta and hexapeptide, respectively. Conventionally, a peptide consisting of a few amino acid residues (2-20) is called an oligopeptide, while the one having about 100 or more amino acid residues is called a polypeptide. Proteins can be made up of thousands of amino acid residues having molecular weights as high as a few thousands kDa. Peptide bond is a covalent bond formed by a condensation reaction in which a molecule of water is removed from the –COOH group of one amino acid and the –NH2 group of another. During dehydration, the –OH comes from carboxyl group and –H from amino group (Figure 5.17). Thus, a polypeptide chain has polarity because its one terminal has an B-amino group while the other terminal has an B-carboxyl group. Conventionally, the sequence of a peptide chain starts with the amino acid with free –NH2 group and continues until the last residue with free –COOH group. For example, Fig. 5.17 Peptide bond formation in a tetrapeptide Ala-Tyr-Gly-Phe, alanine is the

Proteins-I 113

amino-terminal (N-terminal) residue and phenylalanine is the carboxyl-terminal (C-terminal) residue.

5.6.1

Formation of Peptide Bond

Chemical synthesis of peptide bond seems to be quite unfavourable as the equilibrium of this reaction favours hydrolysis instead of synthesis, and requires an input of energy. The intracellular synthesis of peptide bond occurs through an intensive process involving a series of events under the guidance of genetic information. The sequence of nucleotides in DNA determines a complementary sequence of nucleotides in RNA, which, in turn, specifies the amino acid sequence of a protein. Each of the 20 amino acids is encoded by one or more specific sequences of three nucleotides, known as genetic code (Table 5.4). Under this process, the amino acid is first recognized by a transfer RNA molecule on the basis of this genetic code. For every triplet code, there exists a specific tRNA in the cytosolic pool. The specific amino acid is attached to the t-RNA through an ester bond forming an activated form aminoacyl-tRNA; this reaction is catalysed by an enzyme, aminoacyl-tRNA synthetase with the expenditure of energy derived from ATP. The aminoacyl-tRNA is acted upon by ribosome which facilitates the peptide bond formation between two consecutive amino acids. The mechanism is precise and all proteins are synthesized starting at their N-terminus and moving towards their C-terminus (Figure 5.18).

     

      

       



     

      

                  

Fig. 5.18 The process of protein synthesis in a cell

114 Textbook of Biochemistry

Genetic Codons The genetic information remains stored in DNA in the form of specific sequence of four nucleotides, adenine, guanine, cytosine and thymine (T). Each functional gene sequence is transcribed into a complementary sequence in mRNA. A set of three nucleotide bases on mRNA encodes for one amino acid; this triplet of bases is called a codon. All the 20 standard amino acids are encoded by the genetic codons. Except two amino acids– methionine and tryptophan. All other amino acids can be coded by more than one codon, i.e., the codons are degenerative. The codon AUG is called start codon as it codes for methionine which initiates the protein synthesis. Besides these codons do not code for any amino acid and function as stop codons which are UAA(ochre), UGA(opal) and UAG(amber). These codons help to terminate the polypeptide chain during protein synthesis. Thus there are 64 codons in all, out of which 61 encode for amino acids while 3 are stop codons. Table 5.4

Genetic codons for 20 standard amino acids

Glycine-GGU,GGC, GGA,GGG Valine-GUU,GUC,GUA,GUG Leucine-CUU,CUC,CUA,CUG,UUA,UUG Isoleucine-AUU, AUC, AUA Arginine-CGU,CGC,CGA,CGG,AGA,AGG Cysteine-UGU,UGC Lysine-AAA,AAG Proline-CCU,CCC,CCA,CCG Threonine-ACU,ACC,ACA,ACG Methionine-AUG Histidine-CAU,CAC Tyrosine-UAU,UAC Serine-UCU,UCC,UCA,UCG,AGU,AGC Alanine-GCU,GCC,GCA,GCG Phenylalanine-UUU,UUC Glutamine-CAA, CAG Glutamate-GAA, GAG Asparagine-AAU, AAC Aspartic acid-GAU, GAC Tryptophan-UGG

5.6.2

Characteristic Features of Peptide Bond

The amino acid units are joined together to form a polypeptide chain through a condensation reaction in which –COOH group of one amino acid reacts with –NH2 group of next adjacent amino acid. This reaction involves removal of a molecule of water while joining two molecules of amino acids (dehydration synthesis) and generating one amide. The bond thus formed by this condensation is called peptide bond (–CO–NH–).

Proteins-I 115

Considering the structural orientation of atoms, the peptide bond yields a chain of repeating units of –C–C–N–C–C–N which is called the backbone of polypeptide and R groups of amino acid residues form the side chains. Due to the presence of carbonyl (C=O) group, the backbone has appreciable hydrogen-bonding capacity. The interactions among the functional groups and side chains help to stabilize the protein structures. Peptide bonds are planar in nature, i.e., all amino acids lie in the same plane. The rigidity of the polypeptide chain is contributed by the amide bonds and its limited conformational flexibility is chiefly due to the rotations along the bonds involving alpha-carbon atoms. The amide bond accounts for the rigidity due to nitrogen electron pair delocalization into the carbonyl group that results in significant double bond character between the carbonyl carbon and the nitrogen. This causes resonance between carbonyl C and amine N which prevents rotation around this bond and makes it planar. The C–N distance in a peptide bond is 1.32 Å, which lies between the values of C–N, i.e., 1.49 and C=N, i.e., 1.27 Å. Due to these planar peptide bonds two conformational configurations are possible, i.e., cis and trans. In cis form, two alpha carbons are on the same side whereas in trans form they are on opposite side with respect to each other in peptide bond. Almost all the peptide bonds in protein are trans rather than cis (1000:1 – trans-cis ratio) because of steric hindrance in between the groups attached to the two alpha carbons which are on the same side. Exceptionally, peptide bonds involving proline may frequently be found in cis configurations because its cis and trans isomers often have equivalent energy. The flexibility in the polypeptide chain exists because of the presence of single bonds between the amino group and the B-carbon atom and between the B-carbon atom and the carbonyl group. These bonds allow some degree of freedom for rotation of two adjacent rigid peptide units about the peptide bond. This helps in acquiring different orientations and protein folding. Because of the formation of peptide bond in between the B-amino and B-carbonyl groups of all the non-terminal amino acids, they are not able to ionize and thus do not contribute to the gross acid-base behaviour of the peptide. Thus, the acid-base character is only dependent upon the R group because they are free and are able to ionize easily. Like amino acids, all peptide bonds have their specific isoelectric point (pI) and a specific titration curve which defines its electrochemical properties. The free amine and carboxylic acid groups on a peptide chain form a zwitterionic structure at their isoelectric pH.

5.7

PROTEIN SEQUENCING

Each protein is unique owing to the distinctive number and sequence of amino acid residues. The particular function of the protein depends upon its three-dimensional structure which, in turn, is determined by its primary structure. Thus, the primary structure of a protein can yield lot of information regarding its biological roles and can be highly valuable in biochemical investigations. The very first attempt of elaborating the protein sequence was done by Frederic Sanger in 1953, when he determined the amino acid sequence of bovine insulin

116 Textbook of Biochemistry

and was awarded the Nobel Prize in 1958 for this work. This was a landmark achievement in the field of biochemistry, paralleled by the discovery of double-helical structure of DNA by James D. Watson and Francis Crick in the same year, suggesting a relationship between the nucleotide sequence in DNA and the amino acid sequence in proteins that defines a gene function. The proteins can be of various sizes and may attain different levels of structural complexity due to the presence of many polypeptide chains. The elucidation of primary structure of a protein thus requires its degradation into simpler forms. The process of sequencing of proteins is explained below. (a) Denaturation of proteins: Complex proteins are first denatured to convert their globular compact structures into simple linear forms. Denaturation can be done by heating the protein or treating it with denaturating agents like urea (Figure 5.19).

Figure 5.19

Denaturation of protein on heating

(b) Breaking of disulphide bonds: Inter- and intra-chain disulphide bonds interfere with the enzymatic or chemical cleavage of the polypeptide and interrupt the sequencing procedure, therefore disulphide bonds are cleaved by using the reducing agents dithiothreitol (DTT) or 2-mercaptoethanol. Disulphides are reduced thiols or sulfhydryl groups, which are highly susceptible to oxidation and must be protected to prevent them from reforming the disulphide bond. In order to prevent their reoxidation, the thiols are further treated with alkylating agents such as iodoacetic acid that reacts with cysteine residues to form S-carboxymethylcysteine (Figure 5.20). (c) Determination of number of polypeptide chains or subunits: The number of polypeptide chains or subunits can be determined by end group analysis. The end group of an amino acid sequence helps us to determine that on which side the Nterminal is facing and on which side the C-terminal is placed. End group analysis can be done for both N-terminal and C-terminal. The methods for both are given here. (i) N-terminal analysis: The N-terminal amino group is free and can act as a nucleophile, whereas the B amino groups of all the other amino acids form the amide linkages and are much less nucleophilic. The amino-terminal amino acid residue can be labelled and identified through various methods. Sanger’s method for N-terminal residue analysis involves treating a peptide with 1-fluoro2,4-dinitrobenzene (FDNB), which is very reactive towards nucleophilic aromatic substitution. The fluoride from 1-fluoro-2,4 dinitrobenzene is displaced

O

O

C

Fig. 5.20

Cysteine

C

O

O

C O

HC—CH2—S—H

O

C

O

–

HN

C

O

HN

Fig. 5.21

N-terminal analysis by Sanger’s method

C

–

Carboxymethylation by lodoacetate (ICH2COO )

OOC—CH2—S—CH2—S—CH2—CH2—CH

O

H—S—CH2—CH

O

Carboxymethylated Cysteine residues

HC—CH2—S—CH2—COO–

HN

Reduction by Dithiothreitol (DTT)

HN

Cleavage of disulphide bonds and fixation of cysteine residues through carboxymethylation

HN

HC—CH2—S—S—CH2—CH

HN

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118 Textbook of Biochemistry

by amino group of the N-terminal amino acid and a peptide is generated in which the N-terminal nitrogen is labelled with a 2,4-dinitrophenyl group (DNP) (Figure 5.21). Another method uses 1-dimethyl aminophthalene-5-sulfonyl chloride or dansyl chloride for N-terminal analysis. The amino-terminal residue is labelled with dansyl chloride to yield dansyl polypeptide chain, which is further subjected to acid hydrolysis to liberate all amino acids along with the N-terminal dansyl amino acid. Amino acids are separated and fluorescence of the dansyl amino acid is detected. Dansyl derivatives are comparatively easier to detect than the dinitrophenyl derivatives. The acid hydrolysis destroys the polypeptide chain so this method cannot be used to sequence it beyond the amino-terminal residue. However, it can be used to determine the number of polypeptides in a protein that have different amino-terminal residues (Figure 5.22).

Fig. 5.22

N-terminal analysis by dansyl chloride method

Proteins-I 119

(ii) C-terminal analysis: C-terminal analysis can be done either by using hydrazine or by using carboxy-peptidase enzyme. In hydrazine method, a peptide is heated with anhydrous hydrazine at high temperature (~ 100°C) so that all amino acid residues are converted to hydrazides except for the terminal amino acid. The terminal amino acids however, still have a free carboxy terminal, thus can be separated and identified. The hydrazine treatment products are often subjected to reverse phase HPLC for identification (Figure 5.23).

Fig. 5.23

C-terminal analysis by hydrazine method

In carboxypeptidase method (Figure 5.24), a polypeptide is enzymatically degraded in a successive manner using different carboxy-peptidases. Commonly used carboxy-peptidases are A, B, C and Y. These enzymes function in a specific manner such that each one acts upon certain specific amino acids. Carboxypeptidase A hydrolyzes all residues at C terminal except proline, arginine, and lysine, while carboxypeptidase B is effective when only Lys and Arg are present at C terminal. If carboxypeptidase A and B are used together, all amino acid residues towards C terminal are hydrolyzed except proline. Carboxypeptidase C is not specific and is able to hydrolyze any amino acid residue at C terminal.

Fig. 5.24

C-terminal analysis by carboxy-peptidase method

(d) Cleavage of the polypeptide chain: Sequencing of large polypeptides does not often give accurate results due to several reasons such as long and incomplete reactions, interference due to impurities from side reactions and intra-chain disulphide bonds. Direct sequencing can be efficiently carried out for peptides having up to about 50-100 residues only. Polypeptide chain thus requires to be

120 Textbook of Biochemistry

further fragmented by enzymatic method or chemical method. Enzymes that catalyze the hydrolytic cleavage of peptide bonds are called proteolytic enzymes or proteases. Interestingly, proteases cleave certain peptide bonds adjacent to particular amino acid residues, which allow the fragmentation of polypeptide chain in a predictable manner. Proteases can be classified as endopeptidases and exopeptidases. Among endopeptidases, pepsin is relatively nonspecific, preferably cleaves at the amino end of hydrophobic aromatic residues Phe, Tyr, Trp. The chymotrypsin which is also highly specific for hydrophobic amino acids, cleaves at the carboxyl end of Phe, Trp, Tyr and also Leu & Met, provided that the next residue is not proline. Trypsin is the most commonly used proteolytic enzyme. It is highly specific and only cleaves the peptide bonds in which the carbonyl group is contributed by the basic amino acids Lys and Arg irrespective of the length sequence of amino acid in the polypeptide chain. Exopeptidases cleave at the terminal end of a polypeptide chain and sequentially remove one amino acid at a time. For example, leucine aminopeptidase cleaves the N-terminal leucine residue. Fragmentation of polypeptide can also be done by chemical methods, for example, a number of chemical reagents cleave the peptide bond adjacent to specific residues. Cyanogen bromide (CNBr) specifically cleaves Met residues at the C-end, 2-nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues, formic acid cleaves at aspartic acid-proline (Asp-Pro) peptide bonds; hydroxylamine cleaves at asparagine-glycine (Asn-Gly) peptide bonds breaking imide link to Gly.

Fig. 5.25

Phenylisothiocyanate reaction in Edman degradation

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5.8

PROTEIN SEQUENCING BY EDMAN DEGRADATION

The amino acid sequence of each peptide fragment is determined through repeated cycles of Edman degradation. This method was introduced by Pehr Edman in which he used an innovative method for sequencing that involved labelling and cleaving or degrading the peptide. In Edman degradation the amino-terminal residue from a peptide is removed, leaving rest of the peptide bonds intact. This process uses phenylisothiocyanate or PITC that specifically reacts with the N-terminal amino acid residue of the polypeptide fragment under mild alkaline conditions to yield a phenylthiocarbamoyl (PTC) polypeptide (Figure 5.25). The PTC polypeptide is then exposed to anhydrous trifluoroacetic acid that cleaves the N-terminal peptide bond, while causing the N-terminal residue to cyclize to form a thiazolinone derivative. The N-terminal residue is now cleaved while the remaining polypeptide remains unchanged. The thiazolinone derivative is then extracted with an organic solvent and is converted to the more stable phenylthiohydantoin (PTH) derivative under acidic conditions, which is then identified by the chromatographic method. The next exposed amino-terminal residue now labelled to PITC, is removed and identified. The remaining polypeptide can then be subjected to further rounds of Edman degradation until the entire sequence is determined (Figure 5.26).

Fig. 5.26

Edman degradation

Reconstructing Polypeptide Sequence After determining the sequences of each segment by Edman method, all the peptide fragments are ordered to reconstruct the complete amino acid sequence of polypeptide chain. Trypsin digestion specifically generates fragments with C-terminal lysine or arginine. The segment lacking lysine or arginine at its C-terminal end can be assigned to the C-terminal side of the original polypeptide. Further, another enzyme or chemical with

122 Textbook of Biochemistry

different specificity is used for cleavage of samples, for example, cyanogen bromide specifically breaks the peptide bonds after methionine residue at the C end. The sequences thus generated are overlapped with those resulting from the trypsin cleavage and the correct order of the peptide (Figure 5.27).

Fig. 5.27

Specific cleaving action of trypsin

Locating the Positions of Disulphide Bonds Identifying the locations of disulphide bonds in the proteins is the final step in sequencing. This is done by again fragmenting the polypeptide with trypsin or other reagent, but before reducing and alkylating its disulphide bonds. The peptides obtained this time are compared with those obtained after reducing disulphide bonds. The separation of peptide fragments and their comparison can be done with the help of electrophoresis. The fragmentation pattern of peptides generated by trypsin before breaking the disulphide bonds will be different than earlier because the polypeptide linked by disulphide bond will not be fragmented by trypsin and will remain intact.

5.9





SUMMARY s 0ROTEINS ARE HIGHLY DIVERSE MACROMOLECULES WHICH SERVE AS ESSENTIAL STRUCTURAL and functional components of a cell and drive all the metabolic activities of living organisms. s !LLPROTEINSAREPOLYMERSOFAMINOACIDS/UTOFLARGENUMBEROFAMINOACIDSFOUND in nature, only 20 amino acids are encoded by the genetic code and used for the synthesis of proteins. s !MINOACIDSAREVARIOUSLYCLASSIFIEDONTHEBASISOFHUMANNUTRITIONALREQUIREMENT eight amino acids are called essential amino acids which must be obtained from the diet, these are isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Rest of the 12 amino acids can be produced within the body. s !MINOACIDSARECOLOURLESSCRYSTALLINESOLIDSANDHAVEHIGHMELTINGPOINT # and are stereospecific in nature due to the presence of a chiral carbon (B-C). Stereoisomers of amino acids are also designated as D or L. All naturally-occurring proteins consist exclusively of the L forms of amino acids.

Proteins-I 123







s )N SOLUTION THE AMINO ACIDS EXIST IN THEIR IONIC FORM !T A CERTAIN P( KNOWN AS ‘isoelectric point’, an amino acid exists as a dipolar ion called a zwitterion which acts both as a proton donor (an acid) and proton acceptor (a base). s !MINO ACIDS CAN PERFORM THE CHEMICAL REACTIONS CHARACTERISTIC OF BOTH AMINES AND carboxylic acids such as esterification of the carboxyl group and acylation of the amino group. s !MINO ACIDS CAN POLYMERIZE TO FORM PEPTIDES AND PROTEINS BY FORMING A PEPTIDE bond, a covalent linkage formed by a condensation reaction in which a molecule of water is removed from the –COOH group of one amino acid and the –NH2 group of another. s 4HE INTRACELLULAR SYNTHESIS OF PEPTIDE BOND OCCURS THROUGH AN INTENSIVE PROCESS OF protein synthesis involving a series of events under the guidance of genetic information. This process is also called translation. s 4HESPECIFICSEQUENCEOFAMINOACIDRESIDUESINAPOLYPEPTIDECHAINAREDECIPHERED through protein sequencing. This process involves denaturing of proteins, disrupting disulphide bonds, determining number of polypeptide chains and reconstructing polypeptide sequence.

MULTIPLE-CHOICE QUESTIONS 1. All proteins are polymers of .................... . (a) amino acids (b) monosaccharides (c) organic acids (d) nucleotides 2. Proteins and polypeptides are biosynthesized by using .................... standard amino acids. (a) 10 (b) 20 (c) 30 (d) 40 3. Enzymes, the biological catalysts, are essentially made up of .................... . (a) proteins (b) carbohydrates (c) lipids (d) organic acids 4. Sulphur-containing amino acids are .................... and .................... . (a) alanine and lysine (b) valine and glycine (c) glutamine and histidine (d) methionine and cysteine 5. Non-proteinogenic gamma amino butyric acid (GABA), ornithine and citrulline are called .................... amino acids. (a) standard (b) unimportant (c) non-standard (d) non-essential 6. Nine amino acids which cannot be formed inside the human body and are acquired dietarily are called .................... amino acids. (a) essential (b) non-essential (c) unimportant (d) non-standard 7. Amino acids exhibit the property of an ampholyte and act both as a proton donor or proton acceptor by forming .................... . (a) strong acid (b) weak acid (c) zwitter ion (d) alkali 8. A set of three nucleotide bases on mRNA forming a triplet that encodes for an amino acid during protein synthesis is called a .................... . (a) genome (b) proteome (c) nucleotide (d) genetic codon

124 Textbook of Biochemistry 9. The three codons UAA (ochre), UGA (opal) and UAG (amber) are called .................... as they terminate the process of protein synthesis. (a) stop codons (b) start codons (c) chain elongation codons (d) chain initiation codons 10. Reducing agents dithiothreitol (DTT) and C-mercaptoethanol are used to cleave .................... bonds in proteins and polypeptides. (a) ester (b) disulphide (c) glycosidic (d) peptide

Answers 1. (a) 9. (a)

2. (b) 10. (b)

3. (a)

4. (d)

5. (c)

6. (a)

7. (c)

8. (d)

Short-Answer Type Questions 1. 2. 3. 4. 5.

Why is proline known as imino acid? Mention three letter abbreviations for each of the 20 amino acids. Some non-proteinogenic amino acids play important physiological roles. Justify. Classify standard amino acids on the basis of their metabolism. What are genetic codons? Give their significance.

Long Answer Type Questions 1. Explain the general structure of amino acids and explain stereoisomerism in amino acids. 2. Classify the amino acids on the basis of the properties of their R-groups. 3. Discuss the acid-base properties of amino acids giving an example of titration curve of a standard amino acid. 4. Describe the general chemical reactions of B amino acids. 5. What is a peptide bond, how is it formed? Mention the characteristic features of a peptide bond. 6. Give a detailed account of the process of protein sequencing.

6 Proteins-II 6.1

INTRODUCTION

Proteins in cells act as building blocks. They maintain the structural integrity of the cell along with diverse functions like storage, transport, immunity, etc. Proteins are actually small fragments of amino acids. This fragment joins together in different forms, forming different conformations for various biological functions of proteins. X-ray diffraction and crystallization of proteins are the best known methods for the analysis and structural identification of proteins. These methods have some limitations and are also time taking. Involvement of computerization in biological systems leads to development of novel methods for protein structural analysis like 3D structural modelling of proteins. These methods are Homology modelling, ab initio method and threading. Nowadays protein 3D structures are created and deposited along with its coordinates in a specialized protein database PDB (Protein Data Bank). On the basis of structural organization proteins can be divided into different levels, like primary, secondary and tertiary (Figure 6.1). The three-dimensional structure of a protein depends upon many factors and all are interlinked to each other; like structures of a protein largely depends upon amino acid sequence, the structure of a protein is responsible for its function of protein, isolation of protein depends upon ability of protein to form stable structure. Among all the forces, non-covalent forces are responsible for the stabilizing proteins specific structure. The relationship of amino acids and its connections to form polypeptides, this finally leads to function of the protein being a big puzzle for biologists. Arrangement of atoms in an amino acid (proteins) is called conformation of protein. Change in the conformations of proteins is actually change in the state of proteins without breaking or damaging the covalent bonds of the protein. Generally, these conformation changes occur due to change in the rotation of bonds. These conformational changes take place with every atom of the protein but only a few conformations exist and function because every conformation of the protein is not stable, so only those conformations are functional in a protein which are stable. Stable conformation is actually conformation having lowest Gibbs free energy (G). Proteins of any folded conformation which is functional is known as native protein. Protein stability is very crucial for functioning of a protein and generally it is achieved through hydrophobic residues, that are largely buried in the protein

126 Textbook of Biochemistry

interior and always stay away from water molecules, similar to this hydrogen bonds must be maximum within the proteins.

Fig. 6.1 Organization of protein: structural level

6.2

PRIMARY STRUCTURE

The primary structure of proteins is simple and it forms only peptide bonds for joining amino acids. There are 20 different amino acids that help in the formation of protein in the cell. The amino acid contains both acidic carboxylic group and basic amino group. These two different (acidic and basic) groups makes amino acids unique and different amino acids join together to form a long chain of amino acid linked by peptide bond. So peptide is actually amide bond formed between the –NH2 of one amino acid and the –COOH of another. The peptide bond is actually a type of covalent bond. A small chain up to fifty amino acids is called peptide and the terms, polypeptide and protein are generally used for longer sequences. A protein can be made up of one or more polypeptide molecules. Every end of a peptide or protein sequence has a free carboxyl group called the carboxyterminus or C-terminus while the next end has an amino-terminal or N-terminus. The amino acids differ from each other by means of their side chains. These side chains are actually responsible for different physical, chemical and structural properties of protein or final protein, and are also used to classify the proteins like acidic, basic and neutral. The amino acid information is encoded in the DNA after transcription (DNA strand to make a complimentary messenger RNA strand, mRNA) and translation (the mRNA sequence is used in the synthesis of the chain of amino acids which make final protein). For a proper functioning of proteins, it must undergo post-translational modifications, such as glycosylation or phosphorylation. The amino acid sequence makes up the primary structure of the protein and three-dimensional or tertiary structure of the protein.

6.3

NATURE OF PEPTIDE BOND

In 1930s Linus Pauling and Robert Corey laid the foundation of protein structure which was based on their experimentations on proteins (Figure 6.2).

Proteins-II 127

Fig. 6.2

Structure showing formation of peptide bond

They started with the analysis that the B carbons of adjacent amino acid residues were arranged like CB-C-N-CB separated by three covalent bonds (Figure 6.3(a)). C-N bonds of dipeptides and tripeptides are shorter than the C-N bond in a simple amine and atoms form coplanar peptide bonds. This conclusion was based on the X-ray diffraction studies of amino acid crystals.

Fig. 6.3(a)

Resonance caused due to positive and negative groups

This proves that carbonyl oxygen and amide nitrogen are involved in partial sharing of two pairs of electrons between them. A small dipole develops between the partial negative charge of oxygen and partial positive charge of nitrogen. The six atoms of the peptide group lie in a single plane, while oxygen atom of the carbonyl group and the hydrogen atom of the amide nitrogen lie at the trans position to each other. The rotation is allowed to N-CB and the CB-C bonds while C-N bonds are unable to rotate freely because they possess partial double-bond character (Figure 6.3(b)). The backbone of a polypeptide appears as a rigid plane with consecutive planes sharing a common point of rotation at CB. Rotation of bond angles with respect to CB are two types named K
(phi) for the N-CB bond and Z (psi) for the CB-C bond. The K and Z are known as dihedral angles. When all peptide groups lie in a same plane and polypeptide in a fully extended conformation, the values to K and Z are defined as 180°. The value of K and Z can have any value between –180° and +180°, but this value is limited due to steric interference between polypeptide

128 Textbook of Biochemistry

Fig. 6.3(b)

Common rotation point of CB (picture taken from Lehninger)

backbone and amino acid side chains. G. N. Ramachandran introduced a plot based on the allowed values for K and Z. The graph is plotted between K versus Z and it clearly indicates the stability of a polypeptide. This plot is named as Ramachandran plot and used for the structural stability of the polypeptide.

Ramachandran Plot A Ramachandran plot, is a plot between K and Z angles developed in 1963 by G. N. Ramachandran and his colleagues. This plot is used to explain the geometrical features of the backbone. Backbone dihedral angles K against Z of amino acid residues in the protein structure are unique for specific secondary structural elements. The plot is usually used to determine which conformations are possible for a protein but its usage is very important in structure validation of proteins. Some regions in the map are forbidden because of geometrical restraints in the backbone, the calculations of the dihedral angles serve as a measure of the structural integrity of the protein structure. (Ramachandran, G.N.; Ramakrishnan, C.; Sasisekharan, V. (1963). “Stereochemistry of polypeptide chain configurations”. Journal of Molecular Biology, 7: 95–99).

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6.4

SECONDARY STRUCTURE

In 1951 first of all, Pauling and Corey on the basis of their experiments and observations predicted the existence of secondary structures of proteins. Secondary structures are actually formed due to local folding conformation within a polypeptide. This folding is due to formation of bonds between atoms of the backbone (polypeptide chain apart from the R groups – so secondary structure does not involve R group atoms.) Among different secondary structures of proteins, B helix and the C pleated sheet are the most common and stable as well. Secondary structures are formed due to repeating amino acids with the same (K, Z) angles. Turns, coils and 310 helices are the other secondary structure elements. Secondary structure refers to a local spatial arrangement of the polypeptide backbone. Two regular arrangements are common, B helix and C sheets as discussed below.

6.4.1

B Helix

Linus Pauling in 1951, predicted the B-helical structure of protein as one of the major secondary structures. It is the simplest helical arrangement of polypeptide chain having rigid peptide bonds, while other single bonds are free to rotate. Helical structure is formed due to the polypeptide backbone being tightly wound around a virtual (imaginary) axis in longitudinal direction at the middle of the helix. All R groups of the amino acids residues are protruding in outward direction of the helix. A single repeating unit of the helix turns 5.4 Å. The amino acids in the helix have Z angles from – 45° to – 50° and for K angle, it is equal to – 60° and each helical turn includes 3.6 amino acids residues. H-Bonds are formed between the carbonyl CO-atom of residue n and the N-H of residue n+4. The complete B-helical structure has a dipole moment aligned along its helix, which is negative at the C-terminus and positive at the N-terminus. This dipole is stabilized, by capping the N- and C-terminus, by placing acidic residues at the N-terminus and basic residues at the C-terminus. The amino acid sequence and length are the two important factors that decide the formation of B-helical structure of a protein. B helix are found in two forms, right- and left-handed helix. The simplest method is to understand it as our right hand thumb pointing in upwards direction and four fingers curled pointing in clockwise direction, this is right-handed helix (Figure 6.4). Similar to this left-handed thumb showing clockwise direction is leftFig. 6.4 Right-handed and left-handed handed helix. B-helix stability depends upon helix.

130 Textbook of Biochemistry

few factors like (1) the electrostatic repulsion or attraction between residues of successive amino acids and charges of R groups; (2) bulkiness of adjacent R groups; (3) interactions between different R groups spaced three (or four) residues apart; (4) presence of Proline and Glycine residues; and (5) interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the B helix.

6.4.2

C Sheet

Pauling and Corey in 1951 by using X ray methods, found that C sheets are extended conformations of polypeptides. C conformations form zigzag sheets rather than a helix. These sheets join each other side by side forming C sheets. The sheets are formed when two or more polypeptide chains run along side of each other linked by hydrogen bonds in a regular fashion. Side chains protrude from the sheet alternating in up and down directions. These hydrogen bonds are formed between the main chain, C=O and N-H groups. In B helix hydrogen bonds are formed in the same element of secondary structure, while in C sheet hydrogen bonds are formed between different segments of the polypeptide. The side chains of neighbouring residues in a C-strand point in opposite directions. On the basis of amino and carboxyl terminal, C sheets can be parallel or antiparallel and bonding in parallel C sheet is stronger as compared to antiparallel sheet (Figure 6.5). In parallel strand, the CO and –NH group between which the hydrogen bonds forms lie closer to each other than antiparallel strands. In parallel C sheets, the H-bonded strands run in the same direction. In antiparallel C sheets, the H-bonded strands run in opposite directions so it forms linear hydrogen bonds. Blended hydrogen bonds are weaker as compared to straight bonds.

Fig. 6.5

Antiparallel and parellel C sheets

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6.4.3

Random Coils

Few secondary structure do not have any specified pattern. However, they play major role in the structural integrity of the proteins in the form of coils, loops and sheets. These structures are formed due to mixing of different amino acids. These structures are other than B helix and C sheets. In most of the globular proteins, these random coils are found along with B helix and C sheets.

6.4.4

C Turn

Protein structures contain a few specified structures which allow the peptide to turn back and it is called C turn. They are the connecting units, connecting B helix and C sheets. It leads to the formation of a tertiary structure, held together by noncovalent interactions. These turns are found at the surface of proteins, where the two central amino acid peptide groups form hydrogen bond with water. Proline and glycine residues are often associated with C turns, as the former is smaller and more flexible than the latter because peptide bonds involving the imino nitrogen of proline readily assume the cis configuration, a form that is particularly amenable to a tight turn.

6.5 TERTIARY STRUCTURE In a protein, three-dimensional atomic arrangement leads to formation of tertiary structure of proteins. The tertiary structures of proteins generally have single polypeptide with one or many structures differentiated into different domains. In a tertiary structure, amino acid present far from each other comes closer after folding of the peptides and it forms a compact structure of protein. Interacting parts of polypeptides are held together by different types of weak and strong interactions. Covalent bonds are the common type and different polypeptides join together by means of disulphide crosslinks. Proline, Glycine, Threonine and Serine are the residues found at the locations where bending and specific angle is required to turn a protein. Tertiary structures are mainly represented by “Ribbon models”. This model represents the geometry of secondary structures like spring-like ribbons represent B helices and the flat side-by-side ribbons represent C sheets. Often arrows are used at the ends of ribbons, indicating the direction of polypeptide from N-terminus to C-terminus.

6.5.1

Classification of Tertiary Structures

SCOP (Structural Classification of Proteins) is a database (scop.mrc-lmb.cam.ac.uk/) that includes entries of Protein DataBase (PDB), which is a protein 3D structural database. Mostly all proteins have structural similarities with other proteins and, with this it can be concluded that proteins share a common evolutionary origin. SCOP database provides a detailed survey of all known protein folds and information of their close relatives. SCOP divides a tertiary structure of proteins into four classes. 1. B−helix includes all the proteins having B−helices usually wrapped around common hydrophobic core. Approx. 22% of proteins in PDB fall into this category.

132 Textbook of Biochemistry

2. C−protein mainly contains C−strands arranged into C−sheets, which are stabilized by hydrogen bonds (HB network). These proteins have structures in different layers with hydrophobic core. About 16% of PDB proteins belong to this class. 3. B/C proteins contain alternating pattern of B− and C−structures (15% of PDB database). 4. B+C proteins also contain mixed B− and C−structure, but it is spatially separated (29% of PDB structures).

6.5.2 Tertiary Structures Stabilizing Forces There are many different forces responsible for the stability of tertiary structures: 1. Hydrogen bonding: It is formed between polar side chains of the amino acids of the peptides. 2. Hydrophobic interactions: London forces attract nonpolar side chains of one residue to the amino acid to the other nonpolar side chains residue of another amino acid and form a water free pocket at the interior of protein giving a very compact and folded structure to the protein. 3. Salt bridges: It is a noncovalent interaction and an attractive force. It is formed between the positive formal charge on polar basic amino acid residue and a negative formal charge on a polar acidic residue. 4. Disulphide bridges: It is a covalent bond formed by oxidation of two thiol (SH) groups. Protein disulphide bonds are known as disulphide bridges. Each cysteine residue contains a thiol group in its side chain that is capable of forming a disulphide bridge with another cysteine residue. 5. Dipole-dipole and ion-dipole forces: It is formed between the polar side chains and/or peptide groups.

6.6

QUATERNARY STRUCTURE

Some proteins contain two or more separate polypeptide chains, or subunits, which may be identical or different. The arrangement of these protein subunits in three-dimensional complexes constitutes quaternary structure. It refers to the organization of subunits in a protein with multiple subunits (an “oligomer”). These oligomeric subunits (may be identical or different) have a defined stoichiometry and arrangement. Subunits are held together by many weak, noncovalent interactions (hydrophobic, electrostatic), example, haemoglobin. Forces of protein folding: (1) Polar side chain interactions occur either through hydrogen bonds or electrostatic interactions. (2) van der Waals interactions, along with hydrophobic forces, ‘favour packing of the amino acid interactions. These forces help in the formation of the hydrophobic core

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of the protein. This exploits the hydrophobic tendency of nonpolar amino acid side chains. (3) Disulphide bond formation: It provides stability to the protein.

6.7

CLASSIFICATION OF PROTEINS

Proteins are classified on the basis of source, function, structure action of protein, etc.

6.7.1

Classification of Proteins on the Basis of Source

1. Animal proteins are derived from animal sources like egg, meat, milk and fish. Animal proteins are treated as high quality proteins as they contain essential amino acids. 2. Plant proteins are actually of low quality due to limitations of essential amino acids.

6.7.2. Classification of Proteins Based on Shape 1. Globular or Corpuscular Proteins have oval or spherical shape due to their length: width (axial ratio) less than 10, commonly not more than 3 or 4. Globular proteins are more complex conformations than fibrous protein; are more dynamic and functional. These are usually soluble in water or in aqueous medium of acids, bases, salts or alcohol. They are usually associated with tertiary and quaternary structures of proteins. Cytochrome C, blood proteins, serum albumin, antibodies (= immunoglobulins), haemoglobin, hormones and enzymes are examples of globular proteins. 2. Fibrous or Fibrillar Proteins look like a fibre as they have length: width (axial ratio) more than 10. They mostly originate from animal sources and do not dissolve in water, organic solvents, dilute acids, alkalis and salts. They can stretch and recoil to their original form. It is a heterogeneous group and includes the proteins of connective tissues, bones, blood vessels, skin, hair, nails, horns, hoofs, wool and silk. Few important fibrous proteins are: 1. Collagens: These are the major proteins of white connective tissues (cartilage and tendons). They are mesenchymal in origin. Collagen is about half of the total protein content of the human body. It produces soluble gelatin after treating with boiling water, dilute acids or alkalies. 2. Keratins: Skin, hair, feathers, horns, hoofs, nails are epithelial tissues, containing a specific protein known as keratin. It is ectodermal in origin majorly found in epithelial tissues. It usually contains sulphur and amino acid, cysteine. 3. Elastins: Also of mesenchymal origin; form the major constituents of yellow elastic tissues (ligaments, blood vessels); differ from collagens in not being converted to soluble gelatins. 4. Fibroin: It is the main constituent of the fibres of silk, composed mainly of glycine, serine and alanine units.

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6.7.3

Classification of Proteins Based on Solubility and Composition

The system divides the proteins into three major groups: (a) Simple (b) Conjugated (c) Derived (a) Simple Proteins or Holoproteins: These are mainly globular type except for scleroproteins which are fibrous in nature. This group includes proteins containing only amino acids, as structural components. On acid decomposition, these liberate the constituent amino acids. On the basis of solubility, they are further subdivided into: 1. Protamines and histones: These are basic proteins, found in animals (sperm cell) and occur almost entirely in animals. They are soluble in water, unlike most other proteins, not coagulated by heat. High content of basic amino acids like lysine, and arginine are found. Protamines are virtually devoid of sulphur and aromatic amino acids. Examples—nucleohistones of nuclei clupeine from herring sperm, salmine from salmon sperm, globin of haemoglobin and sturine from sturgeon. 2. Albumins: It is widely found in nature abundantly in seeds of plants. Water soluble, and dissolve in dilute acid, base and salt solution. Examples ovalbumin from white of egg, lactalbumin of milk whey, leucosine in cereals, legumeline in legumes, serum albumin from blood plasma and myosin of muscles. 3. Globulins: Globulins may be soluble or insoluble in water. On the basis of solubility in water, it is pseudoglobulins (soluble) or euglobulins (insoluble). Euglobulins are more common in nature. Common globulins of various plant seeds include hemp (edestin), soybeans (glycinine), etc. Examples of pseudoglobulins: pseudoglobulin of milk whey. Euglobulins are serum globulin from blood plasma, ovoglobulin from eggwhite, myosinogen from muscle. 4. Glutenins: Glutenin of wheat, corn and oryzenin from rice are actually glutelins. Isolated from seeds of the plants, are insoluble in water, dilute in salt solutions and alcohol solutions but are soluble in dilute acids and alkalies; coagulated by heat. 5. Prolamines: These have also been isolated only from plant seeds, insoluble in water and dilute salt solutions but soluble in dilute acids and alkalies and also in 60–80% alcohol solutions; not coagulated by heat. 6. Scleroproteins or Albuminoids: Commonly known as animal skeleton proteins found in animals; insoluble in water, dilute solution of acids, bases and salts and also in 60–80% alcohol solutions; not attacked by enzymes. E.g., collagen of bones, elastin in ligaments, keratin in hair and horry tissues and fibroin of silk. (b) Conjugated or Complex Proteins or Heteroproteins: It is also a globular protein except the pigment in chicken feathers which is probably of fibrous nature. These proteins

Proteins-II 135

are linked by nonprotein part called prosthetic group. These groups may be either a metal or a compound. On acid decomposition, these liberate the constituent amino acids and the prosthetic group. The conjugate protein is further divided on the basis of presence and type of prosthetic group. The various divisions are metalloproteins (metalloproteids), chromoproteins (chromoproteids), glycoproteins, phosphoproteins, lipoproteins and nucleoproteins. 1. Metalloproteins: These are the proteins linked with various metals. These may be of stable nature or may be more or less labile. Based on their reactivity with metal ions, the metalloproteins may be classified into three groups: A. Metals strongly bound by proteins: Few heavy metals like Hg, Ag, Cu, Zn are strongly bound with proteins like collagen, albumin, casein, etc., through the –SH radicals of the side chains. Few proteins show strong binding affinities for Fe (siderophilin) and Cu (ceruloplasmin). Siderophilin, known as transferrin, is an important metalloprotein and constitutes about 30% of the total plasma protein. It helps in iron transport in cells. Ceruloplasmin is a blue copper-binding protein found in the blood of humans and other vertebrates. This protein contains about 90% of copper in serum. It helps in regulation of copper absorption by reversibly releasing and binding copper at various sites in the body. B. Metals bound weakly by proteins: Calcium (Ca) belongs to this category. Here the binding takes place with the help of radicals possessing the electron charge. C. Metals which do not couple with proteins: Sodium (Na) and potasium (K) belong to this group. These form compounds with nucleic acids where apparently electrostatic bonds are present. 2. Chromoproteins: These proteins are associated with coloured pigment. These pigments have also been found among the enzymes like catalase, peroxidase and flavoenzymes. Similarly, chlorophyll is present in leaf cells in the form of a protein, the chloroplastin. The chloroplastin dissolves in water as a colloid and is readily denatured. E.g., myoglobin, hemoglobin, hemocyanin, hemoerythrin, cytochromes, flavoproteins, catalase, etc. 3. Glycoproteins and Mucoproteins: These are the proteins containing carbohydrate as prosthetic group. Glycoproteins contain small amounts of carbohydrates (less than 4%), whereas mucoproteins contain comparatively higher amounts (more than 4%). E.g., glycoproteins— egg albumin, elastase, certain serum globulins and certain serum albumins; mucoproteins—ovomucoid from egg white, mucin from saliva and Dioscorea tubers, osseomucoid from bone and tendomucoid from tendon. 4. Phosphoproteins: These are proteins linked with phosphoric acid; mainly acidic. E.g., casein from milk and ovovitellin from egg yolk. 5. Lipoproteins: Proteins forming complexes with lipids (cephalin, lecithin, cholesterol) are called lipoproteins; soluble in water but insoluble in organic solvents, e.g., lipovitellin and lipovitellenin from egg yolk, lipoproteins of blood. The lipoproteins

136 Textbook of Biochemistry

are, in reality, the temporary intermediates in the process of transfer of lipids from the site of absorption to the site of utilization. The classification of lipoproteins is frequently based on an operational definition, i.e., the migration of the fraction in a density gradient separation. Lipoproteins have been classified into four categories (a) Very high density lipoproteins (VHDLs), densities greater than 1.21. (b) High density lipoproteins (HDLs), density range of 1.063 to 1.21. (c) Low density lipoproteins (LDLs), densities range between 1.05 and 1.063. (d) Very low density lipoproteins (VLDLs), density range is from 0.93 to 1.05. 6. Nucleoproteins: These compounds are formed by combination of nucleic acid and protein, especially protamines and histones. These are usually the salt-like compounds of proteins, as both have opposite charges bond together by electrostatic forces. They are present in nuclear substances as well as in the cytoplasm. These may be considered the sites for the synthesis of proteins and enzymes, example, yeast nucleoproteins. (c) Derived Proteins: They are actually derivatives of proteins. Form due to action of heat, enzymes or chemical reagents. This group also includes the artificially-produced polypeptides. They may be of two types. 1. Primary derived proteins: These are the derivatives of proteins, having same molecular size as parent molecule. Example, coagulated proteins, proteans metaproteins or infraproteins. 2. Secondary derived proteins: These are derivatives of proteins in which the hydrolysis has occurred and the resulting molecules are of smaller than the original proteins. Examples, proteoses, peptones and polypeptides.

6.8

PHYSICAL PROPERTIES 1. Colour and taste: Proteins are tasteless, colourless, homogeneous and crystalline in nature. 2. Shape and size: Proteins vary in shapes from long fibrillar structures to simple crystalloid spherical structures. Two distinct and common patterns are found. A. Globular proteins: These proteins are spherical in shape, found mainly in seeds and in leaf cells of plants. These are bundles formed by folding of protein chains. E.g., edestin, pepsin, insulin, ribonuclease, etc. B. Fibrillar proteins: These are thread-like or ellipsoidal in shape and found mainly in animal muscles. E.g., fibrinogen, myosin, etc. 3. Molecular weight: Proteins have large molecular weights ranging between 5 × 103 and 1 × 106. It is found that the values of molecular weights of many proteins lie close to or multiples of 35,000 and 70,000. The average molecular weight of the 20 amino acids is about 138. Mainly smaller amino acids predominate in most proteins, hence the average molecular weight of an amino acid is nearer 128. Since

Proteins-II 137

a molecule of water (MW = 18) is eliminated to produce each peptide bond, the average molecular weight of the amino acid residue is about 128 – 18 = 110. 4. Colloidal nature: Because of their giant size, the proteins exhibit many colloidal properties, like slow diffusion rate and may produce considerable light-scattering in solution, resulting in Tyndall effect (visible turbidity). 5. Amphoteric nature: Proteins are amphoteric in nature just like amino acids as it has both acid and alkali in it. They migrate in an electric field and its migration direction depends upon the net charge possessed by the molecule. Protein charge is influenced by the pH value. Each protein has a fixed value of isoelectric point (pl) at which it will move in an electric field.

Isoelectric point It is the pH point at which the number of cations is equal to that of anions. So at isoelectric point, the net electric charge of a protein is always zero. But the total charge on the protein molecule (sum of positive and negative charges) at this point is always maximum. Thus, the proteins are dipolar ions or internal salts or zwitterions (German for ‘ion of both kinds’; amphoteric ions) at pl and exist in solution as: (H3N+) m —R—(COO–) n

6. Ion binding capacity: Proteins are amphoteric in nature and form salts of both cations and anions. At a given pH, mixture of different proteins will include cations and anions both and the salts of protein-protein combinations will be formed. This is found in tissues since both acidic and basic proteins are present. Many ions form insoluble salts with proteins and serve as excellent precipitating agents for proteins. For example, many acid dyes find practical use for colouring the insoluble proteins like silk and wool. 7. Solubility: The solubility of proteins is influenced by pH. Lowest solubility is observed at isoelectric point and it increases with increasing acidity or alkalinity. The reason is that as protein molecules exist as either cations or anions, repulsive forces between ions are high, since all the molecules possess excess charges of the same sign. Thus, they will be more soluble than in the isoelectric state. 8. Optical activity: Proteins are levorotatory in nature; as in a solution they are able to rotate the plane of polarized light to the left.

6.9

CHEMICAL PROPERTIES

A. Hydrolysis: Proteins are hydrolyzed by a variety of hydrolytic agents. Proteins can be hydrolyzed by acidic agents (conc. HCl), by alkaline agents (2N NaOH) and by proteisolytic enzymes (pepsin and trypsin). Alkaline hydrolysis causes loss of optical activity (or racemization) of the amino acids and leads to destruction of certain amino acids like arginine, cysteine, cystine, serine. Enzyme hydrolysis is mainly used for the isolation of certain amino acids like tryptophan.

138 Textbook of Biochemistry

B. Properties due to presence of COOH group 1. Reaction with alkalies (Salt formation): The carboxylic group of amino acids can release a H+ ion with the formation of carboxylate (COO—) ions. These may be neutralised by cations like Na+ and Ca2+ to form salts. Thus, amino acids react with alkalies to form salts.

Sodium salt of glutamic acid (monosodium glutamate) is used commercially as a flavouring agent. It imparts a meat-like flavour to soups, for example. 2. Reaction with alcohols (Esterification): Proteins react with alcohols and esters are produced. The esters, so obtained, are volatile in contrast to the free amino acids.

3. Reaction with amines: Amino acids react with amines to form amides.

C. Properties due to presence of NH2 group 1. Reaction with mineral acids (salt formation): When either free amino acids or proteins are treated with mineral acids like HCl, the acid salts are formed.

The basic amino acids, arginine and lysine react with CO2 in the presence of air to form carbonate salts. Because of this property, they are usually stored and also sold in the form of their monochlorides. 2. Reaction with formaldehyde: Hydroxy-methyl derivatives are formed with reaction with formaldehyde. These derivatives are water insoluble.

Proteins-II 139

3. Reaction with benzaldehyde: Schiff’s bases are formed.

D. Properties due to presence of COOH and NH2 group 1. Reaction with phenyl isocyanate: With phenyl isocyanate, hydantoic acid is formed further concerted to hydantoin.

2. Reaction with phenyl isothiocyanate or Edman reagent: Phenyl isothiocyanate also reacts similarly with amino acids to produce thiohydantoic acid. On treatment with acids in nonhydroxylic solvents, the latter cyclizes to thiohydantoin.

140 Textbook of Biochemistry

3. Reaction with phosgene. With phosgene, N-carboxyanhydride is formed.

E. Properties due to presence of R group 1. Biuret test: Compounds having peptide bonds produce purple colour when treated with an alkaline 0.2% copper sulphate solution (or biuret reagent). This reaction is termed as ‘biuret reaction’ since it is also given by the substance biuret. The colour deepens as the number of peptide bonds is increased and the proteins produce a deep blue-violet colour due to the probable formation of a coordination complex whose structure is given below: The test is, in fact, given by biuret as well as any similar structure having two amide or peptidebonds linked directly or through an intermediate carbon atom. The required unit is shown below between the two broken lines: All proteins except dipeptides, therefore, respond to this reaction. This reaction is widely used both as a qualitative test for the detection of proteins and as a quantitative measure of protein concentration. 2. Xanthoproteic test: Yellow colour develops on boiling proteins with conc. HNO3 due to the presence of benzene ring. This reaction is due to the nitration of the phenyl rings (of tyrosine, tryptophan and phenylalanine) to yield yellow substitution products, which turn orange upon addition of alkali. 3. Millon’s test: Red colour develops when proteins are heated with Hg–NO3 in HNO2. The reaction is specific for tyrosine and takes place between mercuric and mercurous nitrates and tyrosine residues of the protein. Tryptophan also responds to this reaction. 4. Folin’s test. Blue colour develops with phosphomolybdotungstic acid in alkaline solution due to the presence of phenol group. The test is specific for tyrosine. F. Properties due to presence SH group 1. Nitroprusside test: Red colour develops with sodium nitroprusside in dilute NH4. OH. The test is specific for cysteine. 2. Sullivan test: Cysteine develops red colour in the presence of sodium 1,2-naphthoquinone-4-sulfonate and sodium hydrosulphite.

Proteins-II 141

6.10

DENATURATION AND RENATURATION OF PROTEIN

It is a process in which the properties of proteins change. It refers to the loss of biologic function of the protein. In many cases denaturation is followed by coagulation, where the small protein molecules formed after denaturation get collected and form aggregates (Figure 6.7). Denaturation can be achieved by many physical, chemical and mechanical agents. Mechanical agents like shaking, heat treatment, cooling, freezing, high hydrostatic pressure (5000 to 10,000 atm.), UV rays and rubbing, etc. Chemical agents includes organic solvents (acetone, alcohol), ionizing radiations (like X-rays, radioactive and ultrasonic radiations), aromatic anions (salicylates), some anionic detergents (like sodium dodecyl sulphate), etc. Most common example of protein easily denatured by shaking or heat is the albumin of eggwhite. Hsien Wu in 1931 suggested that denaturation leads mainly to the unfolding of the peptide chain and causes disorganization of the internal structure of protein. Denatured proteins are hydrolyzed more easily because of this reason. Due to denaturation peptide chains or the protein molecules unroll, few bonds split and new sites get exposed. These exposed sites provide a platform for certain proteolytic enzymes causing further hydrolysis. So, the H-bonds linking the two peptide chains are partly freed and the disulphide (–S–S–) bonds also linking the two peptide chains split open to yield the free sulphydryl (–SH) groups. Frank Putnam in 1953, gives a list of certain changes followed by a protein undergone denaturation process. These are decrease in their size and shape of the molecule and also decrease in solubility. Cessation of their biochemical activity as enzymes or hormones and increased activity of some radicals present in the molecule such as —SH group of cysteine—S—S— bond of cystine and phenolic group of tyrosine.

Fig. 6.6

Renaturation and denaturation of protein

142 Textbook of Biochemistry

Further, it also underwent changes in optical rotation in the direction and increased levorotation. Denaturation also leads to alteration in surface tension and loss of antigenicity. Few proteins are able to return into their original state and this type of denaturation is known as reversible type, while few are not allowed to revert back into their original state; this is called ‘irreversible’ type. The process of regaining normal protein properties by a denatured protein is called renaturation or refolding (Figure 6.6). During renaturation, certain antibodies may cause a re-rolling of the protein bundles so that most of the original bonds are recovered.

6.11

PROTEIN FOLDING

It is a process which leads to form spatial structure of protein from a linear amino acid sequence of a polypeptide chain. The process of folding is initiated by collapse of the polypeptide chain, which is driven by the desire of hydrophobic amino acids to escape the polar solvent water. Protein folding is a quite fast process and reaches the stable native conformation having minimum energy. After synthesis of protein, folding of protein inside the cell takes place which is more complicated. Proteins are synthesized from the N- to the C-terminus by using ribosomes. The newly synthesized nascent polypeptides do not have complete information necessary for folding. The concentration of cellular macromolecules like ribosomes, nucleic acids and proteins are high. In this crowded macromolecular environment, exposed hydrophobic amino acids of nascent polypeptides and folding intermediates may interact inappropriately leading to misfolding and aggregation. While, as a consequence of this problem, cytosolic proteins should fold as fast as possible, the folding of proteins destined for another compartment has to be delayed in order to allow translocation through the membrane in the unfolded state. Due to Brownian motion and thermal vibrations, even native proteins are always in danger of spontaneously unfolding and losing their active structure. This feature of proteins is probably the evolutionary price for conformational flexibility, which is essential for protein function. For most proteins, there are only small energy barriers between the native and the misfolded state. A number of proteins are specifically thermolabile and their folding status is even more susceptible to changes in the cellular environment. Stress conditions, like a sudden increase in temperature, can therefore lead to unfolding, aggregation, or degradation of many proteins (Figure 6.7).

Fig. 6.7

Denaturation and renaturation of protein

Proteins-II 143

6.12

CHAPERONES

They are the protein repair units found inside the cells. Under sudden stress conditions like increase in temperature proteins can unfold and aggregate. Chaperonins serve as a protective unit, and prevent aggregation by binding to the misfolded proteins. Examples of chaperons are DnaK (Hsp70) and GroEL (Hsp60) along with regulatory co-chaperones both working together and the small heat shock proteins, IbpAB (sHsps). The chaperonins are a class of chaperons that help in folding. All chaperonins can be referred to as chaperones. Chaperonins are classified into two groups that are structurally similar and quite diverse in sequence. Both the groups divide along two distinct evolutionary lines. The group I chaperonins are found in prokaryotes and endosymbiotic organelles such as mitochondria and chloroplasts. Group II chaperonins exist in archaea and eukaryotic cytosol (Figure 6.8) (1). Group I proteins include GroEL in bacterial cytosol, Hsp60 in mitochondria and Rubisco binding protein (RuBisCoBP) in chloroplasts. They consist of double-torous shaped complexes, composed of either 1 or 2 (RuBisCo) subunits. Group II chaperonins constitute Thermosome/TF55 in archaea and TRiC/CCT in the eukaryotic cytosol. They share the double-torous structure with their group I counterparts but are composed of 2-3 subunit types in archaea and 8 (Cct1-8) subunit types in eukaryotes. The members of the two groups function through a similar overall mechanism but differ in the method of substrate encapsulation, which is evident to inspection of their architectures: Group I chaperonins employ a detachable “lid” structure (GroES/Hsp10) that binds in an ATP-dependent fashion, whereas group II chaperonins employ a built-in protrusion structure which may be either marginate or open upward in a polypeptide-accepting state and that then closes when the ring binds ATP to produce the folding-active encapsulated state. Despite the different encapsulation mechanisms, the ATP-directed reaction cycles of the two families of machine appear to be fairly similar, directed by virtually identical equatorial ATP-binding domains.

Fig. 6.8 Classification of chaperones (http://pdslab.biochem.iisc.ernet.in/hspir/hsp60.php)

144 Textbook of Biochemistry

A good example of group I chaperonins is the paradigmatic Escherichia coli GroEL/ GroES system. It consists of 14 subunits arranged in a double heptameric ring complex and requires a ring-shaped co-factor GroES for its function. Each heptamer encloses a central cavity that forms the folding chamber for the polypeptide substrate. The constituent subunits of the heptamer contain three domains; the equatorial domain harbouring the ATP binding site, a substrate binding apical domain and a middle hinge-domain that enables communication between equatorial and apical domain. The equatorial domain is more or less conserved among paralogous subunits. Most of the sequence divergence lies in the apical domain that contains the substrate binding sites. The apical domain forms the opening of GroEL cylinder and exposes a number of hydrophobic residues towards the ring cavity for substrate binding.

Fig. 6.9

Molecular mechanism of chaperone function

The ATP binding to GroEL occurs through allosteric mechanisms (Figure 6.9). ATP binds to the subunits within a ring via concerted mechanism in a positively cooperative manner. However, negative cooperativity exists for sequential binding of ATP between the rings. This ensures asymmetric behaviour of the complex as a two-stroke machine. Prior to ATP binding, both the rings are in the tensed (T) state. Negative cooperativity

Proteins-II 145

allows the nucleotide to bind to cis ring converting it to relaxed (R) state and lowering the nucleotide affinity of the trans ring. T state has high affinity for substrate and R state has a lower affinity. Unfolded polypeptide substrates, exclusively having either B-helical or C-sheets, bind to the exposed hydrophobic patches at the apical domain of GroEL in T state. Nucleotide binding results in elongation of the oligomeric structure and twisting of the apical domains resulting in occlusion of the hydrophobic binding sites. Hence, ATP binding causes substrate release and binding of GroES. This allows establishment of intramolecular interactions in protected aggregation free environment. Hydrolysis of ATP in the cis ring followed by nucleotide binding in the trans ring causes dissociation of GroES and substrate release. Complete folding of the polypeptide to native state prevents further interaction with the hydrophobic sites, otherwise the substrate is primed to the next round of chaperone mediated folding.

6.13











SUMMARY

s %VERY PROTEIN HAS A THREE DIMENSIONAL STRUCTURE THAT REFLECTS ITS FUNCTION s 0ROTEIN STRUCTURE IS STABILIZED BY MULTIPLE WEAK INTERACTIONS (YDROPHOBIC INTERACtions are the major contributors for stabilizing the globular form of most soluble proteins; hydrogen bonds and ionic interactions are optimized in the specific structures that are thermodynamically most stable. s 4HE NATURE OF THE COVALENT BONDS IN THE POLYPEPTIDE BACKBONE PLACES CONSTRAINTS on structure. The peptide bond has a partial double bond character that keeps the entire six-atom peptide group in a rigid planar configuration. s 0ROTEIN IS FOUND IN ALL CELLS IN THE BODY )T IS THE CELLS MAJOR STRUCTURAL component. s 0ROTEIN IS REQUIRED TO FORM BLOOD CELLS IN THE BODY 7ITHOUT IT THE HUMAN BODY cannot survive. s (UMAN HAIR IS MADE FROM KERATIN WHICH IS A TYPE OF PROTEIN )F THERE ARE A LOT OF sulphur links, the person's hair will be curly. s 0ROTEIN IS A MACRONUTRIENT WHICH PROVIDES ENERGY FOR THE BODY &AT AND CARBOHYdrates are also macronutrients. Fat provides nine calories per gram, and protein and carbohydrates each provide four calories per gram. s 0ROTEIN DEFICIENCY CAN LEAD TO A VARIETY OF HEALTH ISSUES SUCH AS KWASHIORKOR decreased immune function, edema, thinning of nails and hair, pain in the muscles and joints, and weakness. s (IGH PROTEIN FOODS INCLUDE COTTAGE CHEESE 'REEK YOGURT EGGS MILK BEEF POULTRY pork, turkey, tuna, halibut, salmon, navy beans, lentils, nuts, peanut butter, quinoa, and wheat germ. s 6EGETABLESHIGHINPROTEININCLUDEPEAS SPINACH KALE SPROUTS BROCCOLI MUSHROOMS Brussels sprouts, artichokes, edamame, potatoes and avocados.

146 Textbook of Biochemistry





s 7HENPROTEINFUNCTIONINTHEBODYSTOPSWORKINGPROPERLYTHISCANRESULTINSEVERAL different diseases such as cancer, Alzheimer's disease, and Creutzfeldt-Jakob disease (the brain looks like a sponge). s 0EOPLE TRYING TO LOSE WEIGHT CAN HELP TO DECREASE THEIR CRAVINGS BY ENSURING THEY are consuming high quality protein at meals. This can make a person feel fuller longer. s %GGS APPEAR TO HAVE THE HIGHEST PROTEIN RATING OF ANY FOOD s 0OULTRY SUCH AS CHICKEN OR TURKEY HAS MORE PROTEIN THAN BEEF

MULTIPLE-CHOICE QUESTIONS 1. Which of the following does not affect the stability of a B-helix? (a) Electrostatic repulsion (b) Bulkiness (c) Interaction between R groups spaced three residues apart (d) Occurrence of alanine and glycine residues 2. Which of the following is not true about secondary protein structure? (a) The hydrophilic/hydrophobic character of amino acid residues is important to secondary structure. (b) The ability of peptide bonds to form intra-molecular hydrogen bonds is important to secondary structure. (c) The B helix, C pleated sheet and C turns are examples of protein secondary structure. (d) The steric influence of amino acid residues is important to secondary structure. 3. Which of the following bonds is not involved in tertiary type of protein structure? (a) Disulphide bond (b) Hydrogen bonding (c) Salt bridges (d) Hydrophilic interactions 4. Which of the following is false? (a) Lysozyme has S-S linkage. (b) Ribonuclease has S-S linkage. (c) Heme group in cytochrome c is covalently linked to the protein on two sides. (d) Ribonuclease has SH-SH linkage. 5. Which of the following forces is favourable for protein folding? (a) Hydrophobic interactions (b) Hydrogen bonding (c) van der Waals forces (d) Ionic bonding 6. Which of the following is false about fibrous protein? (a) It is in rod- or wire-like shape. (b) Keratin and collagen are the best examples. (c) Haemoglobin is the best example. (d) It provides structural support for cells and tissues.

Proteins-II 147 7. Which of the following is a function of chaperone protein? (a) It degrades proteins that have folded improperly. (b) It provides a template for how the proteins should fold. (c) It rescues proteins that have folded improperly and allows them to refold properly. (d) It degrades proteins that have folded properly. 8. Which of the following is chaperone in E. coli? (a) Hsp70 (b) Hsp40 (c) DnaA (d) DnaK and DnaJ 9. The main antibody of both primary and secondary immune response is: (a) IgG (b) IgA (c) IgE (d) IgM 10. Folin’s test is used to identify: (a) lipids (b) proteins (c) carbohydrates (d) urea

Answers 1. (d) 9. (a)

2. (a) 10. (b)

3. (d)

4. (d)

5. (a)

6. (c)

7. (c)

8. (d)

Short Answer Type Questions 1. 2. 3. 4.

Discuss about Ramachandran plot and its uses in protein structure prediction. Describe important bonds for stabilizing tertiary structure of proteins. Discuss few important fibrous proteins. Mention few important chemical reactions, which take place due to the presence of COOH group. 5. What are chaperones?

Long Answer Type Questions 1. 2. 3. 4. 5.

Discuss protein secondary structure and its different forms. Discuss about physical properties of proteins. What is protein tertiary structure and what are the stability factors of tertiary structure? Explain protein denaturation and renaturation in detail. What is immunoglobins? Discuss its types and structure in detail.

References 1. John T. Edsall, Hsien Wu and the First Theory of Protein Denaturation (1931) Advances in Protein Chemistry, Volume 46, 1995, Pages 1-5. https://doi.org/10.1016/S0065-3233(08)60329-0 2. Putnam, F. W. (1953). Protein denaturation. In: The Proteins: Chemistry, Biological Activity and Methods, Vol. 1, pt. b. New York: Academic Press. 807-92.

7 Nucleic Acids 7.1

INTRODUCTION

Nucleic acids are the basic molecules and a remarkable property of the living cells. They have the ability to produce their exact replicas and maintain continuity of life. Nucleic acids have coded information and instructions which are used for making a complete organism. These nucleic acids are found to be major components and are present on chromosomes. Small gene is present on chromosomes and these genes are made up of nucleic acids. Elemental analysis of nucleic acids showed the presence of phosphorus, along with C, H, N & O. Nucleic acid is actually a polymer made up of small monomer units called nucleotides. In any living cell, two types of nucleic acids are found, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is present in nucleus of the cell and helps in transfer of genetic characteristics from parent to child. Similarly, RNA is present in all parts of the cell and its primary function is synthesis of variety of proteins by which biological functions takes place within a cell. The nucleic acids (DNA and RNA) are considered as the ‘molecules of heredity’.

7.2





HISTORICAL BACKGROUND OF NUCLEIC ACIDS s s s s

&ISCHER IN S DISCOVERED PURINE AND PYRIMIDINE BASES IN NUCLEIC ACIDS :ACHARIS IN  IDENTIFIED NUCLEIN WITH CHROMATIN 3ACHS IN  STATED THAT NUCLEINS OF SPERM AND EGG ARE DIFFERENT (ERTWIGINCONFIRMEDTHATNUCLEINISRESPONSIBLEFORTHETRANSMISSIONOFHEREDItary characteristics from one generation to another. s 'EHEIMRAT !LBRECHT +OSSEL IN  OF THE 5NIVERSITY OF (EIDELBERG 'ERMANY identified that histones and protamines are associated with nucleic acids. He also discovered that histones were basic proteins. He identified presence of two purine and two pyrimidine bases in nucleic acids and for this he was honoured with Nobel 0RIZE FOR 0HYSIOLOGY OR -EDICINE IN  s !LTMANN IN  INTRODUCED THE TERM NUCLEIC ACID IN PLACE OF NUCLEIN s 'ARROD IN  STUDIED A RARE GENETIC DISORDER !LKAPTONURIA AND CONCLUDED THAT A specific gene is associated with the absence of a specific enzyme.

Nucleic Acids 149



s 3UTTON IN  GAVE CHROMOSOME STRUCTURE s ,EVENE2USSIANBIOCHEMIST INRECOGNIZEDTHE CARBONRIBOSESUGARANDLATER also discovered deoxyribose in nucleic acids. s -ORGAN IN  GAVE GENE MAPPING s 2OBERT &EULGEN 'ERMAN CHEMIST IN  DEMONSTRATED A COLOUR TEST &EULGEN TEST for the identification of deoxyribonucleic acid. s 3UMNER IN  FOUND 0URIFIED 5REASE AND IDENTIFIED THAT ENZYME ARE PROTEINS s 'RIFFITH IN  GAVE 4RANSFORMING 0RINCIPLE AND CONCLUDED THAT A CHEMICAL transferred from dead bacteria to living cells caused genetically converted strains (“transformation”). s 0! ,EVINE IN  STRESSED THAT THERE ARE TWO TYPES OF NUCLEIC ACIDS VIZ DEOXYribonucleic acid and ribonucleic acid. s #ASPERSSON AND "RACHET IN  IDENTIFIED THAT NUCLEIC ACIDS WERE ASSOCIATED TO protein synthesis. s !VERY -C#ARTY AND-ACLEODINIDENTIFIED'RIFFITHShTRANSFORMATIONPRINCIPLEv as DNA. s #HARGAFF IN  FOUND BASE PAIRING IN GENETIC MATERIAL s S &RANKLINn8 RAYOF$.!/SWALD4!VERY #OLIN--AC,EODAND-ACLYN -C#ARTY IN  DEMONSTRATED THAT $.! IS DIRECTLY INVOLVED IN INHERITANCE OF genetic charecters. s !LFRED $ (ERSHEY AND -ARTHA * #HASE OF #OLD 3PRING (ARBOR ,AB .EW 9ORK IN  DEMONSTRATED THAT ONLY THE $.! OF 4 BACTERIOPHAGE ENTERS THE HOST THE bacterium Escherichia coli, whereas the protein (i.e., capsid) remains behind. They, thus, confirmed that DNA is the genetic material of most living organisms. s *AMES $ 7ATSON AND &RANCIS (# #RICK IN  CONSTRUCTED THE DOUBLE HELICAL model for the DNA molecule which could successfully explain DNA replication. s -ATTHEW 3 -ESELSON AND &RANKLIN ( 3TAHL IN  AT #ALIFORNIA )NSTITUTE OF Technology, presented evidence that nucleic acid forms the genetic material. s !RTHUR +ORNBERG IN  PROVED THE 7ATSON #RICK MODEL AND IN  HE ALSO SYNTHESIZED A MOLECULE OF $.! FROM THE   NUCLEOTIDES











Contribution of Friedrich Miescher (1844-1895) (EWASASTUDENTOFEMINENT'ERMANCHEMIST &ELIX(OPPE 3EYLER(EPURIFIEDNUCLEINANDALLPROTEINS were completely removed; later it was clear that the genetic material was an acid and referred to as nucleic acid.

7.3

NUCLEIC ACID AND ITS COMPONENTS

Nucleic acids are made of three major components:

 .ITROGENOUS "ASES

150 Textbook of Biochemistry



 3UGAR MOIETY  0HOSPHATE

Fig. 7.1

7.3.1

Base organization of DNA and RNA

Nitrogenous Bases

They are organic compounds (carbon-based) having nitrogen containing ring structure. .ITROGENOUS BASES ARE DERIVED FROM PURINE AND PYRIMIDINES &IGURE   (a) Purines: They are heterocyclic compounds having pyrimidine ring and an imidazole ring fused together. The two purine bases are adenine and guanine and both are found in $.! 'UANINE WAS FIRST ISOLATED FROM GUANO BIRD SO NAMED GUANINE 4ABLE   Table 7.1 Name

Formula

Chemical name

Chemical structure and details of purines Nature

Molecular weight

Melting point

Adenine C5H5N5 (A)

6-Amino Purine White crystalline 135.13 g/mol purine base

360° to 365°C

Guanine C5H5ON5 (G)

2-Amino-6-oxy Purine

360°C

Colourless, insoluble crystalline substance

151.129 g/mol

Structure

Nucleic Acids 151

(b) Pyrimidines: Pyrimidine bases consist of six-membered ring with two nitrogen atoms. The pyrimidine bases are cytosine, thymine and uracil. Thymine is found in DNA while CYTOSINE IS FOUND IN BOTH 2.! AND $.! 5RACIL IS FOUND IN ONLY 2.! &IGURE   Sometimes tRNA will contain some thymine as well as uracil (TABLE   Table 7.2 Name

Formula

Chemical structure and details of pyrimidines

Chemical name

Nature

Molecular weight

Melting point

Cytosine (C)

C5H6O2N5

2-Oxy-4-amino pyrimidine

White crystalline

111.1 g/mol

Thymine (T)

C5H6O2N2

2,4-dioxy-5methyl pyrimidine

White crystalline

126.13 g/mol 316° to 317°C

Uracil (U)

C4H4O2N2

2,4-dioxy pyrimidine

White crystalline

112.10 g/mol 338°C

Structure

320° to 325°C

Properties of pyrimidines & purines: Conformation of pyrimidine is planar while purine is puckered. Cytosine, thymine, uracil, guanine, and adenine are more soluble because they have many polar groups that are available for hydrogen bonding. Due to presence of AROMATIC RING PYRIMIDINES AND PURINES CAN ALL ABSORB 56 LIGHT AND THAT IS THE REASON WE can easily measure DNA & RNA concentration in a sample.

7.3.2

Sugar Moiety

In nucleic acid, two types of sugars are found and both are Pentose sugar found in DNA & RNA. It is present in their close five number ring “C-furanose” form and of C-configURATION 4HESE TWO SUGARS ARE DEOXYRIBOSE AND RIBOSE &IVE CARBON PENTOSE SUGAR IN $.! is called deoxyribose, while in RNA, the sugar is ribose. These two are very similar in structure with only one difference, the second carbon of deoxyribose has a hydrogen while in ribose hydroxyl group is present. The carbon atoms of a nucleotide’s sugar molecule are NUMBERED AS a a a a AND a a is read as “one prime”). In a nucleotide, the sugar OCCUPIESACENTRALPOSITION WITHTHEBASEATTACHEDTOITSa carbon and the phosphate group OR GROUPS ATTACHED TO ITS a CARBON &IGURE  

152 Textbook of Biochemistry

Fig. 7.2

7.3.3

Structure of DNA and RNA sugar

Phosphate

In a nucleotide single or up to chain OF THREE PHOSPHATE GROUPS ATTACHED TO THE a carbon OFTHESUGARISPHOSPHATE&IGURE 7HENTHENUCLEOTIDEJOINSTHEGROWING$.!OR2.! chain, it loses two phosphate groups. So, in a chain of DNA or RNA, each nucleotide has just one phosphate group. Monovalent hydroxyl groups and one divalent oxygen atom all are linked to pentavalent phosPHOROUSATOM4HEBASEISJOINEDCOVALENTLYAT.OFPYRIMIDINES AND . OF PURINES AND THE PHOSPHATE IS ESTERIFIED TO THEa-carbon. The N-glycosyl bond is formed by removal of the elements of water (a Hydroxyl group from pentose and Hydrogen atom from the base). Fig. 7.3 Phosphate structure

7.4

NUCLEOSIDES

4HESE ARE THE STRUCTURES FORMED BY COVALENT ADDITION OF 2IBOSE OR  $EOXYRIBOSE SUGAR TO A NITROGENOUS BASE ! 4 ' # &IGURE   #ARBON NUMBER  OF THE SUGAR IS ATTACHED TO NITROGEN  OF A PURINE BASE OR TO NITROGEN  OF A PYRIMIDINE BASE FORMING BETA . GLYcosydic bond. The names of purine nucleosides end in -osine and the names of pyrimiDINENUCLEOSIDESENDIN IDINE&OREXAMPLE !DENOSINE 'UANOSINE 4HYMIDINEAND#YTIDINE &IGURE  

Fig. 7.4

Different nucleosides

Nucleic Acids 153

Fig. 7.5

7.5

(a) Nucleosides and (b) Nucleotides stucture

NUCLEOTIDES

4HEY ARE FORMED BY JOINING PHOSPHORIC ACID TO A NUCLEOSIDE AT THE #a OR THE #a. The PHOSPHATE BONDS WITH ESTER LINKAGE TO CARBON a of the sugar. If more than one phosphate ISPRESENT THENTHEYFORMACIDANHYDRIDELINKAGESTOEACHOTHER&OREXAMPLE a a cAMP INDICATES THAT A PHOSPHATE IS IN ESTER LINKAGE TO BOTH THE a AND a hydroxyl groups of an ADENOSINE MOLECULE AND FORMS A CYCLIC STRUCTURE &IGURE   !-0 ADENOSINE MONOPHOSPHATE #$0 CYTIDINE DIPHOSPHATE D'40 DEOXY GUANOSINE TRIPHOSPHATE C!-0 a a cyclic adenosine monophosphate) and dTTP (deoxy thymidine triphosphate) are few nucleotides.

Fig. 7.6

Structure of nucleotide

&EWNUCLEOTIDESJOINTOGETHERANDFORMACHAINCALLEDPOLYNUCLEOTIDECHAIN4HISSTRUCture is directional and has two ends; both the ends are different from each other. Starting POINT IS a END AND THE a phosphate group of the first nucleotide in point sticks out. At THE OTHER END CALLED THE a END THE a hydroxyl of the last nucleotide exposed at the end. $.! SEQUENCES ARE USUALLY WRITTEN IN THE a TO a direction, showing that nucleotide at THE a END COMES FIRST AND THE NUCLEOTIDE AT THE a end comes last. As new nucleotides are ADDED TO A PREEXISTED STRAND OF $.! OR 2.! THE STRAND GROWS AT ITS a END WITH THE a

154 Textbook of Biochemistry

PHOSPHATE OF AN INCOMING NUCLEOTIDE ATTACHED TO THE HYDROXYL GROUP AT THE a end of the chain. This makes a chain with each sugar joined to its neighbours by a set of bonds called APHOSPHODIESTERLINKAGE&IGURE 0HOSPHODIESTERLINKAGESa OH of the (deoxy) ribose OF ONE NUCLEOTIDE IS LINKED TO THE a OH of the (deoxy) ribose of the next nucleotide via a phosphate. The phosphate is in an ester linkage to each hydroxyl, i.e., a phosphodiester group links two nucleotides.

7.6 TYPES OF NUCLEIC ACIDS Nucleic acids are macromolecules formed by smaller units known as nucleotides. In nature two types of nucleotides are present, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the most common genetic material found in living organisms from single-celled organisms to multicellular mammals. RNA is genetic material of some viruses but never DNA and RNA both. DNA is found mainly in the chromatin of the cell nucleus WHILE 2.! IS PRESENT MAINLY IN CYTOTOPLASM  AND VERY LITTLE IN NUCLEOUS  

7.6.1

DNA

Nucleus is the place where DNA is found both in plants and animals (eukaryotes). In case of bacteria cells like prokaryotes, DNA is found in the form of nucleoid. In eukaryotes, DNA is long, linear pieces called chromosomes while in prokaryotes it is circular (ringshaped) in nature. A chromosome may contain tens of thousands of genes, each providing instructions on how to make a particular product needed by the cell. DNA (deoxyribonucleic acid) is found in every living cell in an organism and it can form exact copies (except for the gametes). Two helically intertwined backbones made up of alternating phosphate and deoxyribose (sugar) molecules support internal base pairs adenine ! PAIRED WITH THYMINE 4 CYTOSINE # PAIRED WITH GUANINE ' &IGURE   $.! IS found in two different helix forms, right-handed and left-handed. On the basis of this DNA ARE OF THREE FORMS ! " AND : FORM 2IGHT HANDED HELIX AND MOST COMMON FORM FOUND IN ALL LIVING CELLS IS " FORM $.!

Chargaff’s Rules #HARGAFF  MADE SOME RULES ON THE BASIS OF HIS OBSERVATIONS ON THE BASES AND COMPONENTS OF DNA. These observations are called Chargaff’s base equivalence rule. (i) The amount of purine and pyrimidine base pairs are in equal amount, like ADENINE  GUANINE  THYMINE  CYTOSINE ;!  '=  ;4  #= IE ;!  '=;4  #=   (ii) Molar amount of adenine is always equal to the molar amount of thymine. Similarly, molar concentration of guanine is equalled by molar concentration of cytosine.



;!=  ;4= IE ;!=;4=   ;'=  ;#= IE ;'=;#=  

(iii) Phosphate and sugar deoxyribose occur in equimolar proportions. IV ! 4 AND #n' BASE PAIRS ARE RARELY EQUAL TO EACH OTHER V 4HERATIOOF;!4=;'#=ISVARIABLEBUTCONSTANTFORASPECIFICSPECIES THISRATIOISUSED to identify the source of DNA. It is low in primitive organisms and higher in advanced ones.

Nucleic Acids 155

Fig. 7.7

Structure of double helical DNA

Watson and Crick model: 7ATSONAND#RICKPROPOSEDAMODELOF$.!STRUCTURE)TPROVIDES A SUPPORT AND EXPLANATION FOR THE #HARGAFFS BASE COMPOSITION DATA )N THE 7ATSON and Crick model, two right-handed helical polynucleotide chains form a double helix around A CENTRAL AXIS 4HE TWO STRANDS ARE ANTIPARALLEL MEANS THEIR a a phosphodiester links are in opposite directions. The bases are stacked inside the helix in a plane perpendicular to the helical axis. These two strands are held together by hydrogen bonds formed between the pairs of bases. Adenine (A) and thymine (T) are connected together by two hydrogen BONDS WHILE CYTOSINE # AND GUANINE ' BY THREE HYDROGEN BONDS Hydrophobic interactions are the second bond type formed between the stacked bases and are helping to maintain the double helical structure of DNA. As per this model, the DISTANCE BETWEEN THESTACKED BASES IS  ¯  NM  ! TURN OF THE DOUBLE HELIX IS COMPLETED IN  ¯  NM A LENGTH THAT CORRESPONDS TO  NUCLEOTIDE RESIDUES 4HE DOUBLE HELIX HAS A MEAN DIAMETER OF ^ ¯  NM AND TWO GROOVES ARE FORMEDnDEEP OR MAJOR and shallow or minor.

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B-DNA: It represents an average conformation of DNA, based on fibre diffraction studies. $IMENSIONSOF" FORMTHEMOSTCOMMON OF$.!IS NMBETWEENBP NMPERTURN ABOUT  BP PER TURN  NM ABOUT  NM OR  !NGSTROMS IN DIAMETER 4ABLE   !S PER 7ATSON #RICK " $.! STRUCTURE SPECIFIC STRUCTURE OF " $.! MAJOR AND MINOR grooves are formed. The major groove is wider as compared to the minor groove in DNA &IGURE -AJARGROOVEISTHESITEWHERESPECIFICAMINOACIDPROTEINS INTERACTS0URINE . AND # GROUPS WHILE 0YRIMIDINE # AND # GROUPS ARE FACING INTO THE MOJOR GROOVE It provides specific contacts with amino acids in DNA-binding proteins. Some amino acids bind to the minor groove also. Amino acids act as source of H donors and acceptors to form Hydrogen bond with nucleotide. A-DNA: )TWASFIRSTDISCOVEREDBYFIBRE DIFFRACTIONSTUDIESOF$.!AT@LOW RELATIVE humidity. It is also right-handed helices DNA. 5NDER DEHYDRATING CONDITIONS AND CERTAIN purine stretches will favour an A-conformation. A stretch of four purines or pyrimidines in a row are able to form a local A-DNA helix. So it might be possible that certain stretch IS HAVING ! FORM WHILE OTHERS HAVE " FORM 4HE MAIN DIFFERENCE IN ! FORM OF $.! IS WIDER AS COMPARED TO " AND : FORMS BECAUSE THE SIZE OF MINOR GROOVE AND MAJOR GROOVE IS ALMOST THE SIZE ! $.! IS LESS STABLE AS COMPARED TO " IT SHOWS MORE RIGIDITY DUE TO PRESENCEOFOFF CENTERBASESTACKING4HEREAREABOUTBPPERTURNFOR! $.! COMPARED WITHABOUTBPPERTURNFORTHE" FORM"ASEPAIRTILTINGIN! $.!ISHIGHERASCOMPARED TO " $.! 4ABLE   Table 7.3

Helix sense

Comparative analysis of A, B and Z DNA B-form

A-form

Z-form

Right-handed

Right-handed

Left-handed

Base pairs per turn

10

11

12

Vertical rise per bp

3.4 Å

2.56 Å

19 Å

Rotation per bp

+36°

+33°

–30°

Helical diameter

19 Å

19 Å

19 Å

Z-DNA: It is a left-handed helix and is the first DNA to be crytallized as an oligomer D'#'#'#  )TS STRUCTURE IS NOT A SMOOTH HELICAL BUT ZIG ZAG LIKE 4HAT IS WHY ITS NAME IS : $.! 4HE : HELIX IS NARROWER THAN THE ! AND " CONFORMATIONS AND IT HAS  BP PER TURN 4ABLE   4HE NUCLEOTIDE BASES ARE FLIPPED UPSIDE DOWN RELATIVE TO THE PHOSPHATE BACKBONE IN : $.! WHEN COMPARED WITH ! $.! AND " $.! "IOLOGY OF ! " AND : $.!BIOLOGYOF! $.!! FORMHELICESARECOMMONFOR$.!n2.!HYBRIDS ASWELLAS for double-stranded RNA; in addition, the A conformation is favoured in triplex DNA. A TRANSITIONFROM" $.!TO! $.!HASBEENPOSTULATEDTOOCCURDURINGTRANSCRIPTION WHERE THE 2.!n$.! HYBRID WOULD BE MORE STABLE IN THE ! CONFORMATION ! $.! ALSO PLAYS A ROLE IN SOME PROCESSES THAT DO NOT INVOLVE 2.! &OR EXAMPLE IN SPORULATING BACTERIA THERE IS A PROTEIN WHICH CAN BIND TO $.! IN THE " CONFORMATION AND INDUCE A CHANGE TO

Nucleic Acids 157

THE ! $.! HELIX : $.! THE MINOR GROOVE IS DEEP AND NARROW AND THE MAJOR GROOVE IS ALMOST NONEXISTENT &IGURE  

Fig. 7.8

Structure of three DNA forms

Stability of DNA – As DNA is a major source of genetic imformation transfer, so it must be highly stable. Aomatic stacking and hydrogen bonding are mainly responsible for the stability of DNA. Aromatic stacking is actually a weak noncovalent force caused by overlapping of Q-orbitals also known as Q stacking. The pyrimidine and purine bases, which bond parallel to each other in DNA, participate in aromatic stacking; this is due to the overlap of their Q-orbitals. Hydrogen bonding is the main structural feature and millions of hydrogen bonds formed in DNA. Although hydrogen bonding is a strong bond, it can easily be broken at the time of DNA replication. Functions of DNA 1. Chromosomes and Genes: 'ENES ARE MINUTE STRUCTURES AND ARE UNITS OF INHERITANCE 'ENE CONTAINS CHARACTERISTICS OF AN ORGANISM AND IT TRANSFERS THE CHARACTERS FROMONEGENERATIONTOTHENEXT'ENESAREPRESENTONCHROMOSOMES CHROMOSOMES AREFOUNDINCELLNUCLEUS'ENESAREACTUALLYSHORTSEGMENTSOF$.!'ENESCONTROL CELLULARCHEMICALREACTIONSBYDIRECTINGTHEFORMATIONOFENZYMES'ENESAREALWAYS found in pairs. Half of each person’s genes come from the mother and half from the father.

158 Textbook of Biochemistry

2. Replication: It is one of the most important function of DNA. All DNA present in a cell divides and becomes double just before cell division. This process is known as replication. DNA replication begins with uncoiling of the DNA strand from one side while other remains in helical form. Topoisomerases are the enzymes which promote initiation of the unwinding of the DNA by nicking a single strand of DNA. Due to this nicking and uncoiling, topoisomerases release tension holding the helix in its coiled and supercoiled structure. DNA helicase is the next enzyme which accomplishes unwinding of the original double strand, once supercoiling has been eliminated by the topoisomerase. These two separated strands have higher affinity to bind again as they bond with hydrogen bond. To prevent binding again, helicases need energy in the form of ATP to break the strands. Now the partially unwounded DNA helix is known as replication fork. It is also called replication bubble, as under electron microscopy it looks like a small bubble. The two exposed DNA strands provide a site of binding for the next enzyme DNA polymerase III as it starts movING FROM ONE POINT TO ANOTHER AND SYNTHESIS OF NEW $.! STRANDS TAKES PLACE "EFORE THIS major step takes place which is synthesis of a short segment of RNA known as RNA primer, its prime role is that it is responsible for the addition of new nucleotide in it. Primase, a type of RNA polymerase synthesizing short RNA sequences that are complementary to a single-stranded piece of DNA, serves as its template. The DNA polymerase on reaching to the starting point of the primer starts adding NUCLEOTIDES ONE BY ONE IN AN EXACTLY COMPLEMENTARY MANNER ! TO 4 AND ' TO # $.! polymerase, a “template dependent” will “read” the sequence of bases on the template strand and then “synthesize” the complementary strand. The template strand is always read in the a TO a direction so the new DNA strand, i.e., complementary must be synthesized in the a TO a DIRECTION "OTH NEWLY SYNTHESIZED AND OLD TEMPLATE $.! STRANDS ARE ANTIPARALLEL to each other. DNA polymerase catalyses the formation of the hydrogen bonds between each arriving nucleotide and the nucleotides on the template strand. It also forms hydrogen bonds between complementary bases on the template and newly synthesized strands. It catalyses SYNTHESISOFPHOSPHODIESTERBONDSBETWEENTHEa phosphate on an incoming nucleotide and THE FREE a OH on the growing polynucleotide. Its result, the new DNA strands grow only IN THE a TO a DIRECTION WHILE STRAND GROWTH MUST BEGIN AT THE a end of the template. "ECAUSE THE ORIGINAL $.! STRANDS ARE COMPLEMENTARY AND RUN ANTIPARALLEL ONLY ONE NEW STRAND CAN BEGIN AT THE a end of the template DNA and grow continuously as the point of replication (the replication fork) moves along the template DNA. The other strand must grow in the opposite direction because it is complementary, not identical to the template strand. This results in this side’s discontinuous replication and produces a series of short SECTIONS OF NEW $.! CALLED /KAZAKI FRAGMENTS ,ATER THESE SHORT SECTIONS ARE JOINED BY the action of an enzyme called DNA ligase, which ligates the pieces together by forming the missing phosphodiester bonds. The last step is for removal of the existing RNA primers and then filling the gaps with DNA. This RNA primer is eventually removed by RNase H and the gap is filled in by DNA polymerase I. Each new strand is complementary to its old template strand and two identical new copies of the DNA double helix are produced

Nucleic Acids 159

during replication. In each new helix, one strand is the old template and the other is newly synthesized, a result described by saying that the replication is semi-conservative.

7.6.2

RNA

RNA is also a genetic material similar to DNA, but in viruses only while it is necessary for eukaryotic organisms as its main function is synthesis of protein known as translation. Ribonucleic acid, or RNA, gets its name from the ribose sugar group present in the backbone of its structure. Similar to DNA, all RNA molecules have a similar chemical organization AND COMPOSED OF THREE SUBUNITS  CARBON SUGAR CALLED 2IBOSE A 0HOSPHATE GROUP THAT IS attached to one end of the sugar molecule and one of several different nitrogenous bases LINKEDTOTHEOPPOSITEENDOFTHERIBOSE"ASE5RASILISFOUNDINPLACEOFTHYMINEWHILEREST ALLISSIMILARTO$.!' !AND# 2.!ISSINGLE STRANDEDSTRUCTUREANDITISITSPRIMARY structure. As RNA is a single-stranded molecule, so its base composition does not follow Chargaff’s rule. There are few secondary structures formed due to formation of hydrogen BONDING BETWEEN ! 5 OR ' # PAIRS 2.! MOLECULES FOLD IN THREE DIMENSIONAL SHAPES AND is known as tertiary structure of RNA. RNA can be generated from DNA process called transcription. RNA acts as genetic messenger as it can easily move around the cells of living organisms. There are three main types of RNA molecules: messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA); they are involved in protein synthesis. Except these there are other RNAs, found in cell known as noncoding RNA. These noncoding RNA are not directly involved in protein synthesis. 1. Messenger RNA (mRNA): *ACOB-ONODINPROPOSEDTHETERMMESSENGER2.! (mRNA) as the fact that this is a template molecule copied from DNA molecule. The information stored in mRNA is used to make new proteins. Precursor mRNAs were first created in eukaryotes and it passed the information for the formation of proteins. After synthesis, newly synthesized protein undergoes modifications like capping and the addition of a poly A tail. Another modification involved the removal of introns and the splicing together of exons (segment of DNA contains information for the synthesis of protein). These exons were interrupted by noncoding DNA called intron, so precursor mRNA contains both the exons and introns. A spliceosome is a complex of proteins and small RNA molecules, where the REMOVAL OF INTRONS AND THE SPLICING TAKES PLACE  TO  OF ALL 2.! IN A NORMAL CELL IS messenger RNA. 2. Ribosomal RNA (rRNA): rRNA becomes a structural part of ribosomes and serves as a genetic link between mRNA and tRNA. Ribosomal RNA is associated with protein-forming bodies called ribosomes. Ribosomes are the sites of protein synthesis. Ribosomal RNA varIES IN SIZE AND IS THE MOST PLENTIFUL 2.! )T CONSTITUTES  TO  OF TOTAL CELLULAR 2.! Ribosomal RNA (rRNA) Ribosomes are made of protein and where translation of RNA to PROTEIN TAKES PLACE )N % COLI RIBOSOMES CONTAIN THREE KINDS OF R2.! 3 3 AND 3 )N EUKARYOTES THERE ARE FOUR KINDS OF R2.! 3 3 3 AND 3 /NE 3 MOLECULE is used to make the small subunit of the ribosome, with the help of several proteins. The 3 3 AND 3 R2.! MOLECULES ARE INVOLVED WITH THE CONSTRUCTION OF THE LARGE SUBUNIT

160 Textbook of Biochemistry

OF THE RIBOSOME 4HE 3 3 AND 3 MOLECULES ARE MADE FROM THE PROCESSING OF A single precursor RNA. 3. Transfer RNA (tRNA): Its main role is to deliver amino acids from the cytoplasm to THE RIBOSOME 4HERE ARE  DIFFERENT T2.!S IN EUKARYOTES BUT THE FUNCTION OF ALL DIFFERent tRNAs is same, i.e., to bring particular amino acid to a ribosome. Structure of tRNA MOLECULES HAS CLOVERLEAF PATTERN HAVING ABOUT  NUCLEOTIDES 4RANSFER 2.!S T2.!S ARE RELATIVELYSMALLERMOLECULES EACHONEISMADEUPOFONLY RIBONUCLEOTIDES4HET2.! is a single strand and it bends around in certain places resulting in some ribonucleotides pairing up with others in the same chain, thus it forms three loops in its structure. Each T2.! MOLECULE HAS ONE AMINO ACID ATTACHED TO ITS a END 3INCE THERE ARE ONLY  AMINO ACIDS AND AROUND  DIFFERENT KINDS OF T2.!S SOME AMINO ACIDS ARE CARRIED BY MORE THAN ONE TYPE OF T2.! /N ONE OF THE THREE LOOPS IS WHAT IS CALLED AN ANTICODON &IGURE   !NTICODONS ARE MADE UP OF THREE BASES AND ARE INVOLVED IN TRANSLATION !TTACHMENT of specific amino acid is determined by its anticodon sequence.

Fig. 7.9

Structure of tRNA

Function of RNA: 2.! IS MOST IMPORTANT AS IT INVOLVES TWO IMPORTANT CELL ACTIVITIESn 1. Translation, i.e., synthesis of proteins 2. Transcription: Synthesis of RNA from DNA.

Nucleic Acids 161

1. Translation: Simply, it is divided into three subprocesses: A. Initiation: Ribosomes are the cellular factory responsible for synthesizing new PROTEINS 4HE RIBOSOME CONSISTS OF STRUCTURAL 2.! AND ABOUT  DIFFERENT PROTEINS In its inactive state, it exists as two subunits; a large subunit and a small subunit. Ribosomes translate the genetic message of mRNA into proteins. The mRNA is TRANSLATEDASaTOa producing a corresponding N-terminal to C-terminal polypepTIDE4HEREARETHREET2.! BINDINGSITESINRIBOSOMES!MINOACYL T2.!BINDING OR! SITE0EPTIDYL T2.!BINDING OR0 SITEAND3TARTCODON!5' OFM2.! ATTACHESTOTHEh2vSITEOFTHERIBOSOME4HE!5'CODONALWAYSINITIATESTRANSLATION and codes for the amino acid methionine. The tRNA has a binding site of three bases called an Anticodon that is complementary to the mRNA codon. The codon of M2.!OF!5'ISRECOGNIZEDBYAT2.!ASITHASA5!'ANTICODON!MINOACIDS bound to tRNAs are inserted in the definite sequence as it has specific binding of each amino acid to its tRNA and specific base-pairing between the mRNA codon and anticodon of tRNA. The tRNA that has this anticodon carries, at its tail, the amino acid methionine. This methionyl-tRNA is in the P site of the ribosome. The A site present next to it, is providing site to the tRNA bearing the next amino acid. There is a specific tRNA for each mRNA codon that codes for an amino acid. B. Elongation: New amino acids are added and connected together to form a polypeptide, and it is specified by the mRNA sequence. An incoming amino-acyl-tRNA recognizes the codon in the A site and binds there. Due to this, peptide bond is formed between the new amino acid and the growing polypeptide chain. Then the amino acid is removed from tRNA as the bonds break between first amino acid and T2.! T2.! PRESENT AT 0 SITE IS RELEASED AND THE T2.! OF ! SITE IS TRANSLOCATED to the P site. This is simple method by which amino acid movement takes place and finally a long chain of polypeptides is formed with peptide bond. C. Termination: This process continues till a special codon, called a Stop Codon, IS REACHED 4HERE ARE THREE 3TOP CODONS 5!! 5!' 5'! 4HE 3TOP CODON ACTS as a stop signal for translation. Release factor is a protein that binds directly to the Stop codon in the A site; this causes addition of water molecule at the end of polypeptide chain. This causes separation of polypeptide chain from the last tRNA. After this, usually mRNA is broken down while ribosome splits into its large and small subunits. The newly synthesized protein is now transported to endoplasmic reticulum and golgi apparatus for final processing. 2. Transcription is the process in which a RNA molecule is generated from existing DNA sequence (Template strand). This process is controlled by a specific enzyme known as RNA polymerase. Transcription begins when RNA polymerase binds to specific promoter sequence and the process ends with termination. A. Initiation: &IRST DNA double helix unwinds near the gene that is getting transcribed. This opened-up region is called a transcription bubble. One of the two exposed DNA strands act as template strand. The RNA product is complementary to the

162 Textbook of Biochemistry

template strand and is almost identical to the other DNA strand, called the nontemplate (or coding) strand. However, there is one important difference: in the newly CONSTRUCTED 2.! ALL OF THE 4 NUCLEOTIDES ARE REPLACED WITH 5 NUCLEOTIDES 4HE SITE ON THE $.! IS CALLED THE  OR THE INITIATION SITE FROM WHICH THE FIRST RNA nucleotide is transcribed. Nucleotides that come before the initiation site are given negative numbers and said to be upstream while nucleotides that come after the initiation site are known as downstream and marked with positive numbers.

RNA polymerase 2.!POLYMERASESAREENZYMESTHATTRANSCRIBE$.!INTO2.!5SINGA$.!TEMPLATE 2.!POLYMERASE builds a new RNA molecule through base pairing.

2.! POLYMERASE ALWAYS BUILDS A NEW 2.! STRAND IN THE a TO a direction, that is, it can only add 2.!NUCLEOTIDES! 5 # OR' TOTHEa end of the strand. RNA polymerases are large enzymes with multiple subunits, even in simple organisms like bacteria. In addition, humans and other eukaryotes have three different kinds of RNA polymerases: I, II, and III. Each one specializes in transcribing certain classes of genes.

A. &IRST 2.! POLYMERASE BINDS TO THE $.! STRAND AT ITS SPECIFIC REGION CALLED THE PROMOTER &IGURE   "ASICALLY THE PROMOTER TELLS THE POLYMERASE WHERE to “sit down” on the DNA and begin transcribing. The promoter region is actually a region just before or few overlapped region from where exactly comes (and slightly overlaps with) the transcribed region whose transcription begins. It contains specific recognition sites for RNA polymerase and for its helper proteins to bind. Once the transcription bubble has formed, the polymerase can start transcribing. In eukaryotes, RNA polymerase does not attach directly to promoters while in bacteria it directly attaches. Helper proteins called basal (general) transcription factors bind to the promoter first, helping the RNA polymerase to bind with the DNA strand. Eukaryotic promoters have a sequence called a TATA box and it is recognized by transcription

Nucleic Acids 163

Fig. 7.10

DNA promorter sites and RNA polymerase

factors. In case of bacteria promoter contains two important DNA sequences, THE n AND n elements. RNA polymerase recognizes and binds directly to these sequences. B. Elongation: Once RNA polymerase binds to a promoter site, the next is transcription elongation. It is the step in which RNA strand gets longer due to addition of new nucleotides. $URING ELONGATION 2.! POLYMERASE hWALKSv IN THE a TO a direction along template strand (DNA). RNA polymerase adds a matching (complementary) RNA NUCLEOTIDE TO THE a end of the RNA strand for each nucleotide in the template &IGURE   C. Transcription termination: RNA polymerase will keep transcribing until it gets signals to stop. The process of ending transcription is called termination, and it happens once the polymerase transcribes a sequence of DNA known as a terminator. There are two major termination strategies of termination, Rho-dependent and Rho-independent. In Rho-dependent termination, the RNA contains a binding site for a protein called Rho factor. Rho factor binds to this sequence and starts “climbing” up the TRANSCRIPTTOWARDS2.!POLYMERASE7HENITCATCHESUPWITHTHEPOLYMERASEATTHE transcription bubble, Rho pulls the RNA transcript and the template DNA strand apart, releasing the RNA molecule and ending transcription. Another sequence found later in the DNA, called the transcription stop point, causes RNA polymerase to

164 Textbook of Biochemistry

Fig. 7.11 RNA polymerases and its action

pause and thus helps Rho catch up. Rho-independent termination depends on specific sequences in the DNA template strand. As the RNA polymerase approaches the end OF THE GENE BEING TRANSCRIBED IT HITS THE REGION RICH IN # AND ' NUCLEOTIDES 4HE RNA transcribed from this region folds back on itself, and the complementary C and 'NUCLEOTIDESBINDTOGETHER4HERESULTISASTABLEHAIRPINTHATCAUSESTHEPOLYMERASE TOSTALL)NATERMINATOR THEHAIRPINISFOLLOWEDBYASTRETCHOF5NUCLEOTIDESINTHE RNA, which match up with A nucleotides in the template DNA. The complementary 5 ! REGION OF THE 2.! TRANSCRIPT FORMS ONLY A WEAK INTERACTION WITH THE TEMPLATE DNA. This, coupled with the stalled polymerase, produces enough instability for the enzyme to fall off and liberate the new RNA transcript.

7.7





SUMMARY s $EOXYRIBONUCLEIC ACID IS A POLYNUCLEOTIDE s $.! FUNCTIONS AS STORAGE FOR GENETIC INFORMATION s $.! POLYMERASE IS USED TO CATALYZE THE SYNTHESIS OF $.! IN THE a TO a direction. s $.! DOUBLE HELIX STRAND IS ORIENTATED IN THE OPPOSITE DIRECTION s .ATURE OF $.! IS ACIDIC DUE TO THE PHOSPHATE GROUPS BETWEEN EACH DEOXYRIBOSE s 0RIMARY STRUCTURE OF $.! CONTAINS SEQUENCES OF ADENINE GUANINE CYTOSINE AND thymine. s 3ECONDARY STRUCTURE IS A DOUBLE HELICAL STRUCTURE STABLILIZED BY HYDROGEN BONDING between base pairs.

Nucleic Acids 165







s 4ERTIARY STRUCTURE IS NUCLEIC ACIDS SUPERCOILED AND WRAPPED AROUND HISTONES (proteins). s %UKARYOTIC CELLS $.! IS FOUND IN NUCLEUS OF CELL WHILE IN PROKARYOTES IT IS FOUND in the form of nucleoid. s 2.! IS A POLYNUCLEOTIDE HELPING IN THE SYNTHESIS OF PROTEINS s 5NLIKE $.! 2.! IS SINGLE STRANDED AND CONSISTS OF A SHORTER NUCLEOTIDE CHAIN s 2.! ISLESS STABLE THAN $.! BECAUSE THE (YDROXYL GROUP ON THE RIBOSE UNDERGOES easier hydrolysis. s 2.! CONTAINS ADENINE GUANINE CYTOSINE AND URACIL s -ESSENGER 2.! BRINGS INFORMATION FROM $.! TO RIBOSOME SITES FOR PROTEIN synthesis. s 4RANSFER 2.! HELPS IN TRANSFERRING OF SPECIFIC AMINO ACID TO A POLYPEPTIDE CHAIN during the translation phase of protein synthesis.

MULTIPLE-CHOICE QUESTIONS













 7HICH CHARACTERISTIC IS SHARED BY PURINES AND PYRIMIDINES A "OTH CONTAIN TWO HETEROCYCLIC RINGS WITH AROMATIC CHARACTER B "OTH CAN FORM MULTIPLE NON COVALENT HYDROGEN BONDS C "OTH EXIST IN PLANAR CONFIGURATIONS WITH A HEMIACETAL LINKAGE D "OTH EXIST AS NEUTRAL ZWITTERIONS UNDER CELLULAR CONDITIONS  7HICH STRUCTURAL FEATURE IS SHARED BY BOTH URACIL AND THYMINE A "OTH CONTAIN TWO KETO GROUPS B "OTH CONTAIN ONE METHYL GROUP C "OTH CONTAIN A FIVE MEMBERED RING D "OTH CONTAIN THREE NITROGEN ATOMS  7HICH CHARACTERISTIC IS SHARED BY BOTH ADENINE AND CYTOSINE A "OTH CONTAIN ONE METHYL GROUP B "OTH ARE ANOMERIC C "OTH CONTAIN ONE KETO GROUP D "OTH ARE HETEROCYCLIC  7HICH CHARACTERISTIC IS FOUND IN BOTH PURINES AND PYRIMIDINES (a) They both have aromatic rings that undergo substantial tautomerization at neutral pH. (b) They both are weak bases that can be positively charged at neutral pH. C 4HEY BOTH HAVE MULTIPLE P+a values that result in zwitterion forms. (d) They both can form stable N-glycosidic bonds with C D-ribofuranose.  7HICH IS A GENERAL PROPERTY OF BOTH NUCLEOSIDES AND NUCLEOTIDES A "OTH CONTAIN A PENTOSE IN THE FORM OF A FURANOSE B "OTH CONTAIN AT LEAST ONE a-phosphate group. C "OTH CONTAIN A NITROGENOUS BASE THAT FORMS COVALENT ( BONDS D "OTH CONTAIN A HEMIACETAL OR HEMIKETAL BOND  7HICH STRUCTURAL FEATURE IS FOUND IN THE SINGLE STRANDED $.! MOLECULE (a) It can have a negatively-charged backbone composed of nitrogenous bases.

166 Textbook of Biochemistry



















 

B %ACH a a-phosphodiester bond will contain one phosphate group linking two deoxyribose sugars. C )T CAN HAVE ONE END WITH A a PHOSPHATE GROUP WHILE THE OTHER END HAS A a-hydroxyl group. (d) Each purine and pyrimidine will be paired with a complementary base. 7HICH CHARACTERISTIC DOES THIS DOUBLE STRANDED MOLECULE HAVE WHEN IT FORMS A " $.! STRUCTURE (a) The two strands will have parallel orientation and identical sequences. B 4HE HELIX WILL BE RIGHT HANDED WITH  BASE PAIRS PER TURN (c) Every base pair will contain one purine and one pyrimidine. (d) There are both covalent and non-covalent bonds between the two chains. 7HICHCHARACTERISTICWILLTHISDOUBLE STRANDED $.!MOLECULE SHAREWITH ADOUBLE STRANDED 2.! MOLECULE OF THE SAME SIZE A "OTH WILL HAVE SECONDARY STRUCTURE B "OTH WILL CONTAIN INVERTED REPEATS C "OTH WILL BE DEGRADED BY BASE D "OTH WILL CONTAIN FOUR TYPES OF BASE PAIRS !SSUME THAT $.! MOLECULES ARE STUDIED IN A VARIETY OF ORGANISMS AND FOUND TO HAVE THE FOLLOWING PROPERTIES 7HICH PROPERTY WOULD BE CONSISTENT WITH THE HYPOTHESIS THAT GENETIC MATERIAL IS COMPOSED OF $.! (a) DNA in all organisms is composed of the same nucleotides. (b) DNA in an organism remains constant as the organism ages. (c) DNA from two different organisms has the same base composition. (d) DNA is different in two different cells of the same organism. (ISTONES (a) are negatively-charged globular proteins. (b) contain both B-helix and C-pleated sheet. C HAVE MOLECULAR WEIGHTS IN EXCESS OF   (d) contain high amounts of basic amino acids. ,EFT HANDED $.! IS A ! $.! B " $.! C : $.! D # $.! 4HE LENGTH OF $.! HAVING  BASE PAIRS IS A   B   C   D  

Answers

 B  B

 A  D D  D  C  D

 A

 B

Short Answer Type Questions

 7RITE DOWN THE IMPORTANT CONTRIBUTION OF &RIEDRICH -IESCHER  7HAT ARE NUCLEOSIDES AND NUCLEOTIDES

 C

 A

Nucleic Acids 167



 7HAT IS #HARGAFFS RULE  $ISCUSS THE BASICS OF ! " AND : FORMS OF $.!  7HAT IS CLOVER LEAF MODEL OF T2.!

Long Answer Type Questions



 %XPLAINTHE7ATSONAND#RICKMODELOF$.!!DDANOTEONDIFFERENTFORMSOF$.!$.! POLYMORPHISM   %XPLAIN THE STRUCTURE AND FUNCTIONS OF DIFFERENT TYPES OF 2.!S  %XPLAIN THE PROCESS OF $.! REPLICATION  %XPLAIN THE PROCESS OF TRANSCRIPTION !DD A NOTE ON ITS INHIBITORS  $ISCUSS DIFFERENT FORMS OF $.!

8 Enzymes 8.1

INTRODUCTION

Living beings demonstrate a remarkable ability to synthesize as well as degrade complex molecules during the various life processes. These reactions are made possible by the catalytic action of the amazing biological catalysts known as ‘enzymes’. Catalysts are the substances that accelerate rate of reaction without undergoing any change during the reaction. The catalytic power of enzymes is immense which enables them to multiply the reaction rate 106 to 1012 times that of the uncatalyzed reactions. Enzymes are highly specific and thousands of enzymes have been identified each of which catalyzes a single chemical reaction or a set of closely related reactions. The enzymes essential for the catalysis of common biological reactions occurring in the cells are located inside the cell whereas some enzymes are secreted outside the cells for specific functions, e.g., digestive enzymes are secreted in the digestive tract or microorganisms produce extracellular enzymes to derive nutrients from the environment. Apart from their vital functions for life, enzymes have emerged as powerful tools for various biotechnology applications ranging from the basic research to process industries. Enzymes have emerged as an important diagnostic tool for diagnosis of many diseases. Deficiency of different types of enzymes is known to cause many disorders some of which may be fatal. Many drugs exert their therapeutic effect through the action of enzymes. The enzymes are being utilized not only for medical purposes but also in research, chemical industry, food processing and agriculture. Enzymes have been known since late 1700s to be present in the stomach and the intestine digestive juices. However, the discovery of the first enzyme is attributed to Anselme Payen and Jean-François Persoz, French chemists who in 1833 were successful in extracting enzyme diastase from malt solution. Louis Pasteur devoted himself to the study of alcoholic fermentation and proved that fermentation of sugar by yeast was catalyzed by a vital force found in the yeast cell. He called this vital force “ferments” which function only within the living organisms. The term enzyme was coined by German physiologist Wilhem Kühne (1837-1900), which in Greek means “leavened”. A major breakthrough in enzyme research came in 1897 when Eduard Buchner proved that sugar could be fermented by yeast extracts that were free of any living yeast cells. In 1907, he received the Nobel Prize in

Enzymes 169

Chemistry “for his biochemical research and his discovery of cell-free fermentation”. The early 20th century witnessed groundbreaking discoveries in enzymology. In 1926, James Sumner isolated and crystallized urease and found that enzymes are proteins. However, his theory was accepted only after John Northrop and Moses Kunitz crystallized pepsin, trypsin, and other digestive enzymes and found them also to be proteins. In 1903, French physical chemist Victor Henri found that enzyme reactions were initiated by weak bonding interactions between an enzyme and substrate. His work was continued by Leonor Michaelis and Maud Menten and they proposed a mathematical model for enzyme substrate reaction which is famously known as Michaelis-Menten equation. G. E. Briggs, Haldane derived a new interpretation of the enzyme kinetics law that was different from the Michaelis–Menten equation which is now accepted by the modern scientific community. Simultaneous to the discoveries made by the academicians, the industrial scientists exploited the catalytic power of enzymes for carrying out reactions of industrial importance. In the far-east, Koji process has been used traditionally for the production of several fermented foods. Inspired by this process, the Japanese scientist Takamine developed a fermentation process for the industrial production of amylase from Aspergillus. At the same time, industrial scientists experimented with crude enzyme extracts for desizing of textiles. Sizing is a process by which the textile yarn is coated with starch to give it strength for the weaving process. After weaving, the textiles are desized to remove the sizing material. The traditional process involved soaking of the cloth in acid or base solution. However, the process was poorly controlled and was liable to cause damage to the cloth or produce discoloration. Boidin & Effront (1917) used the amylase derived from Bacillus subtilis for desizing for the first time. However, the industrial process for large-scale production of bacterial amylase was developed after the Second World War. The use of enzymes for industrial purposes progressed rapidly after 1965, mainly due to the increasing use of enzymes in detergents.

8.2

CHEMICAL NATURE OF ENZYMES

Chemically, all known enzymes are proteins. Interestingly, catalytic RNAs (aptly called ribozymes) are an exception to this rule. They are high molecular weight proteins with molecular weights ranging from 10,000 to 2,000,000. The three-dimensional structures of enzymes have specific sites for substrate binding and for catalysis. The sites responsible for catalysis are usually made of 2-3 amino acid residues and can be far apart in the primary sequence of the enzyme. Enzymes are inactivated when their three-dimensional structure is disrupted due to denaturation. Denaturation can be brought about by heat or action of acids, bases or organic solvents. Some enzymes require presence of either some inorganic ions (cofactors) such as iron, magnesium, manganese, zinc oxide or complex organic substances for their activities. Coenzymes are non-protein organic molecules which are obtained from vitamins. The coenzymes and cofactors utilized by enzymes add diverse functionalities to enzymes, including

170 Textbook of Biochemistry

new functional groups, redox capabilities, electrophillic centres, and sites for coordination of substrates. Some enzymes require both a coenzyme and one or more metal ions for activity. The coenzymes or metal that is tightly bound to the enzyme is known as prosthetic group. The complete enzyme with bound cofactor or coenzyme is known as holoenzyme while only the protein part of such an enzyme is known as apoenzyme (Figure 8.1). A list of cofactors and coenzymes has been given in Tables 8.1 and 8.2, respectively.

Fig. 8.1

Binding of cofactors activates the proteins

Table 8.1 List of inorganic elements that function as cofactors and their respective enzymes Ion Cu

2–

Fe2– or Fe3– K

+

Mn

2–

Mo 2–

Se Zn

Cytochrome oxidase Cytochrome oxidase, catalase, peroxidase Pyruvate kinase

Mg2–

Ni

Enzyme

2

Hexokinase, glucose 6-phosphatase, pyruvate kinase Arginase, ribonucleotide reductase Dinitrogenase Urease Glutathione peroxidase Carbonic anhydrase, alcohol dehydrogenase, carboxypeptidase A and B

Enzymes 171 Table 8.2 Coenzymes derived from dietary vitamins and their respective enzymes Vitamin (Dietary precursor)

Coenzyme

Reaction type

Coenzyme class

B1 (Thiamine)

Thiamine Pyrophosphate (TPP)

Oxidative decarboxylation

Prosthetic group

B2

Flavin Adenine Dinucleotide (FAD)

Oxidation-reduction

Prosthetic group

B5 (Pantothenate)

CoA-Coenzyme

A-Acyl group transfer

Co-substrate

B6 (Pyridoxine)

Pyridoxyl 5a Phosphate PLP

Transfer of groups to and from Prosthetic group amino acids

B12 (Cobolamine)

5a deoxyadenosyl cobolamine

Intramolecular rearrangements

Niacin

Nicotinamide adenine dinucle- Oxidation-reduction otide (NAD+)

Co-substrate

Folic Acid

Tetrahydrofolate

One carbon group transfer

Prosthetic group

Biotin

Biocytin

Carboxylation

Prosthetic group

Lipoic Acid

Lipoate

Transfer of electrons and acyl groups

Prosthetic group

8.3

Prosthetic groups

NOMENCLATURE AND CLASSIFICATION

Early enzymes were assigned arbitrary names when discovered and before specific reaction, their catalysis was known. Pepsin was named after ‘pepsis’ which in Greek means digestion, lysozyme was named so as it leads to ‘lysis’ of bacteria; or trypsin which originated from ‘tryein’ which means ‘to wear down’, this was because it was obtained by washing the pancreatic tissues with glycerol. However, this system of nomenclature was vague and with phenomenal increase in the discovery of new enzymes, this system led to confusion due to ambiguity. Therefore, International Union of Biochemistry (IUB) set up the Enzyme Commission which is responsible for developing a standard nomenclature system for enzymes. Nomenclature committee of the International Union of Biochemistry and Molecular Biology maintains the complete list and description of thousands of known enzymes and it is available at www.chem.qmul.ac.uk/iubmb/enzyme. The committee has laid down general principles for the nomenclature of enzymes. According to these recommendations, the enzyme names should end with ‘-ase’. However ‘ase’ suffix should be used for single enzymes, the systems containing more than one enzyme responsible for catalyzing an overall reaction, the word ‘system’ should be included in the name. The Enzyme Commission number (EC number) gives the systematic names for enzymes based on the reactions catalyzed by these enzymes. The systematic name consists of the letters “EC” followed by four numbers separated by periods. These numbers represent a progressively finer classification of the enzymes. This system divides enzymes into six classes,

172 Textbook of Biochemistry

each with subclasses, based on the type of reaction catalyzed. Table 8.3 gives the broad classification of enzymes in six different classes based on the reaction they catalyze. Table 8.3 Class

Enzyme classification recommended by the Enzyme Commission

Type of reaction

Common examples

Specific example with reaction catalyzed

1.

Oxidoreductases (Transfer of electrons)

Oxidases Reductases Dehydrogenases

Alcohol dehydrogenases (ADH) (EC 1.1.1.1) Alcohol + NAD+ = an aldehyde or ketone + NADH.

2.

Transferases (Transfer of functional groups)

Transaminase Transketolase Transaldolase

Hexokinase (EC 2.7.1.1)

3.

Hydrolases (Hydrolysis reactions)

Amylases Lipases Proteases Nucleases

Alpha-Amylase (EC 3.2.1.1) Endohydrolysis of (1->4)-alpha-D-glucosidic linkages in polysaccharides (starch glycogen) Starch m
maltose + glucose

4.

Lyases (Group elimination to form double bonds without hydrolysis)

Aldolase Decarboxylase Citrate synthase

Histidine decarboxylase (EC 4.1.1.22) L-histidine converted to histamine + CO2

5.

Isomerases

Isomerase mutase Epimerase

Xylose isomerase (EC 5.3.1.5) D-xylose converted to D-xylulose

6.

Ligases or synthetases (Bond formation coupled with ATP hydrolysis)

Synthetases Carboxylases

Glutamine synthetase (EC 6.3.1.2) ATP + L-glutamate + NH3 = ADP + phosphate + L-glutamine

Figure 8.2 illustrates the components of EC name of enzyme. For example, the tripeptideamino peptidases have the code “EC 3.4.11.4”, whose components indicate the following groups of enzymes:

Fig. 8.2

Nomenclature of lactate dehydrogenase

Enzymes 173



8.4

s %#  ENZYMES ARE hydrolases (enzymes that use water to break up some other molecule). s %#  ARE HYDROLASES THAT ACT ON peptide bonds. s %#  ARE THOSE HYDROLASES THAT CLEAVE OFF THE AMINO TERMINAL amino acid from a polypeptide. s %#  ARE THOSE THAT CLEAVE OFF THE AMINO TERMINAL END FROM A tripeptide.

MECHANISM OF ACTION OF ENZYMES

The basic principles of thermodynamics state that a reaction can occur spontaneously only if the change in free energy of a system or %G is negative. When %G is zero, the system is at equilibrium and the rates of forward and backward reactions are equal, therefore no net change occurs in the system. The path of the reactions starts from the energy level of substrates and rises uphill to the energy level of the high energy transition state which is at the top of the ‘energy hill’. At the transition state, decay to substrate or product state is equally probable. The transition state cannot be categorized as any chemical entity as it lacks stability. The difference in energy levels of ground state and the transition state is known as activation energy (Figure 8.3). Activation energy is the energy barrier to the chemical reactions. Only the molecules possessing sufficient energy to cross this uphill energy barrier would be able to react. Therefore, the rate of reaction depends upon the activation energy of the reaction and not on the %G (Figure 8.3). However, we may encounter reactions where the %G is positive. Although such reactions cannot occur spontaneously, they can be made possible by supply of sufficient free energy. The reactions with negative %G occur with the release of free energy and hence are known as exothermic or exergonic reactions since the free energy of the system decreases due to the reaction. On the other hand, the reactions with positive %G absorb heat from the surroundings and are called endothermic. The reactions with positive %G are also known as endergonic reactions as they result in net increase in free energy of the system. The reaction illustrated is the simple conversion of a substrate S to a product P. Since the final energy state of product P is lower than that of substrate S, the reactions proceed in forward direction. However, the reaction path involves a high energy transition state. The energy required to reach this transition state is known as the activation energy which acts as a barrier that determines the rate of the reaction. The enzymes acting as catalysts, the activation energy is lowered and hence the rate of reaction is accelerated. Fig. 8.3

Energy diagrams for catalyzed and uncatalyzed reactions

174 Textbook of Biochemistry

The free energy change of a reaction is a state function and it depends only on the free energy of the reactants and the free energy of the products. Therefore, reactions requiring higher activation energy are slower as compared to the reactions requiring lower activation energy. Increasing the temperature of the reaction mixture leads to increase in reaction rate. When heat energy is supplied to the reacting molecules, a greater number of molecules acquire adequate energy to overcome the energy barrier. Catalysts increase the rate of reaction by lowering the activation energy. The transition state should not be confused with reaction intermediates such as enzymesubstrate (ES) or enzyme-product (EP). A reaction intermediate is any chemical species in the reaction path that has a finite lifetime. A reaction may have several intermediates. In an enzyme catalyzed reaction, enzyme-substrate (ES) and enzyme-product (EP) are considered intermediates. They occupy valleys in the reaction path hill diagram (Figure 8.3). Other less stable chemical intermediates may also be encountered in an enzyme catalyzed reaction. The overall rate of a reaction with more than one step is determined by the step with highest activation energy, i.e., slowest reaction. This step is known as rate-limiting step which is observed as the highest energy point during the reaction course. However, rate limiting step may vary with reaction conditions, and for many reactions several steps may possess similar activation energy and therefore they would all become partially rate limiting steps. Consider a reaction where reactants A and B react to produce C and D as products. A+B

C+D

The %G of this reaction is given by [C][D] %G = %G° + RT ln ______ [A][B]

...(8.1)

where %G° is the standard free-energy change, R is the gas constant, T is the absolute temperature, and [A], [B], [C], and [D] are the molar concentrations. The standard free energy change %G° of a biochemical reaction is the free energy change for this reaction under standard conditions that is, when each of the reactants is present at a concentration of 1.0M (or 1 atmosphere for a gas) at pH 7.0 and temperature of 298 K (25°C). Thus, the %G° of a reaction depends on the nature of the reactants and on their concentrations. At equilibrium, %G = 0. Equation 8.1 becomes [C][D] 0 = %G°a + RT ln ______ [A][B]

...(8.2)

and so [C][D] %G°a = – RT ln ______ [A][B]

...(8.3)

Enzymes 175

The equilibrium constant under standard conditions, Kaeq, is defined as [C][D] Kaeq = ______ [A][B]

...(8.4)

Substituting equation 8.4 into equation 8.3 gives %G°a = – RT ln Kaeq

...(8.5)

%G°a = – 2.303RT log10 Kaeq

...(8.6)

This can be rearranged to give Kaeq = 10 – %G°a(RT)

...(8.7)

Substituting R = 1.987 × 10 –3 kcal mol–1 deg–1 and T = 298°K (corresponding to 25°C) gives Kaeq = 10 – %G°a Thus, the standard free energy and the equilibrium constant of a reaction are related by a simple expression. Enzymes, the biological catalysts, accelerate the rate of reaction significantly by lowering the activation energy of the reaction and hence by giving alternate lower energy path for the reaction to occur. However, they cannot shift the position of the equilibrium. The equilibrium position depends only on the difference in free energy of the reactants and products.

8.4.1

Active Site and Catalysis

The tremendous substrate specificity and highly superior catalytic efficiency implies the existence of a reaction site that is tailored to carry out a specific reaction. This was the seed of the lock and key mechanism proposed by Emil Fisher in the early nineteenth century. He visualized enzyme as a lock and substrate as a key with complementary structure.

Fig. 8.4 Lock and key hypothesis: Enzyme and substrate have complimentary structures which explains the specificity of enzymes

176 Textbook of Biochemistry

While the “lock and key model” accounted for the exquisite specificity of enzyme-substrate interactions, the implied rigidity of the enzyme’s active site failed to account for the dynamic changes that accompany catalysis. Daniel Koshland in 1958 proposed induced fit model that takes into account the flexible nature of enzymes. It states that when substrates bind to an enzyme they induce a conformational change, a change analogous to placing a hand (substrate) into a glove (enzyme) (Figure 8.4). It also follows that the enzyme also induces reciprocal changes in its substrates directing the energy of binding to facilitate the conversion of substrates into products. The induced fit model has been established experimentally by biophysical studies displaying enzyme motion during substrate binding. Although, lock and key model was the first model to propose that the specific interaction between two biological molecules is facilitated by complementarity of surface structures of the two interacting molecules. Though, this hypothesis has greatly influenced the development of biochemistry, it could be proven that enzymes with exact structural complementarity substrate is not beneficial to the reaction to proceed. This is because the hypothetical enzyme-substrate complex would be so stable that further reaction would be impeded. The modern theory of enzymatic catalysis was first proposed by Michael Polanyi (1921) and Haldane (1930) and further explained by Linus Pauling in 1946. They proposed that the enzyme active site is complementary to the reaction transition state. Figure 8.5 demonstrates how such an enzyme can work. An enzyme with a structure complementary to the reaction transition state destabilizes the substrate leading to catalysis of the reaction. The binding energy of the substrate with enzyme compensates for the increase in free energy required to bring about conformational change in the substrate molecule necessary to reach the transition state. This results in an alternative path of reaction with lower activation energy and a faster reaction rate.

Fig. 8.5 The structure of active site of enzyme: Complementarity of enzyme active site to the transition state of the reaction

Enzymes 177

The active site of an enzyme is the part of enzyme where enzyme substrates bind specifically. The active site is made of the specific amino acid residues that actually take part in making or breaking of bonds. These amino acids are known as catalytic groups. Generally, the nature of active site is nonpolar and water is usually excluded. However, polar residue may form the part of the active site and perform specific function during catalysis. The active site is a three-dimensional structure (cleft) formed by amino acids groups that may come from amino acid residues far apart in the primary amino acid sequence of the enzyme. For example, chymotrypsin the active site consists of residues Ser-195, His-57 and Asp-102 (Figure 8.6). It has to be noted that even if these residues are distributed wide apart in the primary sequence of chymotrypsin but they are located in close proximity at the active site. The amino acids other than those forming the active site serve as a scaffold to support the three-dimensional structure and in many proteins act as regulatory sites, sites of interaction with activity modulators and channels to deliver the substrate to the active site.

Fig. 8.6 The crystal structure of chymotrypsin showing the catalytic to triad of amino acid side chain

Substrates bind to the enzymes by multiple weak interactions such as electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions. van der Waals forces become significant in binding only when multiple substrate atoms simultaneously come close to many enzyme atoms. This is possible only when the enzyme and substrate have shapes that are complementary to each other. Binding of substrate to the active site lowers the activation energy resulting in significant increase in reaction rate.

178 Textbook of Biochemistry

The extreme substrate specificity and high catalytic efficiency of enzymes reflect the existence of an environment that is exquisitely tailored to a single reaction. Termed the active site, this environment generally takes the form of a cleft or pocket. The active sites of multimeric enzymes often are located at the interface between subunits and recruit residues from more than one monomer. The three-dimensional active site both shields substrates from solvent and facilitates catalysis. Substrates bind to the active site at a region complementary to a portion of the substrate that will not undergo chemical change during the course of the reaction. This simultaneously aligns portions of the substrate that will undergo change with the chemical functional groups of peptidyl aminoacyl residues. The active site also binds and orients cofactors or prosthetic groups. Members of an enzyme family such as the aspartic or serine proteases employ a similar mechanism to catalyze a common reaction type but act on different substrates. Enzyme families appear to arise through gene duplication events that create a second copy of the gene which encodes a particular enzyme. The proteins encoded by the two genes can then evolve independently to recognize different substrates resulting, for example, in chymotrypsin, which cleaves peptide bonds on the carboxyl terminal side of large hydrophobic amino acids; and trypsin, which cleaves peptide bonds on the carboxyl terminal side of basic amino acids. The common ancestry of enzymes can be inferred from the presence of specific amino acids in the same position in each family member. These residues are said to be conserved residues. Proteins that share a large number of conserved residues are said to be homologous to one another. Among the most highly conserved residues are those that participate directly in catalysis. Amino acid sequences in the neighbourhood of the catalytic sites of several bovine proteases. Regions shown are those on either side of the catalytic site surly (S) and histidyl (H) residues.

8.4.2

Enzymes Employ Multiple Mechanisms to Facilitate Catalysis

An understanding of the complete mechanism of action of a purified enzyme requires identification of all substrates, cofactors, products and regulators. Moreover, it requires information of (1) the temporal sequence in which enzyme-bound reaction intermediates form, (2) the structure of each intermediate and each transition state, (3) the rates of interconversion between intermediates, (4) the structural relationship of the enzyme to each intermediate, and (5) the energy contributed by all reacting and interacting groups to intermediate complexes and transition states. As yet, there is probably no enzyme for which we have an understanding that meets all these requirements. Many decades of research, however, have produced mechanistic information about hundreds of enzymes, and in some cases this information is highly detailed. Five general mechanisms account for the ability of enzymes to achieve dramatic catalytic enhancement of the rates of chemical reactions. Proximity of substrate molecules bound to enzymes favours reaction The primary requirement of any reaction to occur is that the substrate molecules should come close enough for the bond formation to occur. The binding of enzymes with the

Enzymes 179

substrate in its active site creates a region of high local concentration that enhances the rate of reaction. Moreover, it orients the substrates in a position required for the reaction to occur. Enzyme–substrate interaction imposes high strain on the bond to be broken Lytic enzymes catalyze breaking of a covalent bond. Binding of enzymes to such substrates imposes strain on the bond to be broken during the enzymatic reaction. The resulting strain weakens the target bond and makes it susceptible for cleavage. Acid-Base Catalysis In acid-base catalysis of the enzymes, the functional groups such as aminoacyl side chains or those of prosthetic groups themselves act as acids and bases. In specific acid-base catalysis, the reaction rate depends on concentration of (H3O +) or OH– ions. However, the reaction rate is independent of other acids or bases (proton donors or acceptors). When all acids or bases affect reaction rate, the catalytic mechanism is referred to as general acid or base catalysis. Covalent Catalysis Covalent catalysis involves formation of covalent bond between enzyme and one or more substrates. However, this is transient and enzyme acts as a reactant in the subsequent step and the reaction proceeds through an energetically favourable pathway. The enzyme is restored to its original form on the completion of reaction. Covalent catalysis is particularly commonly encountered with enzymes that catalyze group transfer reactions. Generally, the amino acids of the enzyme that contribute to covalent catalysis are cysteine or serine and occasionally histidine. The catalytic cycle is said to follow a ping-pong mechanism in which the first substrate is bound and its product released. Subsequently, the second substrate binds. The proteolytic enzyme chymotrypsin provides an excellent example of this mechanism. Metal ion catalysis Metal ion catalysis involves metalloenzymes which have a metal attached to the protein structure. The most common metal ions that are involved are Fe2+, Cu2+, Zn2+, Mn2+, Co3+, Ni3+, Mo6+ which are found to be tightly bound to the enzyme whereas some of the metals are involved that are not tightly bound (Na+, K+, Mg2+, Ca2+). Metals may activate the enzymes, may facilitate the formation of a nucleophile or may stabilize the transition state.

8.4.3

Examples of Enzymatic Catalysis

Aspartic proteases show acid-base catalysis Enzymes of the aspartic protease family including the digestive enzyme pepsin, the highly specific protease rennin, the lysosomal cathepsins, and the protease produced by the human immunodeficiency virus (HIV) illustrate acid-base catalysis. Aspartyl proteases contain two highly conserved aspartate residues at the active site. The catalytic mechanism consists of the following steps: (i) Aspartate residue (Asp Y) acts as a general base extracts a proton

180 Textbook of Biochemistry

from water molecule, making it more nucleophilic. (ii) The nucleophile then attacks the electrophilic carbonyl carbon of the target peptide bond forming a tetrahedral transition state intermediate. (iii) A second aspartate (Asp Y) donates a proton to the amino group produced by the cleavage of peptide bond and facilitates the decomposition of this tetrahedral intermediate (Figure 8.7). In this mechanism, one aspartate acts as a general base whereas the other aspartate acts as a general acid. This is possible because their immediate environment favours ionization of one but not the other.

Mechanism for catalysis by an aspartic protease such as HIV protease. 1. Aspartate X acts as a base to ionize a water molecule by removing a proton. 2. The activated water molecule attacks the peptide bond, forming a transient tetrahedral intermediate. 3. Aspartate Y acts as an acid to facilitate breakdown of the intermediate and release of the products by donating a proton to the newly formed amino group. Subsequent shuttling of the proton on Asp X to Asp Y restores the protease to its initial state.

Fig. 8.7

Catalytic mechanism of aspartic protease (HIV protease)

One of these targets HIV-1 protease (HIV PR) is an essential enzyme needed in the proper assembly and maturation of infectious virions. Understanding the chemical mechanism of this enzyme has been a basic requirement in the development of efficient inhibitors.

Enzymes 181

Covalent catalysis as shown by serine proteases Bovine pancreatic chymotrypsin is a proteolytic enzyme that belongs to serine protease class of enzyme. These proteases typically illustrate covalent catalysis. Catalysis by the serine protease chymotrypsin involves prior formation of a covalent acyl enzyme intermediate. Chymotrypsin is specific for peptide bonds adjacent to aromatic amino acids Trp, Phe,

Catalysis by chymotrypsin 1 The charge-relay system removes a proton from Ser195, making it a stronger nucleophile. 2 Activated Ser-195 attacks the peptide bond, forming a transient tetrahedral intermediate. 3 Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His57 of the charge-relay system, yielding an acyl-Ser-195 intermediate. 4 His-57 and Asp-102 collaborate to activate a water molecule, which attacks the acyl-Ser-195, forming a second tetrahedral intermediate. 5 The charge-relay system donates a proton to Ser-195, facilitating breakdown of tetrahedral intermediate to release the carboxyl terminal peptide.

Fig. 8.8

Metal ion catalysis by carbonic anhydrase

182 Textbook of Biochemistry

Tyr. Chymotrypsin active site consists of a highly reactive serine, serine 195 that forms a charge-relay network with histidine 57 and aspartate 102 that functions as a “proton-shuttle”. The reaction has two distinct phases. In the phase of acylation, the peptide bond is broken and an ester bond is formed between the peptide carbonyl carbon and the enzyme. In the

Fig. 8.9

Carbonic anhydrase structure and its catalytic mechanism

Enzymes 183

deacylation phase, the ester linkage is hydrolyzed and the enzyme is regenerated in its original form. Binding of substrate initiates proton shifts affecting the transfer of hydroxyl proton of Ser-195 to Asp-102 (Figure 8.8). The enhanced nucleophilicity of the oxygen of serine residue leads to its attack on the carbonyl carbon of the peptide bond of the substrate, forming a covalent acyl-enzyme intermediate. The hydrogen on Asp-102 then shuttles through His-57 to the amino group liberated when the peptide bond is cleaved. The portion of the original peptide with a free amino group then leaves the active site and is replaced by a water molecule. The charge-relay network now activates the water molecule by withdrawing a proton through His-57 to Asp-102. The resulting hydroxide ion attacks the acyl-enzyme intermediate and a reverse proton shuttle returns a proton to Ser-195, restoring its original state. While modified during the process of catalysis, chymotrypsin emerges unchanged on completion of the reaction. The active site is replaced by a water molecule. The charge-relay network now activates the water molecule by withdrawing a proton through His-57 to Asp102. The resulting hydroxide ion attacks the acyl-enzyme intermediate and a reverse proton shuttle returns a proton to Ser-195, restoring its original state. While modified during the process of catalysis, chymotrypsin emerges unchanged on completion of the reaction. CA is a ubiquitous zinc metalloenzyme that catalyzes the reversible hydration of carbon dioxide. Carbonic anhydrase II is a vital enzyme required for CO2 in erythrocytes, lungs and kidney that is essential to maintain acid-base homeostasis; and promoting HCO3− secretion. It has been discovered that the active site cavity of carbonic anhydrase II is approximately 15 Å and approximately 15 Å in depth. The prosthetic group zinc is found to be coordinated to three histidine residues, NF of H94, NE of H96 and NF of H119, and a water molecule in a tetrahedral manner at the bottom of the cavity. These ligands form an extensive hydrogen bond network with other residues. These hydrogen bond acceptors to the direct ligands are called “indirect ligands.” One side of the active site cavity is mainly composed of hydrophilic residues, whereas the other side contains mostly hydrophobic residues. This hydrophobic region is probably the substrate CO2 binding site, as indicated by the structure of the complex of the enzyme with bicarbonate and formate (Figure 8.9). The mechanism of CO2 hydration, catalyzed by CA II, can be separated into two steps. In the first step, zinc-bound hydroxide attacks the carbonyl carbon of CO2 to form zincbound bicarbonate; bicarbonate is subsequently displaced with water by a ligand-exchange step. In the second step, H+ is transferred from zinc-bound water to external buffer via a shuttle group (H64 in CA II) to regenerate the catalytically active species, the zinc-bound hydroxide. EZn-OH+ + CO2 Buffer + EZn-OH2 | H64

EZn-HCO3–

H 2O

EZn-OH+ + Buffer | H64H+

EZn-OH2 + HCO3– EZn-OH– + Buffer H+ | H64

184 Textbook of Biochemistry

Yeast Hexokinase Yeast hexokinase (Mr 107, 862) is a bi-substrate enzyme that catalyzes the reversible reaction in which glucose is converted into glucose 6-phosphate (Figure 8.10).

Fig. 8.10

Reaction catalysed by hexokinase

ATP and ADP always bind to enzymes as a complex with the metal ion magnesium.

Fig. 8.11 Induced fit in hexokinase. (a) Hexokinase has a U-shaped structure (PDB ID 2YHX). (b) The ends pitch towards each other in a conformational change induced by binding of D-glucose(red) [Source: Sinauer Associates, Inc.]

In the absence of glucose, the enzyme is in an inactive conformation with the activesite amino acid side chains out of position for reaction. In the first step, the H phosphoryl of ATP is transferred to the hydroxyl at C-6 of glucose in the hexokinase reaction. When glucose and Mg-ATP bind, the binding energy derived from this interaction induces a conformational change in hexokinase to the catalytically active form. It is noteworthy that this hydroxyl and the hydroxyl of water are similar in reactivity and water is freely available at the active site. Yet hexokinase favours the reaction with glucose by a factor of 106. The enzyme can carry out selective reaction because of a conformational change that occurs due to the binding of the specific substrate (Figure 8.11) which is an apt example of induced fit catalysis. This model has been supported by enzyme kinetic studies. The five-carbon sugar xylose is able to bind hexokinase owing to structural similarity with glucose. It binds to hexokinase but in a position where it cannot be phosphorylated. However, xylose to hexokinase enhances the rate of ATP hydrolysis and hence it leads to

Enzymes 185

phosphorylation of water. This also illustrates that enzyme specificity is not only a function of specific binding of the substrate and formation of ES (enzyme-substrate) complex but it also depends on the rates of reactions that follow the formation of ES complex. Nevertheless, induced fit is only one characteristic of the catalytic mechanism of hexokinase. It also employs other catalytic strategies such as general acid-base catalysis and transition-state stabilization.

8.5

ENZYME KINETICS

Kinetics is the study of rates of chemical reactions. Thermodynamics can be used to predict whether the reactions would occur spontaneously in the given conditions of reactions. Kinetic information is useful for examining possible mechanisms for the reaction. Enzymes are the protein catalysts that speed up the biological reactions considerably without disturbing the equilibrium. Kinetic information is vital to understand the metabolism under different physiological conditions and the regulation of the metabolic reactions. Since many diseases affect enzyme function; kinetic information is useful to understand the pathological states. Moreover, many drugs also affect enzyme function as many drugs function by interacting with enzymes.

8.5.1

Factors that Affect Enzyme Catalyzed Reactions

The substrate concentration [S] is the most important key factor that affects the rate of enzyme catalysed reaction. As the reaction proceeds, the substrate concentration decreases due to conversion into product. Therefore, rate of reaction also changes during the course of reaction. Kinetic determinations can be simplified by measuring the initial rate (or initial velocity), V0. Initial rates are measured when the substrate concentration [S] is much greater than the concentration of enzyme, [E] typically for first 60 seconds or less. Usually, the enzymes may be present in nanomolar quantities as compared to substrate that is present in millimolar quantities and the change in substrate could be regarded as constant. Therefore, the velocity V0 of reaction can be investigated as a function of substrate concentration of [S]. The effect of varying [S] on V0 when the enzyme concentration is held constant is shown in Figure 8.12. At very low substrate concentrations [S], velocity of enzyme catalysed reaction increases with an increase in [S]. In this region, the reaction is first order with respect to the substrate [S]. As the substrate concentration is increased, increase in velocity is smaller with respect to increase in [S]. Finally, the curve approaches a plateau where the increase in V0 is negligible with increase in [S]. This plateau-like V0 region is known as maximum velocity, Vmax. The relationship between [S] and V0 was the basis of hypothesis proposed by Victor Henri, that the formation of ES complex is a necessary step in enzymatic catalysis. Subsequently, Leonor Michaelis and Maud Menten in 1913 elaborated a theory that the enzyme first binds to its substrate to form an enzyme-substrate complex. E+S

k1 k–1

ES

...(8.8)

186 Textbook of Biochemistry

Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction. Vmax is extrapolated from the plot, because V0 approaches but never quite reaches Vmax. The substrate concentration at which V0 is half maximal is Km, the Michaelis constant. Fig. 8.12

Effect of substrate concentration on the initial velocity of freaction catalysed by enzymes

Consequently, ES complex breaks down to regenerate the free enzyme and releases the product. This second step is slower as compared to the first reaction. ES

k2 k–2

E+P

...(8.9)

Since the second step is considerably slower than the first step, it is the limiting step in the reaction and the overall rate of the reaction is proportional to the concentration of ES complex (which is the reactant in the second rate limiting step). When the concentration of substrate [S] is low, most of the enzymes exist in free form and increase in substrate concentration results in proportional increase in reaction rate. However, at very high substrate concentrations most of the enzymes exist in complex with the substrate [ES] and free enzyme concentration [E] is negligible. Under these conditions, the enzyme is saturated with the substrate and further increase in [S] has no effect on rate of the reaction. This is responsible for the plateau-like region observed in Figure 8.12 and therefore, this pattern of kinetics is also known as saturation kinetics. At the start of the enzyme catalysed reaction, there exists a pre-steady state in which the concentration of ES complex increases. However, this period is too short to be of significance and the reaction quickly attains steady state. Here the [ES] remains constant over time. The concept of a steady state was proposed by G. E. Briggs and Haldane in 1925. The steady state kinetics thus describes the early part of the reaction.

8.5.2 The Michaelis-Menten Equation Michaelis-Menten equation can be derived logically and it also includes the steady state assumption of Briggs and Haldane. Consider an enzyme catalysed reaction, during the early phase of reaction the concentration of the product [P], is negligible, therefore it can be assumed that the reverse reaction, E + P gives ES, can be ignored.

Enzymes 187

E+S

k1 k–1

ES

k2


E + P

...(8.10)

Since the second step is the rate determining step, V0 is rate of breakdown of ES complex into the free enzyme and the product. V0 = k2 [ES]

...(8.11)

The rates of formation and breakdown of enzyme–substrate complex can be given as: Rate of ES formation = k1([Et] – [ES])[S]

...(8.12)

Rate of ES breakdown = k–1[ES] + k2 [ES]

...(8.13)

where Et is the total enzyme concentration (the sum of free and substrate-bound enzyme). Therefore, the free or unbound enzyme can then be represented by [Et] - [ES]. Since substrate concentrations of enzyme catalysed reactions are manifold greater than the enzyme concentrations it can also be assumed that the amount of substrate bound to the enzyme at any given time is negligible compared with the total [S]. It can be assumed that the initial stage of reaction is a steady state in which [ES] is constant, that is, the rate of formation of ES is equal to the rate of its breakdown. This is called the steady-state assumption. Therefore, k1[Et][S] –k1[ES][S] = (k–1 + k2)[ES]

...(8.14)

Solving for [ES] k1[Et][S] [ES] = ______________ k1[S] + k–1 + k2

...(8.15)

Dividing by K1 we can simplify the above equation by combining the rate constants: [Et][S] [ES] = ________________ [S] + (k2 + k–1)/k1

...(8.16)

The term (k2 + k–1)/k1 is defined as the Michaelis constant, Km. Substituting this into the above equation and expressing V0 in terms of ES as k2 [Et][S] V0 = ________ Km + [S]

...(8.17)

k2 [Et] can be called the maximum velocity which occurs when the enzyme present in the system is saturated with the substrate. In these conditions, [ES] = [Et]. ...(8.18) The velocity of enzyme catalysed reactions would reach maximum velocity Vmax that could be achieved therefore, k2 [Et] is Vmax that can be achieved in an enzyme catalyzed reaction

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K2 [Et] = Vmax

...(8.19)

Substituting this in equation 8.19 gives Michaelis-Menten equation: Vmax[S] V0 = ________ Km + [S]

...(8.20)

The above rate equation is also known as the Michaelis-Menten equation, the rate equation for a one-substrate enzyme-catalyzed reaction. It establishes the quantitative relationship between the initial velocity V0, the maximum velocity Vmax, and the initial substrate concentration [S]. The Michaelis-Menten constant, Km has units of concentration. A special case arises when the substrate concentration is exactly half of the substrate concentration at Vmax, Vmax[S] Vmax ____ = ________ ...(8.21) 2 Km + [S] This is a very useful, practical definition of Km : Km is equivalent to the substrate concentration at which V0 is one-half Vmax. The Michaelis-Menten equation transformed into a linear form which is useful for the determination of Km and Vmax and also for the analysis of inhibitor action, Km = S Vmax V0 = ____ 2 Taking reciprocal of both sides, Km + [S] 1 ___ = _______ V0 Vmax [S]

...(8.22)

Segregating the terms on the right side Km [S] 1 ___ = _______ + _______ V0 Vmax[S] Vmax[S]

...(8.23)

The above equation can be simplified as Km 1 1 ___ = _______ + ____ V0 Vmax[S] Vmax

...(8.24)

The above form of Michaelis-Menten equation is also known as Lineweaver-Burke equation. This equation describes a line and a ‘double reciprocal’ plot of 1/V0 versus 1/[S] for the enzymes following Michaelis-Menten equation which is a straight line with a slope of Km /Vmax and a Y-axis intercept 1/Vmax. This plot can be used for accurate determination of the Km and Vmax (Figure 8.13).

Enzymes 189

Fig. 8.13

8.5.3

Double reciprocal plot or Lineweaver-Burke plot

Enzyme Kinetic Data can be Used to Compare the Activities of Different Enzymes

Several enzymes demonstrate the hyperbolic relationship between V0 and [S] described by Michaelis-Menten kinetic equation. However, the regulatory enzymes are important exceptions to Michaelis-Menten kinetics. Moreover, it has to be noted that numerous enzymes that follow Michaelis-Menten kinetics have quite different reaction mechanisms involving more than the two-step mechanism proposed by Michaelis and Menten. Enzymatic reactions with six-eight discreet steps often show similar kinetic behaviour. The magnitude and real meaning of Vmax and Km is different for each enzyme. The value of Km varies significantly for different enzymes and for different substrates of same enzyme. For the reactions with simple two-step mechanisms, k2 + k–1 ...(8.25) Km = _______ k1 where k2 is the rate constant for the rate limiting second step and k2>>>k1 the Km reduces to k2 /k1, which can be defined as the dissociation constant, kd. Under these conditions, Km can be said to be a measure of affinity of enzyme towards the specific substrate. However, for many enzymes k2 and k1 are comparable and Km is a function of all three rate constants. Moreover, for the enzymatic reactions with more than two steps, Km becomes a more complicated function. The quantity Vmax also varies greatly from one enzyme to the next. If an enzyme reacts by the two-step Michaelis-Menten mechanism, Vmax = k2 [Et], where the second step with the rate constant k2 is rate-limiting. On the other hand, several reactions proceed through a number of reaction steps with different rate-limiting step(s) enzyme. Let us consider a reaction where product release is the rate-limiting step. Initially, the product concentration is low, and the overall reaction can be given as

190 Textbook of Biochemistry

E+S

k1 k–1

ES

k2 k–2

EP

k3

E+P

...(8.26)

Therefore, at saturation most of the enzymes exist in EP form and Vmax can be given as k3[Et]. Therefore, a more general rate constant, Kcat, turnover number, is used to describe the rate limiting step in the enzyme catalysed reaction at saturation. The turnover number, Kcat can be defined as the number of substrate molecules converted to product on a single enzyme molecule per unit time when the enzyme is saturated with the substrate. Therefore, Kcat is the rate constant for limiting step of the enzyme catalysed reaction. For the simple two-step reaction given in the above equation, Kcat = k2. However, when several steps are partially rate-limiting, Kcat can become a complex function of several of the rate constants that define each individual reaction step. The constant Kcat is a first-order rate constant and hence has units of reciprocal time. The Vmax = Kcat /Et and therefore Michaelis-Menten equation can be rewritten as Kcat [Et][S] V0 = __________ ...(8.27) Km+[S]

8.5.4

Catalytic Mechanisms and Efficiencies of Enzymes

The kinetic parameters Km and Kcat are used to study and compare different enzymes. These values reveal the intracellular environment that is naturally experienced by the enzyme including the concentration of the substrate and chemistry of the reaction being catalysed. Two enzymes catalysing different reactions may have the same Kcat (turnover number), but since the rate of uncatalyzed reactions may be different and hence the rate enhancements brought about would be different. It has been found that, the Km for an enzyme is similar to the intracellular concentration of its substrate. Therefore, the ratio of Kcat /Km is taken as an ideal parameter to compare the catalytic efficiency of an enyzme acting on different substrates. This parameter is also known as specificity constant. It is the rate constant for the conversion of E+S into E+P. When [S] is