Enzymes 9788182092921

Table of contents :
Cover
Contents
1. Introduction to Enzymes
Introduction
Meaning and definition
History
General properties of enzymes
1. Higher Reaction Rates
2. Milder Reaction Condition
3. Greater Reaction Specificity
4. Capacity for Regulations
5. Presence of Species Specificity
6. Variations in Activity and Ability
7. Substrate Specificity
8. Activation and Inhibition
Sources of enzymes
Characteristics of enzymes
Questions
2. Enzyme Nomenclature and Classification
Nomenclature
Classification
Major Classes
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases (Synthetases)
Key to Numbering and Classification of Enzymes
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
Examples
Questions
3. Enzyme Extraction and Purification
Importance of Enzyme Purification
General Procedure
Extraction
Enzyme Histochemistry
Use of Centrifugation
Extraction of Soluble Enzymes
Extraction of Membrane-Bound Enzymes
Nature of the Extraction Medium
Purification of Enzymes
Preliminary Procedure
Fractionation Methods
Partition Chromatography
Electrophoresis
Cation Exchange Chromatography
Gel Filteration
Affinity Chromatography
Hplc Technology
Lyophilization
Determination of Molecular Weight of Enzymes
Examples of Purification Procedure
Adenylate Kinase from Pig Muscles
Ribulose bisphosphate carboxylase from spinach
RNA Polymerase from E.Coli
Arom multi enzyme protein from Neurospora
Glutathione reductase from E.Coli
Adenylate Cyclase
Chymosin
Questions
4. Enzyme Assay
Factors controlling in assays
1. Salt Concentration
2. Temperature
3. pH
4. Substrate Saturation
5. Level of Crowding
Discontinous Assay
Continous Assay
Coupled Assay
Enzyme Assay Techniques
Visible and ultraviolet spectrophotometer methods
Spectrofluorimetric method
Luminescence method
Radioisotope method
Questions
5. Types of Enzyme
Introduction
Coenzymes
Characteristics of coenzymes
Isoenzymes
Lactate dehydrogenase enzyme (LDH)
Monomeric Enzymes
Proteases (proteolytic enzymes)
Oligomeric Enzymes
Lactate Dehydrogenase
Lactose Synthase
Tryptophan Synthase
Pyruvate Dehydrogenase
Metalloenzymes and Metal-activated Enzymes
Metalloenzymes
Metal Activation
Extremozymes
Abzymes
Ribozymes
Synzymes
Bi and Polyfunctional Enzyme
Questions
6. Enzyme Catalysis
Mechanisms in Organic Chemistry
Mechanisms of Enzyme Catalysis
Acid–Base Catalysis
Strain Distortion and Conformational Change
Catalysis by Approximation (Entrophic Contribution)
Covalent Catalysis(Nucleophilic vs Electrophilic catalysis)
Coenzymes in Enzyme-Catalyzed Reactions
Questions
7. Enzyme Specificity
Group Specificity
Absolute Specificity
Stereochemical Specificity
The Active Sites
Fischer’s Lock and Key Hypothesis
Koshland Induced Fit Hypothesis
Non-Productive Binding
Transition State Stabilization
Cryoenzymology
Questions
8. Enzyme Kinetics
Goals of Enzyme Kinetics
Chemical Kinetics
Reaction Order
Classification of Chemical Reactions by Kinetic Order
Kinetics of Bisubstrate Enzyme Reactions
The Michaelis–Menten Equation
The Lineweaver–Burk Plot
The Eadie - Hofstee and Hans Plot
Kinetics of Multisubstrate Enzyme-Catalyzed Reactions
Ping-Pong Bi-Bi Mechanism
Random-Order Mechanism
Compulsory-Order Mechanism
Steady-State Kinetics
Plots For Mechanisms Which Follow the General Rate Equation
Allosteric Enzymes
Monod–Wyman Changeux (MWC) Model
MWC Model and Allosteric Regulation
Questions
9. Enzyme Inhibition
Reversible Inhibition
Competitive Inhibition
Uncompetitive Inhibition
Non-competitive Inhibition and Mixed Inhibition
Partial Inhibition
Substrate Inhibition
Allosteric Inhibiton
Irreversible Inhibition
Questions
10. Enzyme Cofactors
Mode of Action
Cofactors Acting as Carriers
Redox Carriers
Phosphate Carriers
CO2 Carrier
Amino Group Carriers
Acyl Group Carriers
Carrier of One Carbon Group
Sulphate Carrier
Aldehyde Carrier
Questions
11. Enzyme Biology
Introduction
Intracelluar Localization of Enzymes
Histochemical Methods
Separation of Particulate Fraction
Questions
12. Enzyme Engineering
Definition
Objectives
Principle of Enzyme Engineering
Procedure in Enzyme Engineering
Example
Protein Engineering
Addition of Disulphide Bond
Changing Asparagine to other Aminoacids
Reduction of Number of Free Sulphydryl Residues
Increase of Enzymatic Activity
Modification of Enzyme Specificity
Secretion of Cloned Proteins
Large Scale Fermentation
Principles of Microbial Growth
Bioreactors
Harvesting of Microbial Cells
Disruption of Microbial Cells
Downstream Processing
Questions
13. Immobilization of Enzyme
Advantages
Methods of Enzyme Immobilization
Adsorption
Covalent Bonding
Entrapment
Crosslinking
Encapsulation
Applications of Immobilized Enzyme
Immobilized Enzyme Kinetics
Heterogenous Concentrations
Concentration Gradients and Reaction Rates in Solid Catalyst
Effect of External Mass Transfer Resistance
Diffusion Effects in Surface-Bound Enzymes on Non-Porous Support Materials
Design of Immobilized Enzyme Reactors
Batch Reactors
Continuous Flow Reactors
Packed Bed Reactor
Fluidized-Bed Reactors
Membrane Reactors
Questions
14. Biosensors
Principle
General Features
Glucose Electrode
Types of Biosensors
Calorimetric Biosensors
Amperometric Biosensors
Optical Biosensors
Acoustic Wave Biosensor
Potentiometric Biosensors
Whole Cell Biosensors (Microbial Biosensors)
Applications of Biosensors
Questions
15. Clinical Enzymology
Amylase
Transaminases
Clinical Significance
Alkaline Phosphatase (ALP)
Clinical Significance
Acid Phosphatase (ACP)
Clinical Significance
Lactate Dehydrogenase (LD)
Clinical Significance
Creatine Kinase (CK)
Clinical Significance
Gamma-Glutamyl Transferase (GGT)
Clinical Significance
Cholinesterases
Clinical Significance
Questions
Clinical Significance
16 Applications in Enzymes
Applications in Medicine
Applications in Medicine
Assay of Plasma Enzymes
Therapeutic Uses
Application In Industry
In Food Industry
Baking of Bread
Enzymes and Recombinant DNA Technology
In Dairy Industry
Cheese Making
In Starch Industry
In Detergent Industry
In Leather Industry
Wool Industry
Production Syrups
In Sugar Industry
In Brewing Industry
Biotechnological Applications of Enzymes
Analytical Uses
Principles of Enzymatic Analysis
Questions

Citation preview

EnzymEs

EnzymEs

Dr. R. Devika Professor Head, Department of Bioinformatics Aarupadai Veedu Institute of Technology

VIjay Nicole Imprints Private Limited Chennai

Published by Vijay Nicole Imprints Private Limited No. 4, First Floor, Velachery-Madipakkam Main Road, Ram Nagar South, Chennai - 600 091. Phone: 91 44 4281 1452 , 4281 1349 Email: [email protected], www.vijaynicole.co.in

in collaboration with

Tata Mcgraw Hill Education Pvt. Ltd. B-4, Sector-63, Dist. gautam Budh Nagar Noida, UP 201 301 Phone: +91 120 4383400 Fax: +91 120 4393401–403 www.tatamcgrawhill.com EnzymEs IsBn: 978-81-8209-292-1

CopyrIght © 2011, Vijay nicole Imprints private Limited

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Laser typeset at: Maven Learning Private Limited, Chennai - 600 091 Printed at: Novena Offset Printers Co, Chennai 600 005

Contents Preface

1

xiii

Introduction to Enzymes Introduction Meaning and definition History General properties of enzymes 1. Higher Reaction Rates 2. Milder Reaction Condition 3. Greater Reaction Specificity 4. Capacity for Regulations 5. presence of Species Specificity 6. Variations in Activity and Ability 7. Substrate Specificity 8. Activation and Inhibition Sources of enzymes Characteristics of enzymes Questions

2

1 1 2 3 3 4 4 4 4 5 5 5 6 7 8

Enzyme Nomenclature and Classification Nomenclature Classification Major Classes 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases (Synthetases)

9 9 10 11 12 13 13 14 14

vi  Enzymes Key to Numbering and Classification of enzymes 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases examples Questions

3

15 15 18 19 21 21 22 22 24

Enzyme Extraction and Purification Importance of enzyme purification General procedure extraction enzyme Histochemistry Use of Centrifugation extraction of Soluble enzymes extraction of Membrane-Bound enzymes Nature of the extraction Medium purification of enzymes preliminary procedure Fractionation Methods partition Chromatography electrophoresis Cation exchange Chromatography Gel Filteration Affinity Chromatography HpLC Technology Lyophilization determination of Molecular Weight of enzymes examples of purification procedure Adenylate Kinase from pig Muscles Ribulose Bisphosphate Carboxylase from Spinach RNA polymerase from e.Coli Arom Multi enzyme protein from Neurospora Glutathione Reductase from e.Coli Adenylate Cyclase Chymosin Questions

25 25 26 27 28 28 30 31 32 32 32 35 36 36 36 36 37 37 37 38 38 39 39 40 41 42 43 44

Contents  vii

4

Enzyme Assay Factors Controlling in Assays 1. Salt Concentration 2. Temperature 3. pH 4. Substrate Saturation 5. Level of Crowding discontinous Assay Continous Assay Coupled Assay enzyme Assay Techniques Visible and Ultraviolet Spectrophotometer Methods Spectrofluorimetric Method Luminescence Method Radioisotope Method Questions

5

47 47 47 48 48 48 48 49 49 50 50 50 51 51 51

Types of Enzyme Introduction Coenzymes Characteristics of Coenzymes Isoenzymes Lactate dehydrogenase enzyme (LdH) Monomeric enzymes proteases (proteolytic enzymes) Oligomeric enzymes Lactate dehydrogenase Lactose Synthase Tryptophan Synthase pyruvate dehydrogenase Metalloenzymes and Metal-Activated enzymes Metalloenzymes Metal Activation extremozymes Abzymes Ribozymes Synzymes Bi and polyfunctional enzyme Questions

53 53 54 55 55 56 56 57 58 58 58 58 58 59 59 61 61 62 62 63 63

viii 

6

Enzymes

Enzyme Catalysis Mechanisms in Organic Chemistry Mechanisms of enzyme Catalysis Acid–Base Catalysis Strain distortion and Conformational Change Catalysis by Approximation (entrophic Contribution) Covalent Catalysis(Nucleophilic vs electrophilic Catalysis) Coenzymes in enzyme-Catalyzed Reactions Questions

7

Enzyme specificity Group Specificity Absolute Specificity Stereochemical Specificity The Active Sites Fischer’s Lock and Key Hypothesis Koshland Induced Fit Hypothesis Non-productive Binding Transition State Stabilization Cryoenzymology Questions

8

65 67 67 67 68 70 71 72

73 73 74 74 75 75 76 77 77 77

Enzyme Kinetics Goals of enzyme Kinetics Chemical Kinetics Reaction Order Classification of Chemical Reactions by Kinetic Order Kinetics of Bisubstrate enzyme Reactions The Michaelis–Menten equation The Lineweaver–Burk plot The eadie - Hofstee and Hans plot Kinetics of Multisubstrate enzyme-Catalyzed Reactions ping-pong Bi-Bi Mechanism Random-Order Mechanism Compulsory-Order Mechanism Steady-State Kinetics plots For Mechanisms Which Follow the General Rate equation

79 79 80 80 81 83 87 89 90 91 91 92 92 95

Contents  ix Allosteric enzymes Monod–Wyman Changeux (MWC) Model MWC Model and Allosteric Regulation Questions

9

Enzyme Inhibition Reversible Inhibition Competitive Inhibition Uncompetitive Inhibition Non-competitive Inhibition and Mixed Inhibition partial Inhibition Substrate Inhibition Allosteric Inhibiton Irreversible Inhibition Questions

10

103 103 106 106 106 107 107 108 108

Enzyme Cofactors Mode of Action Cofactors Acting as Carriers Redox Carriers phosphate Carriers CO2 Carrier Amino Group Carriers Acyl Group Carriers Carrier of One Carbon Group Sulphate Carrier Aldehyde Carrier Questions

11

97 97 99 102

109 111 111 113 114 114 115 115 116 116 117

Enzyme Biology Introduction Intracelluar Localization of enzymes Histochemical Methods Separation of particulate Fraction Questions

119 120 120 120 127

x 

12

Enzymes

Enzyme Engineering definition Objectives principle of enzyme engineering procedure in enzyme engineering example protein engineering Addition of disulphide Bond Changing Asparagine to other Aminoacids Reduction of Number of Free Sulphydryl Residues Increase of enzymatic Activity Modification of enzyme Specificity Secretion of Cloned proteins Large Scale Fermentation principles of Microbial Growth Bioreactors Harvesting of Microbial Cells disruption of Microbial Cells downstream processing Questions

13

129 129 130 130 131 132 132 134 135 137 138 140 140 141 145 147 149 151 152

Immobilization of Enzyme Advantages Methods of enzyme Immobilization Adsorption Covalent Bonding entrapment Crosslinking encapsulation Applications of Immobilized enzyme Immobilized enzyme Kinetics Heterogenous Concentrations Concentration Gradients and Reaction Rates in Solid Catalyst effect of external Mass Transfer Resistance diffusion effects in Surface-Bound enzymes on Non-porous Support Materials design of Immobilized enzyme Reactors Batch Reactors Continuous Flow Reactors packed Bed Reactor

153 154 154 155 156 156 156 160 163 163 164 165 167 168 168 169 169

Contents  xi Fluidized-Bed Reactors Membrane Reactors Questions

14

Biosensors principle General Features Glucose electrode Types of Biosensors Calorimetric Biosensors Amperometric Biosensors Optical Biosensors Acoustic Wave Biosensor potentiometric Biosensors Whole Cell Biosensors (Microbial Biosensors) Applications of Biosensors Questions

15

173 177 180

181 182 184 185 185 185 186 187 187 187 189 190

Clinical Enzymology Amylase Clinical Significance Transaminases Clinical Significance Alkaline phosphatase (ALp) Clinical Significance Acid phosphatase (ACp) Clinical Significance Lactate dehydrogenase (Ld) Clinical Significance Creatine Kinase (CK) Clinical Significance Gamma-Glutamyl Transferase (GGT) Clinical Significance Cholinesterases Clinical Significance Questions

193 193 194 194 195 195 195 196 196 196 197 197 197 197 198 198 198

xii 

16

Enzymes

Applications in Enzymes Applications in Medicine Assay of plasma enzymes Therapeutic Uses Application In Industry In Food Industry Baking of Bread enzymes and Recombinant dNA Technology In dairy Industry Cheese Making In Starch Industry In detergent Industry In Leather Industry Wool Industry production Syrups In Sugar Industry In Brewing Industry Biotechnological Applications of enzymes Analytical Uses principles of enzymatic Analysis Questions

199 199 201 203 203 204 205 207 207 207 207 209 209 209 211 211 212 213 214 222

PrefaCe This book "Enzymes", caters to the needs and demands of the present day students. The book portrays the salient features of Enzymology, now a part of Biochemistry, which has a wide range of applications in life sciences. Sincere efforts have been taken to highlight its potentialities in the field of Biotechnology, in a concise manner so that the students may not feel alien to the subject when they pursue advanced studies and research. All the fundamental concepts of Enzymes such as its characteristics, nomenclature, types, coenzymes, cofactors, kinetics, inhibition, etc. have been covered. The book also covers in detail, topics such as Biosensors, Immobilization, Enzyme Engineering, Applications etc. Intensified efforts have been made to cover the syllabi of both UG and PG courses. We shall be thankful to those who point out the errors and infirmities in the illustrations. Constructive suggestions are welcome, which will be taken into account and incorporated in the future editions. Dr. R. Devika

1

CHAPTER

Introduction to Enzymes

IntroductIon Life depends on a complex network of chemical reactions brought about by specific enzymes and any changes in enzyme pattern have far reaching consequences for the living organisms. In living organisms, these chemical reactions occur to obtain energy, to synthesize protoplasmic and other structured materials, to absorb required substances for life from the external environment and to eliminate unwanted materials into the environment. These enormous variety of biochemical reactions are mediated by a series of remarkable biological catalysts known as “Enzymes”.

MeanIng and defInItIon Enzymes are proteins with specific catalytic functions that are produced by all living cells. They are biocatalysts, which accelerate biological reactions without themselves undergoing any change. In general, “catalysts” are effective in small amounts, and they remain unchanged after the reaction. They do not affect the position of equilibrium of a reversible reaction, but simply increase the speed at which equilibrium is achieved. However, the concept of “Biocatalyst” is very wide. It includes the pure enzymes, crude cell extract, viable plant cells, viable animal cells, viable microbial cells and intact non-viable microbial cells. Enzymes are defined as soluble, colloidal organic catalysts formed by living cells and accelerates the rates of reaction without themselves undergoing any change. The general characteristic

2 

Enzymes

features of enzymes are proteinaceous in nature, inactive at 0OC and they have high degree of specificity and efficiency. The study of enzymes is referred to as “Enzymology”. Enzymology has become a large and rapidly developing subject with ramifications in many directions with many science subjects.

HIstory •

•

• • •

•

• •

•

The beginning of the subject was in the early nineteenth century, but great developments in this field came during the last few decades. Initially Louis Pasteur and others assumed that living systems were endowed with a vital force that permitted them to evade the laws of nature governing inanimate matter. Justus Von Liebig argued that biological processes were caused by the action of chemical substances known as “Ferments”. The name “Enzymes” (Greek: en, in + zymes, yeast) was coined in 1878 by Friedrich Wilhelm Kuhne. It means “in yeast”. In 1833, Payer and Persoz found that alcoholic precipitate of malt extract contained a thermolabile substace, which converted starch into sugar and named it as “ Diastase” (now called as “Amylase”). In 1897, Edward Bucher succeeded in extracting the enzymes from yeast cells and demonstrated their activity outside the cells. He showed that a cell-free yeast extract could, infact, carry out the synthesis of ethanol from glucose (alcoholic fermentation). C6H12O6 → 2CH3CH2OH + 2CO2 This chemical transformation actually proceeds in 11 enzyme catalyzed steps. In 1898, Duclaux proposed the use of the last three letters, “ase” as suffix, to be attached to a root indicating the nature of the substance on which the enzyme acts and few names ending in “in” had been given to digestive enzymes. Towards the end of the nineteeth century, Emil Fischer demonstrated steric relationship between enzyme and substrate. Fischer’s “Lock and Key” model developed an important branch

Introduction to Enzyme

•

•

• •

•

 3

of enzyme study. A necessary consequence of the close fit between enzyme and substrate is that each enzyme acts on a very limited range of substrates. This implied the existence of a large number of different enzymes. The chemical composition of enzymes was not firmly established until 1926, when James Sumner crystallized Jack bean Urease, which catalyzes the hydrolysis of urea to NH3 and CO2. He also confirmed that enzymes are proteins. Purification of enzymes was carried out by Willstatter and his co-workers between 1922 and 1928. Dixon and Kodama purified Xanthine oxidase. In 1930, Northrop crystallized several enzymes such as “Pepsin” from swine stomach and “Trypsin” from beef pancreas. In 1937, Sumner isolated “catalase” from beef liver and kunitz isolated “RNAase” from beef pancreas in 1940 and “DNAase” from beef pancreas in 1950. The number of purified enzymes were very less, 35 years ago, but now, the number has increased to 1500, in pure and crystalline form. The availability of enzymes in the pure state had made possible to great developments in structured enzymology in recent years.

general propertIes of enzyMes Enzymes differ from ordinary chemical catalysts in several important respects.

1. Higher reaction rates The rates of enzymatically catalyzed reactions are typically 106 to 1012 times greater than those of the corresponding uncatalyzed reactions. (Table 1.1)

4 

Enzymes

Table 1.1

Reaction Rates of Enzymes

Non Enzymatic enzymatic Rate Enzymes reaction rate reaction enhancement (S-1) rate (S-1) 7.7 × 10-6 1 × 10-6 Carbonic anhydrase 1.3 × 10-1 50 1.9 × 10-6 Chorismate mutase 2.6 × 10-5 Triose phosphate isomerase Carboxypeptidase AMP nucleosidase

4.3 × 10-6 1.0 × 10-9 1.7 × 10-13

4300 578 95

1.0 × 10-9 6.0 × 10-12 5.6 × 10-14

2. Milder reaction condition Enzymatic reactions occur under relatively mild conditions such as i Temperature below 100oC ii Atmospheric pressure iii Nearly neutral pH

3.  Greater Reaction Specificity Enzymes have greater degree of substrate specificity and they rarely produce side products.

4.  Capacity for Regulations Enzymes involved reactions vary according to the concentration of substances other than substrates. They include: i Allosteric control ii Covalent modification of enzymes iii Variation of the amounts of enzymes

5.  Presence of Species Specificity Enzyme types (protease, α-amylase, lactase) have the properties, which vary as much as the other properties of the organisms. e.g. Protease of two closely related species differ in several ways inspite of some similarities.

Introduction to Enzyme

 5

6.  Variations in Activity and Ability Enzymes are influenced externally by temperature and pH. e.g. Optimum temperature for amylase activity of a thermophilic species (Bacillus coagulans) differ from that of mesophilic species (Bacillus licheniformis). Xylose isomerase is stable at the range from 4.0 to 8.5 but shows optimum activity at pH between 5.5 to 7.0. In addition to pH and temperature, the stability of enzyme is also influenced by many factors such as i High concentration of respective enzymes. ii Presence of their substrate or product. e.g. Amylase shows more stability in the presence of starch than in its absence. iv Presence of ions e.g. α-amylase is denatured within 4 hours in the absence of Ca++. v Reduction of water content in the reaction.

7.  Substrate Specificity Organic matter contains various constituents such as (complex matrix). In nature, they are decomposed and mineralized by a variety of microorganisms. (It is not possible for a single microbe to decompose all the constituents). e.g. A cellulose enzyme will fail to decompose lignin.

8.  Activation and Inhibition Enzymes obtained from different sources show differences in response to a given activator or inhibitor. e.g. β-galactosidase isolated from fungi does not require cobalt, but β-galactosidase isolated from bacteria require cobalt. (Thus cobalt activates β-galactosidases isolated from bacteria but inhibits when obtained from fungi.

6 

Enzymes

Some activators of enzymes commercially used are : Proteins : Proteases Starch : α-amylases Cellulose : Cellulases Pectin : Pectinases

sources of enzyMes The sources of enzymes are microorganisms, higher plants and animals. Animal enzymes: e.g. Lipase, Trypsin, Rennet, etc. Plant enzymes : e.g. Papain, Proteases, Amylases, Soyabean lipoxygenases, etc. These enzymes are used in food industries. e.g. Papain, extracted from papaya fruit, is used as a meat tenderizer. Pancreatic protease is used in leather softening and in the manufacture of detergents. Microbial enzymes have advantages over the animal and plant enzymes. They include: 1. They are economical and can be produced on large scale within the limited space and time. (The amount produced depends on the size of fermenter, types of microbial strains and growth conditions) 2. They are capable of producing a wide variety of enzymes. 3. They can grow in a wide range of environmental conditions. 4. They show genetic flexibility. (They can be genetically manipulated to increase the yield of enzymes). 5. They have a short generation time. At present, more than 2,000 enzymes have been isolated and characterized, out of which about 1,000 enzymes are recommended for various applications. In 1981, the total world production of enzymes was estimated about 65,000 tonnes which valued about 4 × 108 US dollars. Table 1.2 shows some examples of enzymes produced by bacteria.

Introduction to Enzyme

Table 1.2

 7

Enzymes produced by Bacteria

Bacteria Bacillus cereus Bacillus coagulans Bacillus licheniformis Bacillus megaterium Citrobacter sp. Escherichia coli Klebsiella pneumoniae

Enzymes Pencillinase α-amylase α- amylase and protease Penicillin acylase L-asparaginase Penicillin acylase, β- galactosidase Pullulanase

cHaracterIstIcs of enzyMes Enzymes possess many characteristic features. Since enzymes are proteins, they exhibit all the properties of proteins: 1. Enzymes are large in size, therefore they possess extremely low rate of diffusion and form colloidal system in water and are non-dialyzable. Urease, papain and trypsin can be crystallized and they are dialyzable. 2. Enzymes are subjected to denaturation due to change in pH or by increase in temperature of their solution. They are inactive at 0oC. 3. Enzyme have their own isoelectric point at which they are least soluble. 4. Enzymes can be precipitated by different precipitating agents such as alcohol, ammonium sulphate and alkaloidal reagents. 5. Chemical analysis of purified crystalline enzymes give composition typical of proteins (such as C, N, H and S). 6. Turnover number: The enzyme activity is represented by a turnover number, which is the number of moles of substrate converted to the product in one minute, by one mole of the particular enzyme. Recently, The International Enzyme Commission has defined a new unit known as Katal (briefly Kat) which is defined as the number

8 

Enzymes

of substrate molecules converted into product per unit time. The enzyme carbonic anhydrase is known to have the highest turnover number. CO2 + H2O Carbonic anhydrase

H2CO3

7. The rate of forward and backward reactions is increased in the right proportion in the presence of the enzyme. This does not alter the equilibrium of that reaction. 8. Enzyme has a number of active sites where the substrate is actually bound. If the active site is destroyed or altered the substrate can no longer bind itself to the enzyme and the formation of the product is stopped.

QuestIons 1. 2. 3. 4. 5. 6. 7. 8.

Define enzyme. What is a catalyst? What is a biocatalyst? How will you state that enzymes are proteins? Write down the different sources of enzymes. What is a substrate? Define turnover number. Explain the importance of enzyme distribution?

CHAPTER

2 Enzyme Nomenclature and Classification

noMenclature Enzymes are commonly named by the suffix “ase” to the name of the enzyme’s substrate or to a phrase describing the enzyme’s catalytic action. e.g. Urase catalyzes the hydrolysis of urea. Alcohol dehydrogenase catalyzes the oxidation of alcohol to their corresponding aldehyde. The names of some enzymes also denote the type of reactions along with the substrate. e.g. Lactic acid catalyzes the dehydrogenation (removal of hydrogen) of lactic acid. Some enzymes retain their trival names. e.g. pepsin, ptylanin, rennin, etc.

classIfIcatIon There is no perfect method for the classification of enzymes. However, in 1961, The Enzyme Commission of The International Union of Biochemistry (IUB) devised a more comprehensive system for the classification of enzymes. This system is called as IUB system of enzyme classification. Here, each number is given a systematic code number called Enzyme Commission number (E.C number). The major features of these system of classification are as follows: 1. According to the general type of chemical reaction they catalyze the enzymes are classified into six major classes.

10   Enzymes

2. Each class is further classified into sub-classes on the basis of i Type of bond spliced. ii Bond formed. iv Chemical group removed. v Chemical group transferred. vi In some cases, by simple reaction type. 3. The third number is used for more detailed subdivision of the substrate. 4. The fourth number is used to indicate the serial number of the specific enzyme within its own sub-class. On the basis of the above points, each enzyme is given a systematic code number commonly called as “Enzyme Commmission Number or E.C Number”. e.g. 1.1.1.1 is the E.C number for alcohol dehydrogenase. The first digit denotes the reaction type (major class) The second digit denotes the sub-class The third digit denotes the sub-sub-class The fourth digit denotes the particular enzymes

MaJor classes There are six major classes in enzyme classification. 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases (sythetases)

Enzyme Nomenclature and Classification

1.

 11

oxidoreductases

Oxidoreductases are those enzymes which catalyze oxidationreduction reactions between two substrates. For example, consider two substrates A and B. The reaction is as follows: A reduced + B oxidized

A oxidized + B reduced

Oxidation refers to • Addition of oxygen (or) • Removal of hydrogen (or) • Removal of electrons Reduction refers to • Removal of oxygen (or) • Addition of hydrogen (or) • Addition of electrons The enzymes in this class catalyze oxidation-reduction reactions CHOH, C = O, CH – NH2 and CH = NH groups. There are three types of oxidoreductases. They are 1. Dehydrogenases 2. Oxidases 3. Oxygenases Dehydrogenase: The dehydrogenases are enzymes that catalyze the transfer of hydrogen atoms or electrons from one substrate to another substrate. e.g. Alcohol dehydrogenase Alcohol dehydrogenase (ADH) (1.1.1.1) which facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD+ to NADH. Oxidases: Oxidases are enzymes which catalyze the removal of hydrogen from the substrate. Flavin adenine dinucleotide (FAD). In FAD, flavin serves as oxidizing agent or hydrogen carrier.

12   Enzymes

Oxidase is an enzyme that catalyses an oxidation-reduction reaction involving molecular oxygen (O2) as electron acceptor, oxygen is reduced in water (H2O) or hydrogen peroxide (H2O2). e.g. Glucose oxidase enzymes (C9OX) (EC.1.1.3.4) is an oxidoreductase that catalyses the oxidation of glucose to hydrogen peroxidase and D - glucose L - lactone. Oxygenases: Oxygenases are enzymes which catalyze the incorporation of oxygen directly into the substrate. These enzymes require the presence iron or copper. There are two types of oxygenases (i) Monoxygenases (ii) Dioxygenases. i) Monoxygenases or fixed function oxidases: Transfer one oxygen atom to the substrate and reduce the other oxygen atom to water. ii) Dioxygenases or oxygen transferases: They incorporate both atoms of molecular oxygen (O2) into the products of the reaction. e.g. P450 involves in insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water.

2.  Transferases Transferases are enzymes which catalyze the transfer of a group (X) other than hydrogen from one substrate to another. A.X + B

Transferase

A + B.X

These enzymes are further classified into various sub-classes on the basis of the nature of the transferring group (X). 1. Transaminase: Transfers amino group. e.g. glutamate–pyruvate transaminase, aspartate aminotransferase. 2. Transphosphorylase: Transfers phosphate group. e.g. hexokinase, phosphoglucomutase, phosphoglycerokinase. 3. Transacylase: Transfers acyl group. E.g. choline acyltransferase, acetoacetate transacetylase, amino acid transacetylase, etc.

Enzyme Nomenclature and Classification

 13

3.  Hydrolases Hydrolases are enzymes which catalyse the hydrolysis of ester, ether, peptide, glycosyl, acid anhydride by the direct addition of water molecules (this takes place across the bond, which is cleaved). The sub-classes of this class are esterases, etherases, peptidases, glycosidases, phosphatases, thioesterases. The name of these enzymes are usually the name of substrate with the suffix. e.g. penicillinases, ureases, dipeptidases etc. Some hydrolases do not indicate the type of reaction they catalyze. e.g. lysozyme (muramidase). Many peptidases ends with “in’’. e.g. chymotrypsin, renin and papain. This group includes the extracellular digestive enzymes and many intracellular enzymes: 1. Enzyme acting on peptide bonds. e.g. pepsin, rennin, chymotrypsin, etc. 2. Enzymes acting on amide bonds other than peptide bonds. e.g. urease, arginases, glutaminase, etc. L-ornithine + Urea L-arginine + Water 3. Hydrolytic deaminases e.g. guanine deaminase. Xanthine + HNO2 Guanine 4. Esterases e.g. lipases, phosphatases, sulphatases 5. Enzymes acting on glycosyl compounds. e.g. β–galactosidase β-D-galactoside + H2O Alcohol + D-galactose

4.  Lyases These are the enzymes which catalyze the removal of small groups from larger substrate by mechanism other than hydrolysis (usually leaves double bond). Since these reactions are reversible, lyases also catalyze the addition of small molecules to the substrate molecules.

14   Enzymes

They are further classified on the basis of the linkage they attack (viz) C–C, C–O, C–N, C–S and C–halide bonds. e.g. aldoses, fumarases, hydratases, pyruvate decarboxylase, enolase, deaminases, etc. 1. Fructose-1, 6-phosphate aldolase Glyceraldehyde-3phosphate + Dihydroxyacetone phosphate 2. L-Histidine deaminase Uroconate + NH 3

5. Isomerases These enzymes catalyze the interconversions of optical, geometrical or positional isomers by intramolecular rearrangement of atoms or groups. The sub-classes are: 1. Racemases and epimerases e.g. alanine racemases L-alanine D-alanine 2. Cis-trans isomerases e.g. retinene isomerase II-trans-retinene II-cis-retinene 3. Intramolecular oxidoreductase e.g. glucose phosphate isomerase D-glucose-6-phosphate D-fructose-6-phosphate 4. Enzymes catalyzing the interconversion of aldoses and ketoses e.g. triose phosphate isomerases D-glyceraldehyde-3-phosphate Dihydroxyacetonephosphate

6.  Ligases (Synthetases) These enzymes catalyze the linking together of two compounds coupled with the breaking of a phosphate bond in ATP or a similar compound.

Enzyme Nomenclature and Classification

 15

This class includes enzymes catalyzing reactions forming C–O, C–S, C–N and C–C bonds. The subclasses are: 1. Enzymes catalyzing the formation of C–S bonds. e.g. acetyl CoA ligase(AMP) or acetyl-CoA synthetase ATP + Acetate + CoA → AMP + Pyrophosphate + acetyl-CoA. 2. Enzymes catalyzing the formation of C–N bonds. e.g. L-glutamate-ammonia ligase (ADP) or glutamine synthetase. ATP + L-glutamate + NH3 → ADP + Orthophosphate + L-glutamine. 3. Enzymes catalyzing the formation of C–C bonds. e.g. acetyl CoA-CO2 ligase (ADP) or acetyl CoA carboxylase. ATP + Acetyl-CoA + CO2 + H2O → ADP + Orthophosphate + Malonyl-CoA.

Key to nuMBerIng and classIfIcatIon of enzyMes 1.  Oxidoreductases 1.1 Acting on the CH–OH group of donors 1.1.1 With NAD+ or NADP+ as acceptor 1.1.2 With a cytochrome as acceptor 1.1.3 With oxygen as acceptor 1.1.99 With other acceptors 1.2 Acting on the aldehyde or oxo group of donors 1.2.1 With NAD+ or NADP+ as acceptor 1.2.2 With a cytochrome as acceptor 1.2.3 With oxygen as acceptor 1.2.4 With a disulphide compound as acceptor 1.2.7 With an iron–sulphur protein as acceptor 1.3 Acting on the CH–CH group of donors 1.3.1 With NAD+ or NADP+ as acceptor 1.3.2 With a cytochrome as acceptor

16   Enzymes

1.3.3 With oxygen as acceptor 1.3.7 With a disulphide compound as acceptor 1.3.99 With other acceptors 1.4 Acting on the CH–NH2 group of donors 1.4.1 With NAD+ or NADP+ as acceptor 1.4.2 With a cytochrome as acceptor 1.4.3 With oxygen as acceptor 1.4.4 With a disulphide compound as acceptor 1.4.7 With an iron–sulphur protein as acceptors 1.4.99 With other acceptors 1.5 Acting on the CH–NH group of donors 1.5.1 With NAD+ or NADP+ as acceptor 1.5.3 With oxygen as acceptor 1.5.99 With other acceptors 1.6 Acting on NADH or NADPH 1.6.1 With NAD+ or NADP+ as acceptor 1.6.2 With a cytochrome as acceptor 1.6.4 With a disulphide compound as acceptor 1.6.5 With a quinone or related compound as acceptor 1.6.6 With a nitrogenous group as acceptor 1.6.7 With an iron–sulphur protein as acceptor 1.6.99 With other acceptors 1.7 Acting on other nitrogenous compounds as donors 1.7.2 With a cytochrome as acceptor 1.7.3 With oxygen acceptor 1.7.7 With an iron–sulphur protein as acceptor 1.7.99 With other acceptors 1.8 Acting on a sulphur group of donors 1.8.1 With NAD+ or NADP+ as acceptor 1.8.2 With a cytochrome as acceptor 1.8.3 With oxygen as acceptor

Enzyme Nomenclature and Classification

 17

1.8.4 With a disulphide compound as acceptor 1.8.5 With a quinone or related compound as acceptor 1.8.6 With a nitrogenous group as acceptor 1.8.7 With an iron–sulphur protein as acceptor 1.8.99 With other acceptors 1.9 Acting on a haem group of donors 1.9.3 With oxygen acceptor 1.9.6 With a nitrogenous group as acceptor 1.9.99 With other acceptors 1.10 Acting on diphenols and related substances as donors 1.10.1 With NAD+ orNADP+ as acceptor 1.10.2 With a cytochrome as acceptor 1.10.3 With oxygen acceptor 1.11 Acting on hydrogen peroxide as acceptor 1.12 Acting on hydrogen as acceptor 1.12.1 With NAD+ or NADP+ as acceptor 1.12.2 With a cytochrome as acceptor 1.12.7 With an iron–sulphur protein as acceptor 1.13 Acting on single donors with incorporation of molecular oxygen (oxygenases) 1.13.11 With incorporation of two atoms of oxygen 1.13.12 With incorporation of one atom of oxygen (internal mono oxygenases or internal mixed function oxidases) 1.13.99 Miscellaneous (requires further characterization) 1.14 Acting on paired donors with incorporation of molecular oxygen 1.14.11 With 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors 1.14.12 With NADH or NADPH as one donor, and incorporation of two atoms of oxygen into donor 1.14.13 With NADH or NADPH as one donor, and incorporation of one atom of oxygen

18   Enzymes

1.14.14 With reduced flavin or flavoprotein as one donor and incorporation of one atom of oxygen 1.14.15 With reduced iron–sulphur protein as one donor, and incorporation of one atom of oxygen 1.14.16 With reduced pteridine as one donor, and incorporation of one atom of oxygen 1.14.17 With ascorbate as one donor, and incorporation of one atom of oxygen 1.14.18 With another compound as one donor and incorporation of an atom of oxygen 1.14.99 Miscellaneous (requires further characterization). 1.15 Acting on superoxide radicals as acceptor 1.16 Oxidizing metal ions 1.16.3 With oxygen acceptor 1.17 Acting on –CH2–groups 1.17.1 With NAD+ or NADP+ as acceptor 1.17.4 With a disulphide compound as acceptor 1.18 Acting on reduced ferredoxin as donor 1.18.1 With NAD+ or NADP+ as acceptor 1.18.2 With dinitrogen as acceptor 1.18.3 With H+ as acceptor 1.19 Acting on reduced flavodoxin as donor 1.19.2 With dinitrogen as acceptor

2. Transferases 2.1 Transferring one-carbon groups 2.1.1 Methyltransferases 2.1.2 Hydroxymethyl formyl and related transferases 2.1.3 Carboxyl and carbamoyl transferases 2.1.4 Amidinotransferases

Enzyme Nomenclature and Classification

 19

2.2 Transferring aldehyde or ketonic residues 2.3 Acyltransferases 2.3.1 Acyltransferases 2.3.2 Aminoacyl transferases 2.4 Glycosyltransferases 2.4.1 Hexosyltransferases 2.4.2 Pentosyltransferases 2.4.99 Transferring other glycosyl groups 2.5 Transferring alkyl or aryl groups other than methyl groups 2.6 Transferring nitrogenous groups 2.6.1 Aminotransferases 2.6.3 Oxaminotransferases 2.7 Transferring phosphorus containing groups 2.7.1 With alcohol group as acceptor 2.7.2 With a carboxyl group as acceptor 2.7.3 With a nitrogenous group as acceptor 2.7.4 With a phosphate as acceptor 2.7.5 With regeneration of donors 2.7.6 Diphosphotransferases 2.7.7 Nucleotidyltransferases 2.7.8 Other substituted phosphate groups 2.7.9 With paired acceptor 2.8 Transferring sulphur containing groups 2.8.1 Sulphurtransferases 2.8.2 Sulphotransferases 2.8.3 CoA-transferases

3. Hydrolases 3.1.1 Carboxylic ester hydrolases 3.1.2 Thiolester hydrolases 3.1.3 Phosphoric monoester hydrolases

20   Enzymes

3.2

3.3

3.4

3.5

3.6

3.1.4 Phosphoric diester hydrolases 3.1.5 Triphosphoric monoester hydrolases 3.1.6 Sulphuric ester hydrolases 3.1.7 Diphosphoric monoester hydrolases 3.1.11 Exodeoxyribonucleases producing 5-phosphomonoester 3.1.13 Exoribonucleases producing 5- phosphomonoester Acting on glycosyl compounds 3.2.1 Hydrolyzing O-glycosyl compounds 3.2.2 Hydrolyzing N-glycosyl compounds 3.2.3 Hydrolyzing S-glycosyl compounds Acting on ether bonds 3.3.1 Thioester 3.3.2 Ether hydrolysis Acting on peptide bonds (peptide hydrolases) 3.4.11 Alpha amnioacyl peptide hydrolases 3.4.13 Dipeptide hydrolases 3.4.16 Serine carboxypeptidases 3.4.17 Metallo-carboxypeptidases 3.4.21 Serine proteinases 3.4.22 Thiol proteinases 3.4.24 Metalloproteinases Acting on carbon–nitrogen bonds (other than peptide bonds) 3.5.1 In linear amides 3.5.2 In cyclic amides 3.5.3 In linear amidines 3.5.4 In cyclic amidines 3.5.5 In nitriles Acting on acid anhydrides 3.6.1 In phosphorus containing anhydrides 3.6.2 In sulphonyl containing anhydrides

Enzyme Nomenclature and Classification

3.7 Acting on carbon–carbon bonds 3.7.1 In ketonic substances 3.8 Acting on halide bonds 3.8.1 In C–halide compounds 3.8.2 In P–halide compounds 3.9 Acting on phosphorus–nitrogen bonds 3.10 Acting on sulphur–nitrogen bonds 3.11 Acting on carbon–phosphorus bonds

4. Lyases 4.1 Carbon–carbon lyases 4.1.1 Carboxy lyases 4.1.2 Aldehyde lyases 4.1.3 Oxo-acid lyases 4.1.99 Other carbon–carbon lyases 4.2 Carbon–oxygen lyases 4.2.1 Hydro lyases 4.2.2 On polysaccharides 4.3 Carbon–nitrogen lyases 4.3.1 Ammonia lyases 4.3.2 Amidine lyases 4.4 Carbon–sulphur lyases 4.5 Carbon–halide lyases

5. Isomerases 5.1 Racemases and epimerases 5.1.2 On aminoacids and derivatives 5.1.2 On hydroxyacids and derivatives 5.1.3 On carbohydrates and derivatives 5.2 Cis–trans isomerases

 21

22   Enzymes

5.3 Intramolecular oxidoreductases 5.3.1 Interconverting aldoses and ketoses 5.3.2 Interconverting keto and enol groups 5.4 Intramolecular transferases 5.4.1 Transferring acyl groups 5.4.2 Transferring phosphoryl groups 5.4.1 Transferring amino groups 5.5 Intramolecular lyases 5.99 Other isomerases

6. ligases 6.1 Forming carbon–oxygen bonds 6.1.1 Forming aminoacyl-tRNA and related compounds 6.2 Forming carbon–sulphur bonds 6.2.1 Acid-thiol ligases 6.3 Forming carbon–nitrogen bonds 6.3.1 Acid-ammonia ligases 6.3.2 Acid-amino acid ligases 6.3.3 Cyclo ligases 6.3.4 Other carbon–nitrogen ligases 6.4 Forming carbon–carbon bonds 6.5 Forming phosphoric ester bonds

eXaMples 1.1.1.1 1.1.1.6 1.1.1.19 1.1.1.27 1.1.1.37 1.1.1.47 1.1.1.49

Alcohol dehydrogenase Glycerol dehydrogenase Glucuronate reductase Lactate dehydrogenase Malate dehydrogenase Glucose dehydrogenase Glucose-6- phosphate dehydrogenase

Enzyme Nomenclature and Classification

2.1.1.18 Polysaccharide methyl transferase 3.1.3.2 Acid phosphatase 2.3.1.9 Acetyl-CoA carboxylase 2.6.1.2 Alanine aminotransferase 3.1.3.1 Alkaline phosphatase 3.5.1.14 Aminoacylase 3.2.1.1 Alpha-amylase 3.5.3.1 Arginase 3.5.1.1 Asparaginase 3.1.1.1 Carboxylesterase 1.11.1.6 Catalase 3.4.21.1 Chymotrypsin 4.2.1.11 Enolase 2.7.1.4 Fructose kinase 3.5.1.2 Glutaminase 1.1.3.4 Glucose oxidase 2.7.1.1 Hexokinase 3.2.1.17 Lysozyme 3.4.22.2 Papain 3.5.1.15 Pectinase 1.11.1.7 Peroxidase 2.4.1.1 Phosphorylase 3.2.1.41 Pullulanase 3.4.23.15 Renin 2.7.7.6 RNA polymerase 3.4.21.4 Trypsin 3.5.1.5 Urease 5.3.1.5 Xylose isomerase 2.4.1 Hexosyltransferases 2.4.2 Pentosyltransferases

 23

24   Enzymes

QuestIons 1. Define the term nomenclature. 2. What are the types of reactions catalyzed by enzymes? 3. Why are microbial enzymes are significant than other types of enzymes. 4. List out the various microbial enzymes? 5. Expand IUB and briefly explain their role in the study of enzymes. 6. How are enzymes classified? 7. Explain the significant reactions of the six major classes. 8. Explain oxidoreductase reactions. 9. Explain E.C. number with an example. 10. What are the different sub-classes of transferases? 11. Differentiate lyases and ligases. 12. Give few examples of isomerases.

CHAPTER

3 Enzyme Extraction and Purification

IMportance of enzyMe purIfIcatIon Enzymes are found in nature as complex mixtures in cells (hundred or more different enzymes). To study a particular enzyme, they have to be properly purified. In case if they are not purified, other enzymes present will interfere, by attacking the substrate to give side reactions or by transforming the product into some other substance, or by attacking the coenzyme or even the enzyme itself. To study the properties and behavior of an enzyme as a chemical catalyst, it is necessary to have it in the pure state. Purification may affect the activity of an enzyme in various ways. It may remove any cofactors that may be essential for the reaction, but these can be restored in testing. Purified 3-hydroxybutyrate dehydrogenase (E.C.1.1.1.30) requires the addition of lipids for their activity. In few cases, the properties of enzymes themselves have been found to be altered by purification process. E.g. succinate dehydrogenase (E.C.1.3.99.1) is altered when it is solubilized.

general procedure Highly purified preparations are essential for the investigation of enzyme structure, characteristics and reaction mechanisms without complications arising from the presence of other enzymes. Tissue slice technique involves cutting a tissue into a thin slice (usually 0.5 mm thick) using a sharp implement known as microtome. Due to thinner size, large portion of the cell gets damaged and leads to diffusion. The tissue is incubated in a suitable medium at controlled pH and temperature.

26   Enzymes

Another approach to study tissue is by perfusion techniques. An intact tissue is removed from an animal and placed in a fluid environment which provides sufficient oxygen. To isolate tissue from each other, the tissues are treated with collagenase or other proteolytic enzymes (to break the matrix). All the transport occurs through plasma membrane and this can be overcome by cell-free system which includes homogenization of tissue with suitable isotonic medium (for example, 0.25 M sucrose). Homogenates of soft tissues such as liver, can be prepared by forcing the tissue and medium through narrow gap (0.3) between a glass tube and a close fitting pestle (made of Teflon or glass), which moves up and down the tube by electric motor. The concentration of the enzyme in solution is expressed as units per ml. The amount of a given enzyme may be different in different tissues. In case of bacteria , the enzyme is not usually available in large quantities. Enzymes are normally present in the intracellular particles such as mitochondria. Therefore, in such cases, mitochondrial extract is prepared by high-speed centrifugation. The enzymes and other constituents of the cytoplasm are removed by ordinary fractionation methods.

eXtractIon When suitable source has been found, the main thing is to bring out the enzyme in solution. For this, the cell wall is ruptured to extract enzymes from animal tissues (minced tissue or liver). For this procedure, water is sufficient. There as, for a strongly glycolysing tissue, care has to be taken, where buffer solution is used. The extraction is carried out using high-speed homogenizer or a waring blender. For yeasts, autolysis (either alone, or with toluene, ethyl acetate or sodium sulphide) is the only method of attacking the cell membrane. This method results in an active dry powder, which can be stored in bulk and it forms a convenient starting material from which the enzyme can be extracted with water or buffer solution. [The acetone treatment must be carried out at low temperature for enzymatic lysis of Micrococcus lysodeikticus by lysozyme (E.C.3.2.1.17).

Enzyme Extraction and Purification

 27

enzyMe HIstocHeMIstry The subcellular location of many enzymes may be revealed by microscopy by suitable fixation and staining procedure. The procedure is as follows: 1. Tissues are frozen to –20o C. 2. They are then sliced using cryostat (a refrigerated microtome). 3. The slice must be about 10 µm in thickness. (This minimizes the loss of enzyme activity.) 4. The slices are then fixed in formaldehyde (Formalin) to prevent any subsequent diffusion of proteins. (Formaldehyde brings about cross-linking between side chain amino groups of proteins). Protein

Protein

NH 2

NH CH 2

+

HCHO

NH 2

NH

Protein

Protien

5. To retain the activity after treatment, the tissue section is treated with a buffered solution of a staining mixture containing the substrate of enzyme. For example, Lead–salt method is used for retaining the activity of acid phophatases enzymes. (Sodium Beta-glycerophosphate and lead ions in buffer at pH 5.5.) (Any acid present in the tissue hydrolyzes the glycerophosphate and the phosphate liberated reacts immediately with lead ions to form insoluble lead phosphate (PbPO4). Some enzymes are more firmly bound to the structural framework of mitochondria (as lipoprotein complex) and requires special methods for their extraction. The methods include drying with acetone, treatment with butanol-water mixture treatment, with aqueous solution such as detergents e.g. Such as, cholate, Tween,

28   Enzymes

Triton, Emascol, etc. The hydrolytic enzymes such as lipases, nucleases or proteolytic enzymes are exposed to treatment with chaotropic agents such as perchlorate.

use of centrIfugatIon The sedimentation characteristics of the various subcellular organelles are different. So, it is possible to separate them by centrifugation of a tissue homogenate and then investigate which enzyme is associated with each cell fraction. The simplest and most widely used method of separating the various subcellular organelles from each medium is differential centrifugation. A tissue homogenate is suspended in a medium of low density (0.25M sucrose) and centrifuged in a series of stages (the centrifugal field of each step is higher than the previous one). Liver homogenate

Pellet (nuclei, unbroken cells)

Supernatant

Pellet (nuclei, unbroken cells) Pellet

Supernatant

Supernatan

Fig. 3.1 General Procedure of Centrifugation.

When the suspending medium is not uniform throughout, density-gradient centrifugation can help to overcome some or all of these problems, that is layering of one solution on top of the other, in the centrifuge tube e.g. on to a tissue homogenate, 0.25 M sucrose is layered and then 0.5 M sucrose one above the other and then centrifuged.

eXtractIon of soluBle enzyMes Soluble cytoplasmic enzymes are the simplest to extract. Soluble enzymes present in the organelles of the eukaryotic cells are also easy to liberate. For this, cell fractionation may be carried out prior to the disruption of organelles. The extraction procedures used depend on the type of organism, acting as source.

Enzyme Extraction and Purification

 29

1. Animal tissue: Animal tissue is removed soon after death and kept in cold to minimize autolysis. Fat deposits and connective tissues are removed. Then, homogenization in an isotonic medium disrupts the plasma membrane but leaves most organelles intact. 2. Higher plants: The extraction of soluble enzymes from higher plants varies considerably according to the type of source material. Enzymes may be extracted from cereal flours by placing in an aqueous medium and stirred, and from potato, by mincing and pressing out the juice through muslin or cheese cloth. Extracts from leaves, dried seeds etc., can be obtained by grinding with a suitable medium in a pestle homogenizer. Leaves and plant tissues may be homogenized directly in a medium by the use of waring blender (in the presence of several volumes of cold acetone) to displace water, followed by careful drying to produce an acetone powder. These powders may be treated with suitable medium. Tougher tissues, such as fibrous roots are disrupted by grinding in a mortar with sand or ground glass. 3. Microorganisms: In general, methods of extraction fall into two main categories: i. Drying ii. Mechanical disruption i. Drying: Drying may cause the cell to become permeable, enabling soluble enzymes to be extracted. In air drying (particularly for yeast) the cells are allowed to dry in air for several days at around 30oC. In vacuo drying involves placing the cell suspension in vacuum desiccators for one or more days. Lyophilization: (freeze drying): It is a method where a frozen cell suspension is dried by sublimation in vacuo. Soluble enzymes are less easily extracted from lyophilized cells than from air-dried or vacuum-dried preparations. Microorganisms may also be dehydrated with acetone to produce powders containing easily extractable enzymes.

30   Enzymes

Mechanical disruption: Microbes can be disrupted by a. Grinding with carborundum beads. b. Other abrasives. c. Applying pressure (with Hughes or French process.) d. Combinations of these methods. Bacterial cell: The bacterial cell wall is weakened by enzymes such as lysozyme and glucuronidase (they are available in an insolubilized form and can be removed) e.g. Bacillus subtilis. The gram-negative bacterium E.coli is prepared by addition of EDTA.

eXtractIon of MeMBrane-Bound enzyMes Enzymes, which are bound to plasma or intracellular membranes cannot normally be extracted into the surrounding medium simply by disruption procedure. Some enzymes are loosely bound and some are tightly bound. Membranes, beside enzymes and proteins, contain amphipathic lipids, that is, lipids containing both hydrophilic and hydrophobic regions. The hydrophilic portion of the molecule is small compared to the hydrophobic part. Some membranes in eukaryotic cells contain sterols. An amphipathic lipid may therefore be represented as a molecule with hydrophilic head and one or more hydrophobic tails. Peripheral proteins are apparently linked by electrostatic or hydrogen bonds to the polar heads of lipids or to integral proteins and can easily be dissociated from the membrane. e.g. By treating with a solution of high ionic strength, such as 1M NaCl, by freezing and thawing or by sonication. The integral proteins can only be extracted by breaking the hydrophobic interactions between lipids and proteins, that is, by dissociating a lipoprotein complex. Detergents have been used to disrupt membranes and extract lipoprotein fragments into aqueous media. These detergents include natural bile salts such as cholate, non-ionic synthetic detergents like Triton X-100 and ionic synthetic detergents, which may be Zwitter ionic.

Enzyme Extraction and Purification

 31

Lipoproteins may also be extracted by the use of organic solvents, where free proteins may be liberated on subsequent dispersion in aqueous media. e.g. Xanthine oxidase. The solvent most widely used for the extraction of enzymes from membranes is n-butanol. This has both lipophilic and hydrophilic character, so it can act like detergent in removing lipoproteins from membranes. n-butanol is used at different conditions for different groups of enzymes. e.g. Alkaline phosphatase has been extracted at pH 5–6 alpha-glutamyltransferase at pH 7 and urate oxidase at pH 10.

nature of tHe eXtractIon MedIuM A considerable amount of debris (mainly membrane fragments) are likely to be present. The solubility of proteins depends on four main factors. 1. Salt concentration 2. pH 3. Organic solvent content 4. Temperature The temperature of the medium is usually kept below 4oC, to minimize the loss if activity of the enzyme despite the reduction in solubility. Proteins are least soluble at their isoelectric point. So the extraction of an enzyme must be carried out at a pH value far from its isoelectric point. e.g. Glutamate dehydrogenase, arginase, alkaline phosphatase and acid phosphatase from bovine liver homogenate are soluble in 19% ethanol at pH 5.8 and ionic strength 0.02. But catalase, peroxidase and D-amino acid oxidase are not. However, these enzymes are soluble at pH 5.8 in the absence of ethanol and at ionic strength of 0.15. Proteinases are insoluble in both of these media at pH 7.4. Stabilizers such as dithiothreitol (Cleland’s reagent) or mercapto ethanol are added to the extraction media to prevent oxidation of sulphydryl groups and thus loss of enzyme activity. Inhibitors of proteolytic enzymes, such as DFP, may also be added. Polyols (e.g. glycerol) or chelating agents (e.g. EDTA) may be used in stabilizing solubilized enzymes.

32   Enzymes

purIfIcatIon of enzyMes Preliminary Procedure Extracted nucleic acids may be precipitated by treatment with basic substances such as streptomycin or protamine or with MnCl2 or MgCl2. All precipitates and cell debris are then removed by centrifugation and discarded. Polysaccharides may also be removed by high-speed centrifugation. The next stage is to precipitate the enzyme of interest from solution, thus separating it from mono and oligosaccharides, nucleotides, free amino acids etc. This may be achieved by altering the pH and the organic or salt concentrations of the medium. e.g. The pH may be adjusted to isoelectric point of the enzyme. To alter high salt concentration, ammonium sulphate may be used (extremely soluble in water). The residual ammonium sulphate is removed by dialysis. Considerable degree of purification can be achieved but proteins may still be present because of overlap of solubility range. This can be achieved by raising the temperature of the medium for a few minutes.

fractionation Methods The enzyme extract contains numerous other substances of both large and small molecular weight. The small molecules can be removed by dialysis or gel filteration and the remaining substances may be predominantly proteins and some polysaccharides. There are different types of fractionation method. pH and electolyte concentration are the two factors affecting fractionation methods. Each fractionation step consists of the separation of total protein into a series of fractions by gradual increase of salt concentration or the amounts of adsorbent added or acidity or concentration of organic solvent. In this method, a portion of enzyme solution is taken and each fraction is removed before the next is precipitated. In fractionation methods, precipitation starts when the ammonium sulphate concentration reaches 66% of saturation.

Enzyme Extraction and Purification

 33

1. Fractional precipitation by change of pH: This is the most important fractionation method which is more advantageous in the extraction of animal tissues, where the pH is adjusted to 5 and then centrifuged. The nucleoprotein and particulate materials are removed and clear solution results. 2. Fractional denaturation by heating: By heating the solution for a definite time to a temperature just below the temperature which the enzyme is destroyed, it is sometimes possible to cogulate unwanted protein which is then centrifuged and discarded. In some cases, the presence of the substrate frequently has a specific stabilizing effect on the enzyme which may be possible to heat the solution higher, in its presence, than in its absence, without the loss of enzyme. Heating may be carried out in three subsequent water baths, the first at the desired temperature, the second at few degrees hotter and the third cold. The solution is placed in a round flask, which should not be more than half full and immersed in the hottest bath and immersed in the hottest bath and the contents of the flask are kept rapidly swirling round. The rise in temperature is checked by thermometer and when the solution reaches the desired temperature, the flask is removed and placed at a desired temperature. After the desired heating period (10–15 minutes) the flask is then transferred to the cold bath and reduced by swirling. 3. Fractional precipitation with organic solvents: This is the most standard method of fractionation where ethanol is used as the solvent for separation (Cohn et al. For separation of blood proteins). The enzyme separation from muscle extract was carried out by Askonas with various solvents and concluded that acetone gave the sharpest separation with lowest loss. To get a good separation, a low electrolyte concentration (less than M/30) is required at pH 6.5. Since most enzymes are inactivated by solvents at room temperature, the whole procedure has to be carried out at low temperature (–5o C) with efficient stirring. The solvent is added when the temperature reaches 0oC (to prevent freezing) and is continued until the desired amount of precipitate is obtained. The solution is then centrifuged in a refrigerated centrifuge at the same temperature (addition of acetone is then continued). The precipitate

34   Enzymes

is either freeze-dried or immediately dissolved in a sufficient quantity of cold water or buffer solution to dilute the acetone or it may be dialyzed at the freezing point. A few enzymes are very stable to acetone. In these cases, it is a positive advantage to allow the solution to stand at room temperature, which cause extensive denaturation and inactivation of other proteins and enzymes, which can be removed. 4. Fractional precipitation by salts: This is a very widely used method employing ammonium sulphate on amount of its high solubility in water and absence of harmful effects on most enzymes. This has a stabilization action on many enzymes and usually not necessary to carry out the fractionation at low temperature. The recovery of the enzymes is 100% but separations are not always sharp (a small percentage of the enzyme remain in discarded fraction). Precautions i. Pure quality ammonium sulphate (to avoid toxic impurities) must be used. ii. The pH should be controlled. iii Ammonium sulphate must be added as solids and not as solution. Ammonium sulphate is weighted (boiling tube) and enough of the salt is added to the enzymes solution to bring down and amount of precipitate suitable for one fraction (care must be taken to dissolve all crystals). The boiling tube is then weighed for the difference. After 15 minutes, centrifugation is carried out at 10,000 rpm (high speed) and then filtered. After centrifuging the first fraction, the solution is poured off into another centrifuge tube and the addition of salts is continued. The precipitate is dissolved in a small volume of water or buffer solution. The precipitate range is determined not only by the pH, temperature and the nature of salt, but also by the concentration of the enzyme. It is usually not worthwhile to repeat an ammonium sulpahte fractionation under the same conditions, several times in successions. However, on varying the pH or temperature of the fractionation, the order in which the different conditions may be well worthwhile.

Enzyme Extraction and Purification

 35

5. Fractional adsorption: This is one of the most important and successful methods of fractionation. Two distinct ways can be adopted: i By stirring successive amounts of the adsorbent into the enzyme solution and removing each portion by centrifugation. ii By passing the enzyme solution through a column of adsorbent into a fraction collector. In the first method, if the enzyme becames adsorbed, it may thus be removed and separated form other components of the solution and afterwards extracted or eluted form the adsorbent. If the enzyme is not absorbed, treatment with the adsorbent may be used to remove much unwanted material form the enzyme solution. E.g. Calcium phosphate gel and Zn (OH2). Charcoal can be used to remove unwanted components. Adsorption occurs best in slightly acid solution (pH 5 or 6) and low electrolyte concentration. Even considerable amount of salt will interfere in the adsorption. In the interest of economy, a preliminary dialysis is beneficial. Elution of the enzyme can often be accomplished by slighly alkaline buffer solution, e.g, phosphate buffer at pH 7.6, in some cases, with phosphate containing about 10% of ammonium sulphate. Precautions: The volume of eluent used should not be too large and is generally not greater than the volume of centrifuged gel. It is better to elute several times in succession with small volumes than once with a larger volume.

Partition Chromatography This is used to separate mixtures of enzymes. For example ribonuclease was purified by Martin and Porter (1951) on column packed with Kieselguhr using a two-phase system consisting of ammonium sulphate, ethyl cellosolve and water. The sample was applied to a column which had been pre-equilibrated with the organic phase and then eluted by aqueous phase. Separation of components occurs on the basis of relative solubility in the two phases.

36   Enzymes

electrophoresis This is ideally suited for the separation of small amount of materials. The rate and direction of migration of proteins in the electric field depends on their net charge at particular pH and the size of the molecules. Zone electrophoresis

In this method, a mixture of proteins is introduced at a common point (the origin). Each molecule travels in the same zone with same charge and size. To minimize diffusion, the whole process is carried out in a solid support medium which is porous (e.g. starch gel) through which buffer and proteins permeate. After separation, a narrow longitudinal section is cut from the support medium and the protein (e.g. With amido black). Zone electrophoresis may be carried out in a vertical direction in a closed column. e.g. Powdered cellulose is used as support medium. After the completion of electrophoresis, a tap at the bottom of the column is opened to enable the liquid content to be run out and collected in fractions.

Cation Exchange Chromatography Column is packed with carboxylated polystyrene. This method is successful for stable, low molecular weight basic proteins such as lysozyme and ribonuclease.

gel filteration This an important technique in enzyme purification. This is carried out in column packed with swollen gels which separate components of a sample based on the molecular size. Gels commonly used include cross-linked dextrans (sephadex), cross-linked agarose (sepharose) and cross-linked polyacrylamide (biogel).

Affinity Chromatography This is a specific process. It is suited for the separation of one protein from all others. The column is packed with an inert matrix (e.g. agarose) to which ligands of specific enzymes are attached. This results in covalent link means spacer arm (e.g. a hydrocarbon chain).

Enzyme Extraction and Purification

 37

Elution of enzyme is carried out by deforming buffer at a changing pH.

Hplc Technology This is the major technology used for protein purification. Area stainless steel columns and high quality robust packing material of particle size 10 µm are used. The procedure can be completed within a few minutes rather than hours. High performance size exclusion chromatography (HPSEC)

This technique uses conventional gel filteration. Rapid spherical beads of porous silica with bonded hydrophilic polar groups are used. High performance ion exchange chromatography

The technique utilizes amines as anion exchanger and sulphonic or carboxylic acids as cation exchanger. They are bounded by rigid support such as silica. Proteins are separated by reverse phase HPLC on alkylsilica columns. The eluting solvents are buffered aqueous and organic mixtures.

Lyophilization The purified enzymes are quite dilute. (concentration is necessary). Lyophilization is the convenient method and this is followed by redissolving of the enzyme in a small volume of liquid. Freeze drying: A method for preserving foods or biologicals where a substance (eg) Coagulation factor vii is ‘snap frozen’ in liquid nitrogen (–70o C) and placed in a high vacuum to removed the water vapour as it sublines once the water is removed.

deterMInatIon of Molecular WeIgHt of enzyMes Gel filteration: This separates the molecules on the basis of size. A packed column is calibrated by applying proteins of known molecular weight to the top of the column and the volume of buffer requires for the elution of each protein determined. The absorbance peak must

38   Enzymes

be measured at 280 nm for each protein leaving the column. From the data obtained , a graph may be drawn of elution volume against molecular weight. The protein of unknown molecular weight is then passed through the column and its elution volume is determined. Table 3.1

Principal Separation Methods Used in Purification of Enzymes

Property Method Size or mass Centrifugation Gel filteration Dialysis, ultrafiltration Polarity Charge ion exchange chromatography Chromato focussing Isoelectric focussing Electrophoresis Hydrophobic character Hydrophobic chromatography Solubility

Change in pH Change in ionic strength Decrease in dielectric constant Specific Affinity chromatography binding sites Immobilized metal ion or structural chromatography feature Immunoadsorption Covalent chromatography

Scale Small-scale Small-scale Small-scale Large or Small-scale Small-scale Small-scale Small-scale Small-scale Large-scale Small-scale Large-scale Small-scale Small-scale Large or small Small-scale

eXaMples of purIfIcatIon procedure Adenylate Kinase from Pig Muscles Step 1: Minced Muscle is taken. Extraction steps is carried out with 0.01 mol / dm KCl, and strained through a cheese cloth. Step 2: The extract is incubated at pH 3.5, then at pH 7 and centrifuged.

Enzyme Extraction and Purification

 39

Step 3: The supernatant is loaded on to phosphocellular column, and eluted with AMP (5mmol/dm3). Step 4: Pooled fraction containing enzyme activity is concentrated by (NH4)2 SO4 precipitation. Step 5: Pooled fraction containing activity crystallization at 62% saturation with (NH4)2 SO4. Step 6: The next step involves crystallization enzyme to yield the crystalline enzyme.

Ribulose bisphosphate carboxylase from spinach Step 1: Spinach leaves are taken and ground in a buffer (50mM/ dm-3 N,N bis-2-hydroxy ethyl glycine), glycine (1mM/ dm-3 EDTA (10 mM/dm-3), 2-mercaptoethanol, (adjust pH to 8 with KOH) containing 2% w / v insoluble polyvinyl polypyrrolidone to absorb oxidized phenolic substances. Step 2: Polyethylene glycol is added to supernatant to precipitate nucleic acids, chlorophyll and other pigments. Step 3: Precipitation of ribulose bisphosphate carboxylase is achieved by the addition of Mg2+ to final concentration of 20 mM / dm3 to the supernatent. Step 4: The precipitate is eluted with 0–0.4 mol/dm3 sodium bicarbonate. Step 5: Pooled fraction containing 95% activity is subjected to polyacrylamide gel electrophoresis. (Polyacrylamide in the presence of sodium dodecylsulphate (adsorbent). Two bands are seen. Step 6: Two types of polypeptide chain with molecular weight 56,000 and 14,000 daltons are obtained), which is the pure enzyme.

RNA Polymerase from E.Coli Step 1: The frozen cells of E.coli are blended at high speed (glass beads) and add deoxyribonuclease filter (The viscosity of the medium is reduced. This prevents the formation of high M-complex, DNA-RNA, RNA polymerase).

40   Enzymes

Step 2: The extract is centrifuged (100000g/2L) to remove cell debris and ribosome. Step 3: The supernatant is taken and the oligonucleotides produced by deoxyribonuclease are reextracted into NH4SO4 at (33–50%) saturation and reextracted with 42% NH4SO4 saturation. Step 4: Precipitate is redissolved on cellulose column and eluted with KCl gradient. Step 5: Pooled fraction containing activity is chromatographed on phosphocellulase and eluted with 0.35 mol/dm3 KCl. Step 6: Pooled fraction containing activity is then gel filtered on a column of Biogel-A (Agarose gel fractionates in the range of 10,000–2,00,000 is used to remove minor contaminants from RNA polymerase). Step 7: Pure enzyme is obtained (54mg from 200gm of frozen cells).

Arom multi enzyme protein from Neurospora The Arom contains five distinct enzyme activities, which catalyse the biosynthesis of chrorismic acid and the precursors are tyrosine, phenylaline and tryptophan. During this process, proteases interfere during purification, so precautions have to be taken when isolating from fungi (contain large number of proteases). The cells are harvested, dried, stored at 253K and powdered with a blender. Protease inhibitors are added at each step of this procedure. Phenylmethane sulphonyl fluoride (PMSF) is used as an inhibitor of serine protease. Step 1: Powdered cell extract in buffer (0.1 mol dm-3 Tris HCl) containing PMSF is centrifuged and KCl is added. Step 2: To the supernatant cellulose is applied and eluted with buffer containing PMSF and 75 mmol dm3 KCl. Step 3: Pooled fractions containing enzyme activity is obtained. To this, benzamidane (1 mmol dm3) is added

Enzyme Extraction and Purification

 41

and fractionated with ammonium sulphate (40-50%) saturation. (Effective reversible inhibitor of protease like trypsin). Step 4: The precipitate is eluted with KCl. Step 5: The precipitate and redissolved and loaded on to blue dextran-sepharose and then eluted with high ionic strength (1.5 mol dm3 Tris KCl). Step 6: Pure enzyme is obtained with an yield of about 4 mg from 100gm of powdered cells (25%). The purified material appears to be homogeneous on polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. There are no traces of low metal species. The material can be stored satisfactorily for longer periods at 253 K (–20oC) in the presence of 50% glycerol, benzamidine (1.5 mol/dm3) and dithiothreitol (0.4 mmol/dm3).

Glutathione reductase from E.Coli Glutathione catalyzes the NADPH dependent reduction of oxidized glutathione (GSSG). 2GSH + NADP+ GSSG + NADPH + H+ This belongs to flavoprotein oxidoreductases, a family of enzymes (including dihydrolipoamide dehydrogenase) that possess a disulphide bond . Step 1: The procedure uses a French press–to disrupt the cell suspension, followed by centrifugation to remove cell debris. Step 2: Cell extract at pH 7-7.5 by addition of 2 mol/dm3 K2HPO4. The precipitate is redissolved in a low-ionic strength buffer (5 mM / dm3 potassium PO4, pH 7. Containing 1 mM / dm3 EDTA and 1mM / dm3 2-mercapto ethanol. Step 3: Precipitate procion Red HE-7B is crosslinked in agarose column (Specificity for different classes of enzymes). This binds with glutathione reductase strongly. Wash with buffer 0.1 mol dm3 KCl to remove weakly bound proteins, glutathione reductase.

42   Enzymes

Step 4: The pooled fraction containing activity is concentrated by ultra filteration and subjected to gel filteration on sepharose 12 M (1000–300000). Step 5: The yield of pure enzyme is 11.6 mg enzyme from 100 cm3 of cell suspension.

Adenylate Cyclase This is a membrane-bound enzyme which catalyzes the formation of 3, 5' cyclic AMP from ATP and plays a key role in signal transduction. The activities of this are regulated by a large number of factors including Ca2+ ions via the calmodulin system and α and β subunits of G-protein (heterotrimeric guanine nucleotide binding regulatory proteins) This is extracted from spodoptera frugipetra S19. About 1.4 liters of S19 cell cultures were infected with the recombinant baculovirus encoding the histideine tagged type 1 enzyme. The cells were harvested about 50 hours after infection and suspended in a Na-HEPES buffer containing NaCl, EDTA, dithiotheritol and protease inhibitors and lysed by nitrogen pressure at 40 atm. After removing the nuclei by centrifugation, the membranes are harvested by centrifugation at 70,000 g for 30 minutes and resuspended in buffer before being stored at –80oC. Step 1: Cell culture is infected with recombinant baculovirus, harvested and the cells are lysed and centrifuged. Step 2: To the membrane suspension non-ionic detergent, dodecylmaltoside in the presence of glycerol and NaCl, (90% recovery) are added. • The detergent is added dropwise with stirring to give a final concentration of 0.8 % w/v. • It is then homogenized with Teflan homogenizer and centrifuged at 1,50,000g for 30 minutes to remove unextracted membranes.

Enzyme Extraction and Purification

 43

Step 3: The supernatant is subjected to affinity chromatography on forskolin-sepharose (powerful activatior of enzymeextracted from coleus forskohlii). • It is then washed with buffer containing a lower concentration of detergent and high salt (2 mol/dm3 NaCl). • The buffer used usually is a dipolar solvent dimethyl sulphoxide to remove weakly bound contaminants. Step 4: Adenylate cyclase is eluted from column by buffer containing detergent (0.2% w / v) and forskolin (200 µmol / dm3) Step 5: Pooled fraction, containing activity is further purified by chromatography on immobilizer Ni2+ (Ni2+-NTA agarose). and eluted by buffer containing 0.1% (w/v) detergent and 100 mmol/dm3 imidazole. Step 6: 10% recovery of pure enzyme is achieved and the degree of purification is 2100-fold from membrane suspension. On sodium dodecylsulphate – polyacrylamide gel electrophoresis, a single band was observed at the expected molecular mass (1,10,000Da).

Chymosin Chymosin is the active ingredient of rennet and it is extracted from calf stomach which is used to curdle milk of the first step in cheese manufacture. The enzyme specifically cleaves the bond between Phe-105 and Met-106 in α-casein (one of the major phosphoprotein present in milk) Cleavage of this bond leads to a Ca2+ induced aggregation of the casein molecules to form a gel (this has increased the interest over 30 years). Pepsin and aspartic proteinases are used and have generally not proved wholly satisfactory (alteration in the flavour and the texture of the product). Chymosin is first synthesized as preprochymosin (16 amino acid).

44   Enzymes

When this is removed (16 A.A) the zymogen prochymosin is formed under the acid condition of the stomach and this is in turn converted to chymosin by the removal of 42 amino acid propeptides from the N-terminus. Active chymosin could then be formed in vitro by incubation of the prochymosin at pH 6. Step 1: Cell suspension is subjected to lysis (sonication) and centrifuged then (1,2000 g for 5 minutes). Step 2: The Inclusion bodies are washed, solubilized in 8 bmol / dm3 urea, diluted with the buffer at pH 10:7 (PO4 buffer) readjust pH to 8. Step 3: Solubilized prochymosin is loaded on to cellulose (ion exchange chromatography), wash and eluted with 0–0.5 mol / dm3 NaCl gradient. Step 4: Pure prochymosin is incubated at pH 6. Step 5: Active chymosin (54% recovery). Best results were obtained with solubilization of Triton/EDTA washed inclusion bodies in 10 volumes of 8 mol/dm3 urea.

QuestIons 1. Explain the process involved in enzyme histochemistry study. 2. What is the role of formalin in purification process? 3. Describe the various processes involved in fractionation method. 4. Write short notes on fractionation process using heat. 5. How are enzymes extracted from animal tissues? 6. How are enzymes extracted from higher plants? 7. Define lyophilization. 8. Explain the process of extraction of membrane-bound enzymes. 9. Explain the structure of amphipathic lipids. 10. What are the factors necessary, to be an extraction medium? 11. Define stabilizers. 12. Define inhibitors.

Enzyme Extraction and Purification

13. 14. 15. 16. 17. 18.

 45

Write down the preliminary procedure followed during purification. Explain partition chromatography. Explain zone electrophoresis. List out the chemicals used in gel filteration. Explain the functions of HPLC. How will you determine the molecular weight of an unknown enzyme?

CHAPTER

4 Enzyme Assay

Enzyme assay are labortory methods for measuring enzymatic activity for the study of enzyme kinetics and enzyme inhibition. The amount of enzymes can either be expressed as molar amounts or measured in terms of activity in enzyme units. Enzyme activity = Mole of substrate = rate × reation volume. coverted per unit time Enzyme activity is a measure of the quantity of active enzyme present which depends on conditions (which should be specified). The SI unit is katal, 1 katal = 1 mol S–1 A more practical and commonly used value is 1 Enzyme unit (U) = 1 µ mol min-1 1U corresponds to 16.67 nanokatals.

factors controllIng In assays 1. salt concentration Enzyme cannot tolerate extreme high salt concentration (Active at 1–500 mM). The ions interfere with the weak ionic bonds of proteins.

2. Temperature All enzyme work within a range of temperature specific to the organism. Increase in temperature generally lead to increased in reaction rates with a limitation, where at higher temperature there will be a decrease in reaction rates.

48   Enzymes

3. pH Most enzyme are sensitive to pH and have specific ranges of activity (6 to 8). pH can stop enzyme activity by denaturing the three dimensional shape of the enzyme by breaking ionic and hydrogen bonds.

4. Substrate Saturation Increase of substrate concentration increases the rate of reaction. However, the enzyme saturation limits reaction rates since the active sites are occupied most of the time.

5. Level of Crowding Large amounts of macromolecules in a solution will alter the rates and equilibrium constants of enzyme reactions, through an effect called macromolecular crowding. The purpose of enzyme assay is to determine how much of a given enzyme of known characteristics is present in a tissue homogenate, fluid or partially purified preparation. Cellular destruction by natural processes (autolysis) is accompanied by changes in enzymes and breakdown of cofactors. This leads to the death of the organisms. Autolysis is minimized if a tissue is kept in cold (4oC), before and after the preparation of homogenates. It is possible to assay the amount of an enzyme in a given solution quantitatively in terms of catalytic effect it produces.

dIscontInous assay The rate of a particular enzyme-catalyzed reaction can often be measured in a number of ways. One such method is explained with reaction catalyzed by hexokinase. Mg2+ D-glucose + ATP D-glucose-6-phosphate + ADP The rate of this reaction can be monitored by: 1. Taking samples from the reaction mixture at a known time after the addition of the enzyme. 2. Stopping the reaction quickly by the addition of acid to inactivate the enzyme.

Enzyme Assay

 49

3. Measuring the amount of product formed. In this method, there is a “stop and sample”, and therefore, this is called as discontinuous assay procedure. This has disadvantages such as possible sampling errors in the separation and in the estimation of products. This can be corrected by the use of ion-exchange chromatography.

contInous assay This is a more convenient method which involves some changes in the property during the course of the reaction. In the above discontinuous assay, there is no convenient change in absorbance. But such changes occur if we couple the production of D-glucose-6phosphate with the reduction of NADP+ to NADPH using glucose-6phosphate dehydrogenase. D-glucose + ATP

Mg2+

D-glucose-6-PO4 + ADP NADP+

NADPH

D-glucose-L-lactone-6-PO4

Coupled Assay Taking the above reaction, sufficient coupling enzyme and substrate should be added such that D-glucose-6-PO4 formed in the first step is immediately converted to D-glucose-Lactone-6-PO4. So that the coupling reaction is not a limiting one. In a number of assays it is not possible to monitor the reaction continuously so a “stop and sample” is adopted. e.g. Consider a reaction catalyzed by ornithine decarboxylase. If L-[1-C14] ornithine is used, the CO2 liberated will be radioactive. This CO2 can be trapped by a suitable base (e.g. ethanolamine dissolved in 2-methoxyethanol) and the radioactivity is estimated by scintillation counting. Assay procedure involving radioactively labelled substances are very sensitive. Certain precautions must be followed when enzyme assay is done. They are: 1. The substrate, buffers etc.... should have high purity. The contaminants may affect the activity of enzymes. e.g. Commercial preparation of NAD+ contains inhibitors of

50   Enzymes

2. 3. 4.

5.

dehydrogenases. Certain preparation of ATP contains trace amount of vanadate ions, which are powerful inhibitors. Enzyme preparation should not contain any compound or other enzymes that interfere with the assay. The enzyme should be stable during the time taken for assay (break down of substrate should not occur). The activity of enzyme will be affected by the changes in pH, temperature etc. So it is important to ensure that these parameters are stabilized by use of buffer, thermostable bath, etc. It should be ensured that once the steady state has been achieved the measured rate of reaction is constant over a period of interest and is proportional to the amount of enzyme added.

enzyMe assay tecHnIQues Visible and ultraviolet spectrophotometer Methods Many substrates and products absorb light in the visible or UV region, but they do not absorb at the same wavelength. This change in absorbance can be used as the basis for assays of enzyme using spectrophotometer. A double beam spectrophotometer is used with a temperature-controlled cell housing. In most of the assays, NADP or NAD is considered as a substrate and NADPH or NADH as product. Enzymes which are not directly involved in this interconversion may be assayed by coupling reaction. In this case, the enzyme to be assayed is linked to the NAD+/NADH system by means of common intermediates. e.g. Phosphofructokinase (PFK). This has been linked with aldose to glyceraldehyde-3-PO4 dehydrogenase reaction.

Spectrofluorimetric Method This technique is potentially very sensitive. Even trace impurities in the enzyme preparation can stop the emitted radiation. Thus, the compound absorbs radiation in the UV region and emits in visible light. This increase in wavelength is known as “Stoke shift.”

Enzyme Assay

 51

Hydrolases can be assayed by measuring the rate of appearance of fluorescence at 450 nm of the anion of 4-methylumbelliferone.

Luminescence Method In bioluminescent reaction, the intensity of emitted light is used to study enzyme reaction. This method is very sensitive. The reaction can be used to assay ATP and appropriate enzymes via coupled reaction. e.g. Bacterial luciferase uses reduced FMN to oxidizing long chain aliphatic aldehydes. The resulting FMN can be coupled with NAD(P)H.

radioisotope Method This is a highly potential and very sensitive method. This has been restricted to the separation of radiolabelled forms of substrate and product. This method is used in studying the mechanism of enzyme action and in ligand binding studies. If one of the product is a gas then the method is easier. e.g. Assay of glutamate decarboxylase is based on the evolution 14 of CO2. Then the 14CO2 evolved could be trapped in alkali and then measured.

QuestIons What is the significance of enzyme assay? Explain the stop and sampling method with an example. Write short notes on continuous assay. What are the various procedures followed in coupled assay? Explain the function of double beam spectrophotometer in enzyme assay. 6. Define Stoke shift. 7. Write a note on luminescence method in the assay of enzymes. 8. Explain the radioisotope assay method with example. 1. 2. 3. 4. 5.

CHAPTER

5 Types of Enzyme

IntroductIon Enzymes are categorised into three main categories 1) Metabolic enzyme which are produced within the body, 2) Digestive enzyme which body produces and 3) Food enzyme. Metabolic enzymes are responsible for running the body at the blood, tissues and organs. They are required for the growth, repair and maintenance of the body’s organ and tissues. Digestive enzymes aid in the digestion of food and absorption and delivery of nutrients throughout the body. e.g. pancreas, stomach and small intestine. Food enzymes are derived solely from raw fruits, vegetables and supplemental sources, these enzymes enables the body to digest by breaking down the various nutrients.

coenzyMes Many enzymes require the presence of certain non-protein compounds to accelerate their reactions. Unlike enzymes, these are heat stable and are of smaller molecular weight and therefore, are dialyzable. These are called coenzymes or prosthetic groups, depending upon the proximity of their attachment to the enzyme proteins. If the activator is firmly attached to the enzyme protein, then they are called as prosthetic group. e.g. Conjugated protein.

54   Enzymes

Enzyme Protein

Prosthetic group

Fig 5.1 Prosthetic group

If the non-protein compounds are not firmly attached to the enzyme protein, but exist in free state in solution, contacting the enzyme protein only at the instant of enzyme action their they are called coenzymes. Enzyme Protein

Enzyme Protein

Dissociated coenzyme

Fig 5.2 Coenzyme

Nowadays, the term “cofactor” is used in general. The term “coenzyme” is used for those cofactors which are organic molecules and participate in the catalytic processes. The other inorganic cofactors which are not coenzymes help in establishing the three-dimensional structure of the enzyme protein (which is necessary for their catalytic activity). e.g. Nicotinamide adenine dinucleotide (NAD) and Flavin adenine dinucleotide FAD).

Characteristics of coenzymes 1. 2. 3. 4.

They are stable towards heat. They are generally derived from vitamins. They function as co-substrates. They participate in i. Electron transfer reactions e.g. NAD+, NADH+, FMN, FAD, etc. ii. Group transfer reactions e.g. CoA, TPP, Pyridoxal phosphate, Tetrahydro folic acid, etc.

Types of Enzyme

Table 5.1

 55

Coenzymes and their functions

Coenzymes

Functions performed Hydrogen transfer NAD + Hydrogen transfer NADP FAD Hydrogen transfer FMN Hydrogen transfer TPP (thymine pyrophosphate) Acetyl group transfer PP (pyridoxal phosphate) Amino group transfer Biotin Carboxyl group transfer Coenzyme A Acyl group transfer +

IsoenzyMes Many enzymes occur in more than one molecular form in the same species, in the same tissues or even in the same cell. In such cases, the different forms of the enzyme catalyze the same reaction but they differ from each other in their kinetic properties and in amino acid composition. Such multiple forms of the enzymes are called as isoenzymes or Isozymes. They are present in the serum and tissues of mammals, amphibians, birds, insects, plants and unicellular organisms. Wellknown isoenzyme is lactate dehydrogenase (LDH) which catalyzes the oxidation of lactate to pyruvate with NAD acting as coenzymes.

lactate dehydrogenase enzyme (LDH)  1. LDH is found in electrophoretically distinct forms. 2. LDH are tetramers, each made up of two types of units, H and M. 3. H represents the heart LDH and M refers to muscle LDH. Heart LDH is predominant in the heart and is active at lower concentration of pyruvate. This has four identical subunits (HHHH). Muscle LDH is predominant in the skeletal muscles and is active at higher concentrations of pyruvate. The four identical subunits are (MMMM).

56   Enzymes

These two subunits, H and M, have the same molecular weight (35,000Da) but differ in amino acid composition and also differ immunologically. LDH can be formed from H and M subunits to yield a pure H-tetramer and a pure M-tetramer. Only the tetrameric molecule has catalytic activity. The combinations are HHHH – LD1 HHHM – LD2 HHMM – LD3 HMMM – LD4 MMMM – LD5 LDH catalyzes the transfer of two electrons and one hydrogen ion from lactate to NAD +. Lactate

NAD+

Pyruvate

NADH + H+

MonoMerIc enzyMes Monomeric proteins are those which consist of only a single polypeptide chain, so they cannot be dissociated into smaller units. In general, they contain 100–300 amino acid residues with molecular weight between 13,000–35,000 e.g. Carboxypeptidase. Usually these catalyze hydrolytic reactions.

proteases (proteolytic enzymes) They catalyze the hydrolysis of peptide bonds in other proteins. In general, they are often synthesized in an inactive form known as a proenzyme or zymogen and activated when required. e.g. Serine proteases. Serine proteases The serine proteases, chymotrypsin, trypsin and elastase, are produced in an inactive form by the mammalian pancreas, form a closely related group of enzymes. About 40% of the primary structure

Types of Enzyme

 57

is common to all three enzymes and the x-ray crystallography studies show that their tertiary structures are similar. These enzymes have identical mechanism and similar optimum pH of about 8. All are endopeptidases that hydrolyze peptide bonds. Chymotrypsin This enzyme is synthesized in the pancreas as the zymogen chymotrypsin (or pre-chymotrypsin). The enzyme is made up of single polypeptide chain with 245 residues containing five intrachain disulphide binding pocket, which binds phenylalanine, tryptophan and tyrosine. This breaks peptide bond between arginine-15 and isoleucine-16 producing chymotrypsin. Trypsin Trypsinogen lacks nine amino acids residues at the N-terminals and has 1–122 disulphide bridges. The action of the enteropeptidase (or trypsin itself) in the intestine removes a hexapeptide from the N-terminus of trypsinogen to produce the active trypsin. Pepsin It is called acid protease because it functions at low pH. Peptide fragments are removed from the inactive form, pepsinogen, by the action of acid or other pepsin molecules to produce the active enzyme.

olIgoMerIc enzyMes Oligomeric enzymes consist of two or more polypeptide chains, which are usually linked to each other by non-covalent interactions and never by peptide bonds. The component polypeptide chains are termed as subunits and they may be identical. They are sometimes called protomers. Dimeric consists of two, trimeric consists of three and tetrameric protein consists of four subunits. The molecular weight exceeds 3500 Da. e.g. All the enzymes involved in the glycolysis. These enzymes are not synthesized as inactive zymogens but their activities are regulated by feedback inhibition and they may exhibit allostery, (i.e) their different binding sites interact.

58   Enzymes

Lactate Dehydrogenase This an example of oligomeric enzyme where each subunit has the same function. It is a tetramer with a molecular weight of 1,40,000Da. They are of two types each with a different amino acid composition. The M-form, which predominates in skeletal muscles and the H-form, which predominates in the heart. Each monomer is catalytically inactive, but it can combine with others of the same or different type to produce the active tetrameric enzyme.

Lactose Synthase Mammary gland lactose synthase is an example of oligomeric enzyme where non-functional subunit modifies the behaviour of a functional subunit. This enzyme was isolated from milk which contained two subunits, where one is a catalytically inactive protein, alpha lactalbumin, found only in mammary gland and the other is N-acetylactosamine synthase, an enzyme present in most tissues. This enzyme is important in the synthesis of the carbohydrate components of glycoproteins.

Tryptophan Synthase Tryptophan synthase of E.coli is another example of oligomeric enzyme, which contains two different functional subunits. It can be dissociated into two alpha subunits of molecular weight 29,000 Da and two beta subunits of molecular weight 90,000 Da.

Pyruvate Dehydrogenase Pyruvate dehydrogenase of bacteria and animal cells is an example of multienzyme complex. This resembles tryptophan synthase. E.coli enzyme consists of 60 polypeptide chains and has a molecular weight of about 4,600,000 Da.

MetalloenzyMes and Metal-actIVated enzyMes More than a quarter of known enzymes require the presence of metal atoms for full catalytic activity. The metal atoms usually exist as cations and often have more than one oxidation state, as with ferrous (Fe2+) and ferric (Fe3+) ion. This positive charge can stabilize the

Types of Enzyme

 59

transition states by electrostatic interactions. The charged metal ions bind to a particular number of groups (ligands) by accepting free electron pairs to form coordinate bond in a specific orientation. Therefore, in the case of enzyme catalysis, the metal ions involve by 1. Accepting or donating electrons to the activated electrophiles or nucleophiles, even in neutral solution. 2. They themselves act as electrophiles (they mask the unwanted side reactions) 3. They may bring together enzyme and substrate by means of coordinate bonds (causing strain to the substrate in the process).

Metalloenzymes The metal is tightly bound and retained by the enzyme on purification. But in the case of metal activated enzyme, the binding is less tight and the purified enzyme may have to be activated by the addition of metal ions. Mildvan (1970) pointed out that ternary complexes are formed between enzyme (E), metal ions (M) and substrate (S) – the enzyme bridge complex (M–E–S), substrate bridge complex (E–S–M), metal bridge complex (E–M–S). But in the case of metalloenzyme, it cannot form substrate bridge since the purified enzyme exists as E–M. The involvement of metal ions in enzyme may be investigated in NMR, Proton relaxation rate (PRR) enhancement techniques.

Metal activation Activation by alkali metal cations ( Na+ and K+)

K+, the most abundant intracellular cation, is known to activate a great number of enzymes. e.g. Enzymes which catalyze the phosphoryl transfer or elimination reactions. The role of K+ is to bind to negative charge on an inactive form of the enzyme and course in conformational changes and lead to active form.

60   Enzymes

e.g. The carboxyl group of PEP (phosphoenol pyruvate) binds to the enzyme-bound K+, which results in the conformational changes to form a complex. Activation by alkaline earth metal cations (Ca2+ and Mg2+)

Oxygen atoms are often involved in the bonds of both alkali metal and alkaline earth metal cations (the bonds formed are stronger). The divalent cations Ca2+ and Mg2+ can form six coordinate bonds to produce octahedral complexes. Enzymes which require Ca2+ are the extra cellular ones namely, salivary and pancreatic alpha amylases. Ca2+ plays an important role in maintaining the structure required for catalytic activity. Most of intracellular enzymes require Mg2+ for activity and in most cases, this requirement can be replaced in vitro by Mn2+. e.g. Kinases from E–S–M complexes. 1. Let us consider the reaction catalyzed by muscle creatine kinase. Mg–ADP + Phosphocreatine + H+ Creatine + Mg–ATP The true substrate is Mg–ATP. The reaction proceeds via the formation of the complex creatine –E–ATP–Mg. 2. The reaction catalyzed by pyruvate kinase, involved in forming the cyclic metal bridge complex. Mg E

ATP pyruvate

3. Enolase is a dimeric enzyme, which requires Mg2+ (two) to stabilize the active dimer. Activation by Transition Metal Cations (Cu, Zn, Mo, Fe and co Cations)

Transition metal ions bind to enzymes much more strongly than the metal ions and usually from metalloenzymes. They are found in only trace amounts in living organisms (in larger amounts they are toxic).

Types of Enzyme

 61

The trace metals Mo and Fe are found in nitric oxide reductase (The nitrogen activating complex of nitrogen fixing bacteria).Fe is a component of haemoglobin (oxygen-carrier haemoglobin of the erythrocytes of vertebrates. Co is found in vitamin B12. Superoxide dismutase is a copper metalloenzymes which catalyzes the removal of the highly reactive O2– produced. e.g. Bovine erythrocyte superoxide dismutase is a dimeric protein containing two Cu 2+ ions and two Zn2+ ions. Cu2+ is involved in the reactions shown below as _

E – Cu2+ + O2 _ E – Cu+ + O2

E – Cu + + O2 E – Cu 2+ + H2O2

eXtreMozyMes Optimal functions of certain enzymes occur at extreme conditions of temperature, pH etc. Such enzymes are called extremozymes. Hyperthermophiles are found above 100o C and they have a half-life of several days at optimum temperature of 100o C. Such enzymes are called high temperature enzymes. About 30 hyperthermophilic enzymes have been isolated for example, sulphur-reducing bacteria. All hyperthermophiles are strict anaerobes and strict organotrophs which use complex organic mixtures as source of C and N. The enzymes isolated are protease, amylase, α-glycosidase, hydrogenase, glutamate dehydrogenase, DNA polymerase, etc. These enzymes can be used for catalytic reaction under high temperature environment. e.g. A hyperthermostable DNA polymerase essential in PCR, obtained from Pyrococcus furiosus takes 20 hours at 95o C to lose 50% of its catalytic activity, while Thermococcus litoralis takes 7 hours.

aBzyMes Abzymes are antibodies which function as enzymes and catalyze specific chemical reactions. These belong to non-traditional enzyme group because these enzymes have binding sites that preferentially binds to the transition state of their substrate molecules. They are not produced naturally.

62   Enzymes

rIBozyMes If RNA molecules have the capacity to catalyze a chemical reaction, then they are called ribozymes. They are typical enzymes which show kinetics. The two reactions are cleavage of RNA and DNA. These ribozymes cut and splice themselves into a form that can catalyze the cleavage of other RNA/DNA molecules. The catalytic ability of ribozymes is due to their three-dimensional structure, which is able to generate in them the substrate-specific binding site.

synzyMes Synzymes or artificial enzymes are generally synthetic polymers, sometimes proteins, which have enzymatic activities. Synzymes have two functional sites. 1. Substrate binding site 2. A catalytically effective site If the synzyme has a binding site for the transition state of the substrate, then the binding site itself serves as the catalytic site. e.g. Abzymes (antibodies having enzyme activities). Synzymes are derivatized proteins e.g. Myoglobin, (the protein which functions as oxygen carrier in muscle). When [RU (NH3)5]3+ is attached to the three surface histidine residues of myoglobin, it functions as oxidase and oxidizes ascorbic acid and reduces molecular oxygen. They are effective as naturally occurring ascorbate oxidases.

Non-protein synzyme cyclodextrin is an example of this type. They occur naturally with 4 linked D-glucose residues at 6th, 7th, 8th, 9th or 10th position, joined head to tail in a ring. The cavities of cyclodextrin range from 0.5–1 nm but depth is the same (0.7 nm deep). They are hydrophilic but cavity is hydrophobic. e.g. When pyridoxal coenzyme is attached to C-6 hydroxyl group of a β-cyclodextrin, it acts as transaminase specific for L-amino acids and it is not like natural transaminases.

Types of Enzyme

 63

BI and polyfunctIonal enzyMe These enzymes are produced by genes obtained by the fusion of the coding regions of two or more genes encoding different enzymes. Let us consider a gene a producing enzyme A and gene b producing enzyme B. They are combined together to bring about enzyme activity. The stop codon, for example, TAG of gene a is deleted and this modified gene a is then joined with the ATG or initiation codon of gene b. A linker sequence encoding 2–10 amino acids is introduced between the genes a and b. The two genes are then fused in a correct reading frame. The fused gene will produce a single polypeptide which will posses/exhibit the activities of both the enzymes A and B E.g. b-galactosidase–galactokinase, b-galactosidase–galactose dehydrogenase, b-galactokinase–galactose dehydrogenase, galactose dehydrogenase–luciferase. These enzymes are useful in the preparation of biosensors.

QuestIons 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Define monomeric enzymes. What are the various types of protease enzymes? Give a brief account of chymotrypsin. Define oligomeric enzymes with an example. Write a note on lactate dehydrogenase. Explain the various types of oligomeric enzymes. What are extremozymes? Explain the properties of extremozymes with few examples. Define synzyme. Explain the types of synzyme with example. Write a short note on bi and polyfunctional enzymes. How are metals involved in reactions? Define metalloenzyme. Explain the process of Na+ and K+ in the activation of alkali. Write a note about the roles of Ca2+ and Mg2+ in the catalysis.

64   Enzymes

16. 17. 18. 19. 20. 21.

What are coenzymes? Define prosthetic group. What are the characteristic features of a coenzyme? List out some coenzymes and their functions. Write a short on isoenzyme. Explain the different forms of LDH.

6

CHAPTER

Enzyme Catalysis

MecHanIsMs In organIc cHeMIstry A covalent chemical bond involves the sharing of a pair of electrons between the atoms (X:Y). If the bond breaks by homolytic fission, each atom separates as a highly reactive free radical, possessing an unpaired electron (Xo:Yo). These reactions are very uncommon in aqueous solutions where most bonds are broken by hydrolytic fission, leaving one of the atoms with both electrons. If both electrons are retained by a carban atom, a carbonion is produced. R3 C : X

_

Carbonion

R3 C + X+

Carbanions and carbanium ions may be stabilized by delocalization of the charge over other atoms in the molecule. For example, CH2= CH–CH2

CH2–CH=CH2

The curved arrow indicates the flow of a pair of electrons which would take place if one of the canonical structures of the carbanium ion was converted to the other. Conjugated double bond systems (where double and single bonds occur alternately) are of particular importance for stabilization. The formation of carbonions and carbanium ions are likely to occur as part of a complete reaction sequence. Organic compounds can participate in four main types of reactions, namely addition reactions, elimination reactions and rearrangements. Substititution reactions may be nucleophilic. Here, when the group attacking the carbon atom is an electron donor, it is called a nucleophile since it is attracted to nuclei. In an electrophilic reaction, the attacking group is an electron acceptor.

66   Enzymes

Electrophilic substitution reactions often involve the displacement of hydrogen. H + NO2+

NO2 + H+

Benzene

Nitrobenzence

In nucleophilic substitution, an atom other than hydrogen is usually displaced. Such reactions may have a unimolecular or a bimolecular mechanism. In unimolecular nucleophilic substitution reactions, the rate limiting step is the inonization of a single molecule to form a carbanium ion which then reacts with a nucleophile. The carbanium ion is planar and if the resulting product has four different groups attached to that particular carbon atom, a racemic mixture of optical isomers will be obtained. The overall rate of the reaction is the basicity of the leaving groups (Y–). Weak bases are good leaving groups and strong bases are poor leaving groups. The strength of the attacking nucleophile does not usually affect the rate of reaction. R1

R1 R2

C

Y

-Y

+X

C

R1

R3

R3

R2

R2

R2

C

X (or) X

C

R1 R3

R3

In bimolecular nucleophilic substitution reactions, the attacking nucleophile adds to the carbon atom at a point diametrically opposite the leaving group which it displaces in one rapid step. R2 R2 X

C R3

R1 Y

X

R2

R2 C R3

Y

X

C

R1 + Y R3

The rate of the reaction depends on the reactive strengths of X and Y as nucleophiles.

Enzyme Catalysis

 67

MecHanIsMs of enzyMe catalysIs Acid–Base Catalysis Acids can catalyze reactions by donating a proton. Bases accept a proton. Bases may also increase reaction rates by increasing the nucleophilic character of the attacking group. For example, consider specific hydroxide ion catalysis of a reaction in H2O. The rate law will have two terms: 1. The unanalyzed rate. 2. The hydroxide catalyzed term. For more details, the case of the hydrolysis of acetyl imidazole is considered. O H2C

C

N

NH

H2O

_ CH3COO + N

NH

The imidazole may function as a base catalyst in addition to OH. At any given pH, the rate of hydrolysis will increase with increasing concentrations of imidazole. Catalysis by imidazole implies that the energy barrier for hydrolysis is lowered by proton itself, that is proton transfer confers a relative stabilization to the transition state. Therefore, in general, at a given pH, the nucleophilicity of the water molecule is greatly enhanced without the generation of a high concentration of OH. The hydrogen bonding in the transition state may avoid the formation of unstable high energy species thus stabilizing the catalytic transition rate.

Strain Distortion and Conformational Change The strain in starting materials and release of that strain in the transition state to products can provide rate accelerations in chemical reactions. This experiment was carried out by Westheimer and his colleagues with phosphate-ester hydrolysis rates in two compounds.

68   Enzymes

(1)

HO

O O

P

O O

H2O

CH3

O O ()

O )

O (2)

O

CH3

O

CH3

O

O H2O

P

P O

O

O

)

O O )

)

The cyclic example (1) relieves considerable ring strain with ring opening on hydrolysis at a relative rate 108 fold faster than acyclic, example (2). Therefore, strain and distortion effects are important in enzymatic catalysis in which the effect of enzyme can be read during change in the conformation of the three-dimensional structure of the protein which concert a low activity form of the enzyme to a high activity form. On binding of substrate to an enzyme to a portion of the intrinsic interaction energy may be used to accelerate catalysis, (acceleration means reduction of the free energy of activation) and destabilization of the ES complex has some effect, which include geometric distortion of bond angles in the bound substrate or steric compression. This could involve the electrostatic repulsion between a charged group on the substrate and an amino acid side chain of similar charge at the active site. All these mechanisms contribute to the thermodynamic destabilization of bound substrate that lessens the energy barrier to the transition state, provided these destabilization forces are released in the transition state that is, it is only the ES complex that is selectively destabilized.

Catalysis by Approximation (Entrophic Contribution) Approximation literally means to make reactants proximal, that is adjacent to each other. The term “catalysis by approximation” means the reaction rate enhancement is achieved, if two reactants are taken out of dilute solution and held in close proximity to each other, as

Enzyme Catalysis

 69

they will be at the active site of an enzyme. This proximity will raise the effective concentration over that of the reactants, free in solution and lead to a rate acceleration. To quantify some propinquity effects, model studies were carried out to perform comparison rate for similar intra versus intermolecular transformation. e.g. Imidazole catalyzed hydrolysis of p-nitrophenyl acetate, proceeds under a given set of conditions with a rate constant Kobs = 35 min–1 M–1, where Kobs is a biomolecular rate constant. O N + H3C

HN

C

NO2

O

H2O

O NO2 + H3C

HO

) O + NH

C

N+

( pka = 7 )

NO2

O

This second-order rate constant can be compared with the firstorder rate constant for a comparable intramolecular case. HN

N

C

O

H2 O

HN

N

_ COO + HO

NO2

O

NO2

Here, Kobs = 200 min–1, under the same reaction conditions. To assess, the rate of acceleration due to the covalently ensured, we can calculate a value called the effective molarity of the imidazole in this above equation. Thus, (200 min-1)/ (35 min–1 M–1) = 5.7 M

70   Enzymes

Therefore, the effective molarity of imidazole is 5.7 M. Addition of a third methylene group, allowing a six-member rather than fivemember transtition state causes a jump in the effective molarity to 23.9M. HN

N

C _ O

NO2

O 23.9N

Reuben (1971) has postulated that catalytic efficiency of enzymes may be enhanced due to the long of the ES complex relative to the lifetime of a simple biomolecular collisional interaction of chemical reactants. Lifetimes for collisional interactions are thought to be in the order of 10-3 seconds, where as fast kinetic techniques put the mean lifetimes of ES complexes at 10-7 to 10-4 second, where, ES interaction will generate an activated complex or transition state that will go on to products called as “substrate anchoring”.

Covalent Catalysis (Nucleophilic vs Electrophilic catalysis) The side chains of amino acids found in protein present a number of nucleophilic (electron rich) functional groups for catalysis. Their side chains can attack electrophilic (electron deficient) portions of substrates to form a covalent bond between the enzyme and the substrate as a reaction intermediate. Attack by the enzyme nucleophile (En2-X) can produce acylation , phosphorylation or glycosylation of the enzyme nucleophile, as covalent intermediate can then be attacked in a second step by some low-molecular weight nucleophile to yield the observed reaction product. (When nucleophile in water, the overall reaction is hydrolysis). A number of coenzymes form covalent adducts with substrates, these adducts generate new electrophilic groups capable of functioning as electron sinks during catalysis. Adduct-forming coenzymes function to provide routes to low-energy, stabilized, substrate-derived carbonions, therby providing rate acceleration. e.g. This is true for pyridoxal phosphate and thiamine pyrophosphate coenzymes. About 100 enzymes show covalent intermediates during catalysis.

Enzyme Catalysis

 71

coenzyMes In enzyMe-catalyzed reactIons Coenzymes are organic compounds required by many enzymes for this catalytic activity. They are often vitamins or derivatives of vitamins. Sometimes, they act as catalysts in the absence of enzymes, but not so effectively as in conjuction with an enzyme. In this case of metal–enzyme linkage, there is a range of bond strengths at the point of the prosthetic group. Coenzyme with prosthetic groups form an integral part of the active site of an enzyme and undergo no net change as a result of acting as a catalyst. But loosely bound coenzymes are regarded as cosubstrates since they often bind to the enzyme protein together with the other substrates at the start of a reaction and are released in an altered form at the end of it. Some coenzymes are discussed below: 1. Nicotinamide nucleotides: They are the derivatives from the vitamin Niacin (which is nicotinamide or nicotinic acid).The reduction of NAD+ to NADH requires two reducing equivalents per molecule. 1. one electron 2. one hydrogen atom which together may be called as Hybride ion. This is found to be present in the pyridine ring of nicotinamide. At this stage, the pyridine ring is conjugated, so the positive charge may be delocalized, making several points vulnerable to nucleophilic attacks. Therefore, it results in stereochemical specificity. R_N

H H

H

C

R_N

H+ CONH2 C=

72   Enzymes

2. Flavin nucleotides: Flavin nucleotides are derived from riboflavin, vitamin B2. They function in oxidation/reduction reactions. e.g. Glycose oxidase which catalyzes the reaction utilizes FAD as prosthetic group and O2 as hydrogen acceptor. D-glucono-δ-lactone + H2O2 D- glucose + O2 3. Adenosine phosphates: The nucleoside phosphates are involved in phosphate transfer reactions. ATP and ADP may be interconverted by the reaction. ADP + Pi ATP + H2O The importance of ATP in energy metabolism is that when compared to other organic PO4 it is only moderately unstable. In the cell, ADP is stabilized by binding to Mg2+ ions and their metabolism is strictly mediated by enzymes. In some instances, two phosphate groups are removed from ATP to liberate inorganic pyrophosphate. AMP + PPi ATP + H2O

QuestIons 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

What is homolytic fission? Define carbanion. Explain the various types of reactions of organic compounds. What are the types of catalysis? Explain acid–base catalysis. Explain entrophic contribution. What is covalent catalysis? Explain. Explain how strain distortion occurs. Explain the role of nicotinamide nucleotide in catalytic reaction. Write short notes on flavin nucleotide and adenosine phosphate.

CHAPTER

7 Enzyme Specificity

An important characteristic feature of enzymes is that they are specific in action. There are different types of specificity namely 1. Group specificity 2. Absolute specificity 3. Stereochemical specificity The specificity of enzyme action is determined by two separate factors: 1. The relative ability of a potential substrate to bind to the enzyme. 2. After binding, its relative ability to undergo a reaction to form products.

group specIfIcIty Some enzymes exhibit group specificity that is, they may act on several different, but closely related substrates to catalyze a reaction involving a particular chemical group. e.g. Alcohol dehydrogenase catalyzes the oxidation of a variety of alcohols. Hexokinase assists the transfer of phosphate from ATP to several different hexose sugars.

aBsolute specIfIcIty Here, enzymes act only on one particular substrate. e.g. Glucokinase catalyzes the transfer of phosphate from ATP to glucose and to no other sugar.

74   Enzymes

Uncatalyzed reactions give rise to a wide range of products but enzyme-catalyzed reactions are product specific as well as substrate specific.

stereocHeMIcal specIfIcIty Here, enzyme show specificity on substrates that exist in two stereochemical forms, chemically identical but with a different arrangement of atoms in three-dimensional space. e.g. L-amino acid oxidase mediates the oxidation of L-amino acids. A separate enzyme, D-amino acid oxidase is required for the corresponding oxidation of D-amino acids.

tHe actIVe sItes Ogston (1948) pointed out that there must be at least three different points of interaction between an enzyme and its substrate. Binding sites link to specific groups in the substrate, ensuring that the enzyme and substrate molecules are held in a fixed orientation with respect to each other with the reacting group or groups in the vicinity of catalytic sites. R Substrate C R

R

R

Enzyme A"'

A"

A' Active site

Fig 7.1 Diagrammatic representation of three point interaction between enzyme and substrate.

Example: Sites A" and A"' represents the binding sites for R" and R"' respectively and A' a catalytic site for a reaction involving R'. The region which contain the binding and catalytic sites are termed as active site or active centre of the enzyme. This comprises only

Enzyme Specificity

 75

a small proportion of the total volume of enzyme and is usually at or near the surface since it must be accessible to the substrate molecule.

fIscHer’s locK and Key HypotHesIs As early as 1890, Fischer suggested that a substrate might fit into its complementary site on the enzyme as a key fits into a lock. In the lock and key model, all structures remain fixed throughout the binding process. This hypothesis is also known as the concept of intermolecular fit. The formation of ES complex, releases energy which induces the substrate molecule to be in activated state. Certain bonds of the substrate molecule at its activated state become more susceptible in cleavage. Therefore the ES complex is highly unstable now and decomposes to produce the end products and regenerates the free enzyme. RG

CS BG CS

BS

+ BG Substrate

� BS Enzyme

Enzyme-substrate complex

BS = A binding site on the enzyme CS = A catalytic site BG = A binding group on the substrate RG = A reacting group Fig 7.2

Lock and key model

KosHland Induced fIt HypotHesIs The lock and key hypothesis explains many features of enzyme specificity but the flexibility of protein is not taken into account. Thus the binding of a substrate to an enzyme may bring about a conformational change. That is, a change in the three-dimensional structure, but not in the primary structures.

76   Enzymes

Koshland (1958), suggested that the structure of a substrate may be complementary to that of the active site in the enzyme–substrate complex, but not in the free enzyme. A conformational change takes place in the enzyme during the binding of substrate which results in the required matching of structures. The induced fit hypothesis requires the active site to be flexible and the substrate to be rigid, allowing the enzyme to wrap itself around the substrate, in this way, bringing together the corresponding catalytic sites and reacting groups. RG

CS BS CS

BG +

�

BS

BG Substrate

Enzyme

Enzyme-substrate complex

Fig 7.3 Induced Fit Model

In the lock and key mechanism, the active site is always structurally intact with the catalytic sites aligned and freely accessible. e.g. Hand and gloves.

non-productIVe BIndIng Binding group in a molecule other than the substrate might trigger off a conformational change in the enzyme but in general, this would not result in catalytic groups being brought together in the vicinity of an appropriate reacting group, so no reaction will take place. This is termed as non-productive binding. CS

CS

RG BG +

BS CS

�

CS

BS BG Substrate

Enzyme

Fig 7.3

Enzyme-substrate complex

Non-Productive Binding

Enzyme Specificity

 77

transItIon state staBIlIzatIon The structure of the active site is almost complementary to that of a substrate. In such a case, the structure of the active site is rigid, but the substrate must be distorted slightly in order to bind to the enzyme. This distortion might result in stretching and weakening of the bond, which is subsequently cleaved and thus assist the forward reaction. The mechanism for driving the reaction forward is called transition-state stabilization. The substrate bound is in an undistorted form and the resulting enzymes–substrate complex posseses various unfavourable interactions. Therefore, this reaction follows the following reaction sequence: Transition state Products Enzyme–substrate complex e.g. The mechanism for the hydrolysis of peptides by papain appears to cause a conformational change in the enzyme, in addition to transition state stabilization factor.

Cryoenzymology The investigation of transition state structure is difficult and it occurs only under normal conditions. However, if a reaction is carried out at low temperature, for example at –21oC in aqueous dimethyl sulphoxide, the lifetime of intermediates are extended and their structures may be studied by techniques such as NMR. This is termed as cryoenzymology.

QuestIons 1. 2. 3. 4. 5. 6.

What are the different types of enzyme specificity? Explain absolute specificity and stereochemical specificity. Explain lock and key hypothesis. Write short notes on Koshland’s induced fit hypothesis. What is transition state stabilization? Define cryoenzymology.

CHAPTER

8 Enzyme Kinetics

The study of enzymatic reaction rates or enzyme kinetics gained importance only in the twentieth century. Kinetic measurements are most powerful techniques for elucidating the catalytic mechanisms of enzymes. Enzyme kinetics is a branch of chemical kinetics.

goals of enzyMe KInetIcs Enzyme kinetics helps in understanding 1. Cellular metabolism. 2. The mechanism of enzyme-catalyzed reaction. 3. The ligand binding and signal transduction across the cell membrane. 4. Cellular differentiation, cell growth and gene expression.

cHeMIcal KInetIcs Let us consider a simple equation, A P where, A represents the reactant and P represents the product. This reaction may actually occur through a sequence of elementary reactions (simple molecular processes) as shown below: A I1 I2 P Where, I1 and I2 are intermediates in these reactions.

80   Enzymes

reaction order “At constant temperature, the rate of an elementary reaction is proportional to the frequency with which the reacting molecules come together”. The proportionality constant is known as the rate constant (K). In the case of elementary reaction, A to P, the instantaneous rate of appearance of the product or disappearance of the reactant is called the velocity (V).

Classification of Chemical Reactions by Kinetic order In a biochemical reaction, the rate of formation of product from its substrate is directly proportional to time and enzyme concentration. The chemical reactions are classified into three groups by kinetic order: 1. Zero-order reactions: The rate or velocity (V) is constant and is independent of the reactant concentration (A). V = K [ A]0 = K 2. First-order reactions: The velocity is proportional to the reactant concentration. V = K [ A ]1 = K [ A] 3. Second-order reactions: The velocity is proportional to the product of the concentration of the reactants. V = K [ A ]2 The rate constant has the unit of reciprocal seconds. (S–1). The reaction order of an elementary reaction corresponds to the molarity of the reaction (the number of molecules responsible for the formation of the products). Thus the first-order reaction is a unimolecular reaction.

Enzyme Kinetics

 81

KInetIcs of BIsuBstrate enzyMe reactIons In 1967, W. W. Cleland proposed four types of mechanisms for bisubstrate enzyme reactions and he represented it diagrammatically known as “Cleland diagrams”. In bisubstrate enzyme reactions, two or more substrates are involved in which to the substrates bind to the enzyme at specific sites and participate in the reaction. E.g. Oxaloacetate + Glutamate Aspartate + α - ketoglutarate The Cleland diagrams are of four types and are given below. 1. First type (Orderly sequential mechanism) Two substrates (A) and (B) are involved in this reaction. First (A) binds with enzyme (E) which is followed by the second substrate (B). EAB ternary complex is formed and results in products (P) and (Q) with an intermediate complex EPQ. The product (P) is released first, followed by (Q). A

B

P

Q

E

E (EAB)

(EPQ)

2. Second type (Random sequential mechanism) In this mechanism, there is no definite sequence of substrate binding with the enzymes. Substrates bind in any order and the products are released in a random way. A

B

P (EAB)

Q

(EPQ) E

E (EBA) B

A

(EQP) Q

P

3. Third type (Theorell-Chance mechanism) This is a modified version of the ordered sequential mechanism in which the ternary complex (EAB) is not at all formed.

82   Enzymes

e.g. Reaction calatyzed by alcohol dehydrogenase. A

B

P

Q

E

E EA

EQ

4. Fourth type (Ping-Pong mechanism) In this type, when one substrate binds with enzyme, one product is released immediately before the second substrate binds with the enzyme and then later, the second product is released. In this mechanism, the enzyme acts as a board and the substrate acts as ping-pong balls and therefore, this type of reaction is called pingpong mechanism. e.g. Reaction catalyzed by glutamate dehydrogenase. A

P

B

EP)

F

Q

E

E (EA

(FB

EQ)

where, A and B - substrates E - Enzyme P and Q - Products F - for intermediary stable forms of enzymes. Enzymes catalyze a tremendous variety of reactions using different combinations of certain basic catalytic mechanisms. Some enzymes act on only a single substrate molecule, others act on two or more different substrate molecules. Some enzymes form covalently-bound intermediate complexes with their substrates and other do not. The study of enzyme kinetics begun in 1902 when Adrian Brown investigated the rates of hydrolysis of sucrose by the yeast enzyme β-fructo furanosidase. Brown found that when the sucrose concentration is much higher than that of the enzyme, the reaction rate became independent of the sucrose concentration and the rate is of zero order. Later, he found that the overall reaction is composed

Enzyme Kinetics

 83

of two elementary reactions in which the substrate forms a complex with the enzyme and then subsequently decompose to product and enzyme.

The Michaelis–Menten Equation  The sequence or events in an enzyme-catalyzed reaction is represented as: K1 K2  ES  E + S  → E+P K –1

(Equ 1)

where, E - Enzyme S - Substrate P - Product ES - Enzyme-Substrate complex. K1 and K–1 - The forward and reverse rate constants for the formation of ES. K2 - Rate constant for decomposition of ES to P. In a complex kinetic scheme, the rate of formation of product can be expressed as: 1. The product of the rate constants of the reaction, yielding product. 2. The concentration of its immediately preceding intermediate. The velocity (rate) of the previous reaction is given by V=

d[P] = K 2 [ES] dt

The overall rate of production of ES is the difference between 1. the rates of elementary reactions leading to its appearance. 2. those resulting in its disappearance. d[ES] = K1[E][S] – K –1[ES] – K 2 [ES] dt

For the above equation, two assumptions are possible: 1. Assumption of equilibrium. 2. Assumption of steady state.

84   Enzymes

Assumption of equilibrium

In 1913, Leonor Michaelis and Maud Menten assumed that K–1 >> K2 So, the first step of the reaction reaches equilibrium KS =

K –1 [E][S] = K1 [ES]

Where, Ks is the dissociation constant of the first step in the enzymatic reaction.Therefore the enzyme-substrate complex [ES] is known as the Michaelis Complex. E = [E 0 ] − [ES] ∴ ∴

([E 0 ] − [ES])[S] = KS [ES] K 2 [ES] = ([E 0 ] − [ES])[S] = [E 0 ][S] − [ES][S]

∴

[ES][S] + K 2 [ES] = [E 0 ][S]

∴

[ES]([S] + KS )

∴

= [E 0 ][S]

[ES] =

[E 0 ][S] [S] + KS

Assumption of steady state

Concentration

[So]

[E]Τ

[P]

[S] [E]Τ = [E] + [ES]

d[ES] dt = 0 [ΕS] [E] Time

Fig 8.1 Progress curve for a simple enzyme catalyzed reaction.

Enzyme Kinetics

 85

A graph was illustrated based on the equation under common physiological conditions, but substrate is in great excess over the enzyme. [S] >> [E] Assumption is made that is ES maintains a steady state and it is treated to have a constant value. Therefore, d[ES] =0 dt

This is called Steady state assumption. In order to be the useful, the kinetic expressions for overall reaction must be formulated in terms of experimental measurable quantities. ET = [E] + [ES] The Eq-1 is combined with the steady state assumption. K1 [E] [S] = K–1 [ES] = K2 [ES] Let [ES] = [ET] – [ES] and rearrangement yields. ([E]T [ES])[S] K −1 + K 2 = [ES] K1

Therefore, Michaelis constant “KM” is derived as KM =

K −1 + K 2 K1

So, rearranging the previous equation it gives. KM [ES] = ([ET] – [ES]) [S] Solving for [ES] [ES] =

[E]T − [S] K M + [S]

The expression for the initial velocity (V0) for the reaction. The velocity at t = 0 thereby becomes. K [E] [S]  d[P]  V0 =  = 2 T   dt  t = 0 K M + [S]

86   Enzymes

Both [E] T and [S] are experimentally measurable quantities. The maximum velocity of a reaction (Vmax) occurs at high substrate concentrations when the enzyme is saturated that is the entire enzyme is in the form of ES Vmax = K2 [ET] Therefore, V0 =

Vmax [S] K M + [S]

This expression is Michaelis–Menten equation, which is the basic equation of enzyme kinetics.

Vmax

V0

Vmax 2

0 0

KM

Fig 8.2

2KM

3KM

4KM

5KM

Velocity vs Substrate [S]

Significance of the Michaelis Constant

The Michaelis Constant KM has a simple operational definition. At the substrate concentration at which [S] = KM yields V0 = Vmax / 2 Where, KM is the substrate concentration at which the reaction velocity is half maximal. Therefore, if an enzyme has a small value of KM, it achieves maximal catalytic efficiency at low substrate concentration.

Enzyme Kinetics

 87

KM is unique for each enzyme substrate pair. 1. Different substrates that react with a given enzyme have different KM Value. 2. Different enzymes that act on a single substrate have different KM values. Therefore, the magnitude of KM varies widely with the identity of the enzyme and the nature of the substrate. It depends on temperature and pH. The Michaelis constant can be expressed as. KM =

K −1 K 2 K + = KS + 2 K1 K1 K1

where, Ks is the dissociation constant of Michaelis complex. As Ks decreases the enzyme’s affinity for substrate increases. Kcat / KM - catalytic efficiency Kcat - catalytic constant of an enzyme This can be expressed as K cat =

Vmax [E] T

This quantity is known as turnover number of an enzyme because it is the number of reaction processes (turnover) that each active sites catalyzes per unit time.

The Lineweaver–Burk Plot The graph of Michaelis–Menten equation V0 against S1 is unsatistfactory. The value of V0 approaches Vmax in a tangential fashion with infinite substrate concentration. So it is very difficult to use a plot of V0 against S is obtain an accurate value of Vmax. The graph being a curve cannot be accurately extrapolated upwards from the values of V0 at non-saturating concentrations. Lineweaver and Burk (1934) overcame this problem without making any fresh assumptions.

88   Enzymes

They simply took the Michaclis-Menten equation. V0 =

Vmax [S] [S] + K M

and inverted it, [S] + K M KM 1 [S] = = = V0 Vmax [S] Vmax [S] Vmax [S] Therefore, K 1 1 1 = M . = V0 Vmax [S] Vmax

This is Lineweaver–Burk equation. (This is of the form Y = Mx + C which is used for the equation of a straight line graph (ie) a plot of Y against x has a slope m and intercept C on the Y axis). 1/V0

Slope =

Intercept = 1/KM

KM V

Intercept = 1/Vmax

1/[S]

Fig 8.3

Lineweaver-Burk Plot

Graph represent the Lineweaver – Burk Plot (1/Vo against 1/[S]) which obeys Michaelis – Menten equation. Since the graph is linear, the line can be extrapolated even if no experiment has been performed even at the saturating substrate concentration. From the extrapolated graph the values of KM and Vmax can be determined.

Enzyme Kinetics

 89

tHe eadIe - Hofstee and Hans plot The Lineweaver – Burk plot has been criticized on several grounds They are : 1. The extrapolation across the 1 / Vo axis to determine the value of –1 / KM sometimes reaches the edge of the graph paper before reacting the 1 / [S] axis. This results in redrawing the graph with altered axis. 2. It is said to give under weight of measurements even at a low substrate concentrations and results inaccuracy. The Eadie – Hofstee plot took the Lineweaver – Burk equation as its starting point (based on Michaelis-Menten equation) Here, in this plot both sides of the equation was multiplied by the factor V0 , Vmax KM L 1 1 (V)Vmax = (V0 ) Vmax + (V0 ) Vmax V0 [Vmax ] [S] [Vmax ] Vmax =

K M V0 + V0 [S]

Therefore, V0 = − K M

V0 + Vmax [S]

This is also a straight line graph from which Vmax and KM can be determined. Vmax V0

Slope = _KM

V0 / [S]

Fig 8.4

Vmax / KM

90   Enzymes

The Hans plot also starts from the Lineweaver–Burk equation. But the whole equation was multiplied by [S] K 1 1 1 [S] M = [S] + [S] V0 Vmax [S] Vmax Therefore, K [S] 1 = [S] + M V0 Vmax Vmax

This also gives a linear plot, from which Vmax and Km can be obtained. [S] / V0

_ Slope = KM

KM / Vmax [S]

Fig 8.5

KInetIcs of MultIsuBstrate enzyMe-catalyzed reactIons Most biochemical reactions involve atleast two substrates, so it is necessary to consider the kinetics of such reactions. We can consider a specific example of two-substrate two-product (bi-bi) reactions. They are often transfer reactions of one type or another (including oxidation / reduction reactions) and can best be represented as, AX + B BX + A. The reaction mechanism may be a sequential one, where both substrates bind to the enzyme to form a teritary complex before the first product is formed, or it may be non-sequential.

 91

Enzyme Kinetics

ping-pong Bi-Bi Mechanism An example of a non-sequential mechanism is the ping-pong bi-bi or double displacement mechanism. E.AX EX.A EX + A AX + E EX + B EX.B E.BX E + BX AX first binds to the enzyme E, forming a binary complex, EAX (X is usually a small group and does not participate in the reaction as a free molecule. So it is not regarded as a separate reaction). An intramolecular reorganization takes place, where the bond E–X is formed and X–A bond being broken. The first product A, then leaves before the second substrate arrives. B cannot bind to the enzyme E but can bind to the modified enzyme EX. Since only one substrate is present on the enzyme at any one time, there may only be a single binding site. Another intramolecular rearrangement takes place. The bond B-X is formed and the bond E–X is broken. The second product, BX is then liberated, leaving the enzyme in its original form. AX

A

B

BX

E

E E.AX

EX.A

EX

EX.B

E.BX

Fig 8.6

random-order Mechanism Random-order mechanism is the one in which any substrate binds first to the enzyme and any product leaves first. It is a sequential mechanism and a two-substrate reaction involves the formation of a ternary complex (the one involving enzyme and both substrates)

92   Enzymes E + AX

E.AX

_BX

+B E.AX.B

E+B

EB

EA

E+A

E.A.BX _A

+ AX

E.BX

E + BX

There are two separate binding sites on the enzymes, one for A/AX and one for B/BX. A

X

A

B

X B

Fig 8.7

Compulsory-Order Mechanism A compulsory-order (or simply ordered) mechanism is a sequential mechanism where the order of binding to and leaving the enzyme is compulsory. For a two-substrate reaction, a ternary complex is involved. The precise order must be specified. - BX

+B E + AX

E.AX

E.AX.B (or)

+ AX E+B

EB

E.A.BX

E.AX.B

E.BX.A

-A

EA

E.BX

E+A

E + BX

The enzyme has a binding site for A/AX and a separate one for B/BX.

Steady-State Kinetics Many two-substrate enzyme-catalyzed reactions obey the MichaelisMenten equation with respect to one substrate at constant concentration of the other substrate. This applies both the reactions catalyzed by enzymes with just one binding site per substrate and to those with several binding sites per substrate, provided there is no interaction between the binding sites.

Enzyme Kinetics

 93

Alberty (1953) derived the general equation: V0 =

(Vmax [AX 0 ][B0 ]) B

(K M [AX 0 ] + K M AX [B0 ] +[AX 0 ][B0 ] + KSAX K M B )

where, Vmax is the maximum possible V0 when AX and B are both saturating. KMAX is the concentration of AX which gives ½ Vmax when B is saturating. KMB is the concentration of B which gives ½ Vmax when AX is saturating. EAX. KSAX is the dissociation constant for E + AX The total enzyme concentration is constant and much smaller than the concentrations of the two substrates. At very large [B] the general equation simplifies to V0 = =

Vmax (1+ K M AX / [AX 0 ]) (Vmax [AX 0 ]) ([AX 0 ] + K M AX )

This is the Michaelis–Menten equation. Similarly at very large [AX0] V0 = =

Vmax (1+ K M B / [B0 ]) (Vmax [B0 ]) ([B0 ] + K M B )

At constant but not saturating [B0], the general equation can be rearranged to give: V0 =

Vmax K1 p [AX 0 ] ([AX 0 ] + K 2 )

94   Enzymes

(which is in the form of the Michaelis–Menten equation) where, K1 =

[B0 ] (K M B +[B0 ])

and K 2 =

(K SAX K M B + K M AX [B0 ]) (K M B +[B0 ])

At constant but not saturating [AX0], a similar expression can be obtained, also of the form of the Michaelis–Menten equation. The reason for the mixed KMAX KMB term (rather than KSB KMAX) can be seen if we consider a compulsory-order mechanism of the form: E.AX EX.AB products E + AX As [B0] tends to be zero, there will be very little formation of EXB from EAX, so E.AX will be very close to equilibrium and KMAX E + AX AX tends to Ks This is consistent with the Alberty equation for a constant but for a very low concentration of B, K1 [B0] / KMB and K2 KSAX (KMB ) / KMB V0 =

Vmax [B0 ][AX 0 ] (K M B ([AX 0 ] + KSAX ))

An expression involving KSAX but not KMAX . For a compulsory-order mechanism, where B binds first to the enzyme, the KSAX KMB term would be replaced in the general equation by KSB KMAX. For a random-order mechanism either term could be used, and would be justified on grounds similar to the above. Many random and compulsory-order reactions involving ternary complexes obey the general rate equation of Alberty, particularly where the rate-limiting step is the inter conversion of the ternary complexes. E.A.BX, the main chemical reaction taking place, (E.AX.B involving the breakage of the A–X bond and the formation of the B–X bond in the forward direction).

Enzyme Kinetics

 95

(A similar observation was made for single-substrate reaction obeying the MM equation, if ES EP was the rate limiting step.) A random-order mechanism, where all the steps except the inter-conversion of the ternary complexes are rapid is said to have a random order rapid equilibrium bi-bi mechanisms. For a ping-pong bi-bi mechanism, the liberation of A from the enzyme in the initial period of the reaction will be irreversible because the concentration of the product A present will be negligible. Hence KS AX = 0 and KS AX.KM B = 0 giving this mechanism a simpler rate equation. V0 =

(Vmax [AX 0 ][B0 ]) (K M B[AX 0 ] + K M AX[B0 ] + [AX 0 ][B0 ])

Plots For Mechanisms Which Follow the General  Rate Equation Two-substrate reactions obeying the general rate equation of Alberty also obeys the Michaelis–Menten equation with respect to one substrate, provided the concentration of the other substrate is maintained fixed. (1 + (KMB / B0)) (1 / Vmax)

Slope = ((KMAX + KSAx.KMB) / [B0]) (1/ Vmax) 1/V0

1/[AX0]

Fig 8.8 (a) B0 is constant and

KMB

96   Enzymes

1 / Vmax 1/V0 Slope = KMAx / Vmax) 1 / AX0

Fig 8.8 (b) B0 is saturating that is pseudo single substrate with regard to AX (1 + (KMAX / [AX0])) (1 / Vmax)

1/V0

Slope = (KMB + (KSAX.KMB / [AX0])) (1/Vmax)

1/[B0]

Fig 8.8 (c) [AX0] is constant and

KMAX

1 / Vmax

1/V0

Slope = KMB / Vmax

1/[B0]

Fig 8.8 (d) [AX0] is saturating that is pseudo single substrate with respect to B

The values of the various constants may be determined by means of secondary plots.

Enzyme Kinetics

 97

E.g. If primary plots of 1/V0 against 1/(AX0) are drawn for a series of values of [B0]then a secondary plot of intercept (on the 1/V0 axis) against 1/[B0], will have an intercept of 1/Vmax and slope of KMB/Vmax, enabling both of these constants to be determined. Furthermore, a secondary plot of primary plot slope against 1 / [B0] will have an intercept of KMAX / Vmax and a slope of KSAX. KMB / Vmax, enabling the values of KMAX and KSAX to be determined.

allosterIc enzyMes Monod–Wyman Changeux (MWC) Model The MWC model is sometimes called as symmetrical model because it is based on the assumption that in a particular protein molecule, all the protomers must be in the same conformational state, that is 1. All in R-form (or) 2. All in the T-form and no hybrids are found because of unfavourable interactions between sub units in different conformational states. The two conformational forms of the protein are in equilibrium in the absence of ligand and the equilibrium is disturbed by the binding of the ligand. Consider a dimeric protein having two identical binding sites for a substrate or ligand (S). In the absence of ligand, there will be equilibrium between the two T2) and the equilibrium conformational forms of the dimer (R2 constant is termed as the allosteric constant and represented by the symbol L. The hybrid RT is found to be unstable and ignored. The ligand can bind to either of the sites on the R2 molecule, each having an intrinsic dissociation constant KR. In the simplest form of hypothesis, it is assumed that S does not bind to T to any appreciable extent. Therefore, the processes to be considered are T2 (equilibrium constant L) R2 R2S (intrinsic dissociation constant KR) R2 + S R2S2 (intrinsic dissociation constant KR) R 2S + S

98   Enzymes

It is diagrammatically represented as: S or T2

S

S R2S2

R2 R2S

Fig 8.9

Consider the binding of a ligand to a dimeric protein (with extra complication of two conformational forms). Assume that the binding of one molecule of S to R2 does not alter the affinity of the other binding site for S. Therefore, The concentration of the bound sub units present = [R2S] + 2[R2S2]. The total subunits present = 2[R2] + 2[R2S] + 2[R2S2] + 2[T2] Therefore, Fractional saturation, Y= =

[R 2S] + 2[R 2S2 ] 2([R 2 ]+[R 2S]+[R 2S2 ]+[T2 ]) [R 2S] + 2[R 2S2 ] 2([R 2 ]+[R 2S]+[R 2S2 ]+ L[R 2 ])

For the first step of binding process R2 + S binding constant is K b1 =

R2S, the apparent

[R 2S] [R 2 ][S]

Therefore, [R2S] =Kb1[R2][S] Since there are two unbound sites which may be filled in the forward reaction but only one ligand to dissociate in the reverse reaction. Kb1 = 2X intrinsic binding constant = 2 / intrinsic dissociation constant = 2 / KR Substituting for Kb1 in the expression [R2S] above [R2S]=2 [R2][S]/KR

Enzyme Kinetics

For the second step of the binding processes. R2S + S

 99

R2S2,

the apparent binding constant is K b2 =

[R 2S2 ] [R 2S][S]

Therefore, [R2S2] = Kb2[R2S][S] = Kb1Kb2[R2][S]2 Since there is only one unbound site, which may be filled in the forward reaction but two bound ligand molecules to dissociate in the reverse reaction. Kb2 = (1/2 ) X intrinsic binding constant = 1/(2 X intrinsic dissociation constant) = 1/2KR Substituting for Kb1 and Kb2 in the expression for [R2S2] above, we get, [R2S2] = (2/KR ) X (1/2KR)[R2][S]2 = [R2][S]2 / [KR]2 Substituting for R2S and R2S2 in the expression for Y above, we get, Y= =

(2[R 2 ][S]/K R + 2[R 2 ][S]2 /[K R ]2 ) (2[R 2 ] +2[R 2 ][S]/K R + [R 2 ][S]2 / (K R ) 2 + L[R 2 ]) ([S]/K R (1+([S]/K R ))) (L+(1+[S]/K R ) 2 )

This is the Monod-Wyman-Changeux equation for a dimeric protein. If a protein consists of N protomers each with a binding site for the substrate or ligand (S), the MWC equation is Y = ((S/KR) (1+ [S] / KR)n-1) / (L + (1 + [S] / KR)n)

According to this equation: 1. The greater value of L, the more sigmoidal is the plot of Y against [S]. 2. If L = 0, a hyperbolic curve is obtained.

MWC Model and Allosteric Regulation MWC model was introduced to explain the phenomenon of allosteric inhibition and activation.

100   Enzymes

In 1956, Umbarger found that isoleucine inhibits threonine dehydratase, an enzyme involved in its biosynthesis in bacteria. In 1963, Monod, Changeux and Jacob put forward the allosteric theory of regulation and stated that they are naturally occurring metabolic regulators called effectors and modifiers and do not resemble the substrate in structure and bind to the enzyme at a separate site and affect the binding of the substrate by heterotropic cooperativity. The word “allosteric” was used to stress the difference in shape between regulator and substrate (“allo” means “others”) and has been used loosely to describe any kind of cooperative effect such as homotropic or heterotropic.. 1. According to the MWC model, allosteric inhibitors bind to the T-form of the enzyme, stabilizing it and increases the value of L. 2. Allosteric activators have the opposite effect, binding to and stabilizing R-form and decreasing the value of L. In either cases, the binding of the modifier to one of the forms of enzyme will disturb the R/T equilibrium and show some degree of sigmoidal character in the absence of substrate. Enzymes subject to allosteric control fall into two categories: 1. K-series 2. V-series. 1. K- series enzymes + Allosteric activator Y In the absence of modifiers + Allosteric inhibitor

[S]

Fig 8.12

Effect of Allosteric Activators and Inhibitors of the Binding of a Substrate to a K-series Enzyme (At fixed concentration of modifier and enzyme).

Enzyme Kinetics

 101

K-series enzymes are those where the presence of the modifier changes the binding characteristics of the enzyme for the substrate but does not affect the Vmax of the reaction. KM does not have meaning in the case of allosteric, but a more appropriate term is S0.5 that is, the ligand concentration. required to produce 50% saturation of a protein. For a K-series enzyme (S0.5) substrate that is the substrate concentration required to half-saturate the enzyme varies with the concentration of modifier The MWC model preferentially binds to R-form giving a sigmoidal binding curve. Allosteric inhibitors, by increasing the value of L, increase the sigmoidal nature of the binding curve for substrate, thus they decrease the fractional saturation of an enzyme with its substrate at low and moderate substrate concentration decreasing the value of V0. Allosteric activators tend to increase the hyperbolic nature of the substrate binding curve. In each case, the degree of allosteric effect depends on the concentration of the modifier but the value of Vmax is not affected. V-series enzymes are those where the presence of a modifier results in a change in the Vmax but not in the value of the apparent KM (or S0.5) for the substrate, the binding curve for the substrate at constant modifier concentration is a rectangular hyperbola, but the binding curve for the modifier is sigmoidal. If the substrate can bind equally well to the R and T form, the reaction catalyzed by the R-form is faster than the T-form. V-series enzymes are much less common than K-series enzymes. E.g. Fructose 1-6 bisphosphatase for which AMP is an allosteric inhibitor and pyruvate carboxylase activated by acetyl-CoA. In this case, allosteric modifiers affect both Vmax and apparent KM values.

102   Enzymes

QuestIons 1. Define enzyme kinetics. 2. List out the advantages of enzyme kinetic study. 3. Write down the classification of chemical reactions with reference to kinetics. 4. Explain the first type. 5. Write short note on the second type. 6. How do theorell chance mechanism works. 7. Explain ping pong mechanism. 8. Define KM. 9. Explain the assumption of equilibrium. 10. Draw and explain the steady state assumption with a graph. 11. Define steady state assumption. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Define Vmax. Write down the significance of Michaelis constant. Define Kcat. Define turn over number. Derive the MM equation with suitable diagram. Explain the LB plot. How do LB plot differ from MM equation. What assumption was made in EH plot. Explain the Hans plot with suitable derivation.

CHAPTER

9 Enzyme Inhibition

Inhibitors are substances, which tend to decrease the rate of an enzymecatalyzed reaction. There are two types of enzyme inhibition, namely, Reversible and Irreversible inhibition. Reversible inhibitors bind to an enzyme in a reversible fashion and can be removed by dialysis (or simply by diffusion) to restore full enzymatic activity. Irreversible inhibitors cannot be removed from an enzyme by dialysis.

reVersIBle InHIBItIon Reversible inhibitors bind to an enzyme in a reversible fashion and can be dissociated and removed by dialysis. The different types of reversible inhibitions are: 1. Competitive inhibition 2. Uncompetitive inhibition 3. Non-competitive inhibition 4. Partial inhibition 5. Substrate inhibition 6. Allosteric inhibition

Competitive Inhibition The inhibitors closely resemble the substrates whose reactions they inhibit and because of their structural similarity, they may compete for the same binding site on the enzyme. Therefore, the enzymebound inhibitor. Lacks the appropriate reactive group or it may be held in an unsuitable position with respect to the catalytic site of the enzyme or to other potential substrates for a reaction to take place.

104   Enzymes

In all the above cases, a dead-end complex is formed. Therefore, the inhibitor must dissociate from the enzyme and it must be replaced by a substrate for the reaction to take place. e.g. Malonate is a competitive inhibitor of the reaction catalyzed by succinate dehydrogenase. CH2COOH

COOH

CH2COOH

CH2

Succinic acid

COOH

Malonate has two carboxyl groups Malonic acid like the substrate succinate. This can fill the succinate-binding site on the enzyme. However, the subsequent reaction involves the formation of double bond and therefore, it cannot react. The effect of competitive inhibitor depends on the following factors: 1. Inhibitor concentration 2. Substrate concentration 3. The relative affinities of the substrates and the inhibitor for the enzymes. If steady state kinetics is studied for a simple single substrate singlebinding site, it gives single intermediate enzyme catalyzed reaction in the presence of a competitive inhibitor I. ES P+E E+S EI E+I The dissociation constant for the reaction between E and I is KI Where, KI =

[E][I] [EI]

KI is called the inhibitor constant.

Enzyme Inhibition

+

�

� S

E

+

ES

+

 105

P

E

� I EI

E

(a) Competitive inhibition, I binding to site S

�

+ S

Ε

�

+ P

E

ES

�

+ I E

EI

(b) Competitive inhibition, I and S binding to different sites +

+ E

� �

S

� ES

+ P

E

+1

ESI

(c) Uncompetitive inhibition

+S +

�

� -S ES

�

�

E

+1

E

+1

+S

� -S EI

ESI

Fig 9.1 Competitive and uncompetitive Inhibition

P

106   Enzymes

Uncompetitive Inhibition Uncompetitive inhibitors bind only to the enzyme-substrate complex and not to the free enzyme. Substrate binding could cause a conformational change to take place in the enzyme and results in inhibitor binding site or the inhibitor could bind directly to the enzyme-bound substrate. In this case, KM and Vmax are altered, but a distinctive kinetic pattern emerges under a steady state condition. Let us consider the equation, ES + I ƒ ESI ESI is the dead-end complex. The inhibitor constant KI = ([ES] [I] / [ESI]) Under steady state conditions, KM =

[E][S] [ES]

Non-competitive Inhibition and Mixed Inhibition A non-competitive inhibitor can combine with an enzyme molecule to produce a dead-end complex, regardless whether a substrate molecule is bound or not. The inhibitor destroys the catalytic activity of the enzyme either by binding to the catalytic site or as a result of a conformational change affecting the catalytic site, but does not affect the substrate binding. EI E+I ES + I ESI A mixed inhibitor binds to enzyme sites that participate in both substrate binding and catalysis. If an inhibitor binds irreversibly to an enzyme, the inhibitor is classified as an inactivator.

Partial Inhibition This is considered only in the situation where enzyme-inhibitor complexes are dead–end ones. ie. where no product can be formed from them. When we consider the general system under partial inhibition, the ESI complex could break down to yield product according to the equation.

Enzyme Inhibition

 107

K'a ESI  →E + P +

Under these conditions, the overall initial velocity is given by V0 = K 2 [ES] + K '2 [ESI] = K 2 [ES] + K '2

[ES][I] K1

 K ' [I]  = K 2 [ES]. 1 + 2  K 2 K1  

Substrate Inhibition For a given enzyme concentration, the initial reaction velocity increases with increasing initial substrate concentration to a limiting value Vmax. At still higher substrate concentration, the initial velocity is found to be less than the maximum value. But in other cases, the substrate in very high concentrations really can inhibit its own conversion to product. e.g. In the reaction catalyzed by succinate dehydrogenase. Fumarate + 2H Succinate For the reaction to take place, both carboxyl groups of the substrate have to bind to the enzyme. But at high substrate concentrations, there is a increased possibility that carboxyl groups from two separate substrate molecules bind to the same enzyme. Proper reaction cannot take place unless one of them has dissociated away again.

Allosteric Inhibiton Allosteric inhibition palys a vital role in metabolic regulation. Consider a biosynthetic pathways. B C D E F A Unnecessary production of excess F may be prevented and supplies of A conserved, by feedback inhibition, where the end product Facts as an allosteric inhibitor of an early enzyme in the pathway e.g. that catalysing the reaction A B.

108   Enzymes

The term allosteric inhibition occurs where the inhibitor rather forming a dead-end complex with the enzymes influences conformational changes which alters. The binding characteristics of the enzyme for the substrate or the subsequent reaction characteristics (or both).

IrreVersIBle InHIBItIon An irreversible inhibitors binds to the active site of the enzyme by a n irreversible reaction and hence cannot dissociate from it. A covalent bond is usually formed between the inhibitor and enzymes. The inhibitor act by preventing substrate binding or it may destroy some component of the catalytic site.

QuestIons 1. 2. 3. 4. 5. 6. 7.

What is the function of inhibitors? Define enzyme inhibitior. What are the types of enzyme inhibition? Explain irreversible inhibition. What is competitive inhibition? Write short notes on uncompetitive and non-competitive inhibition. Explain allosteric inhibition.

8. Define inactivator.

CHAPTER

10 Enzyme Cofactors

Cofactor is an additional substance besides the enzyme and substrate, which is required in many cases in order that the reaction may proceed. Cofactors may take part in the intermediate steps of the reaction catalyzed by the enzyme (or the cycle of reactions catalzed by a system of enzymes). They are not consumed during the process, but are found in their original form at the end of the catalysis. They are regarded as an essential part of the catalytic mechanism.

Mode of actIon The majority of enzyme cofactors act in one of the following ways: 1. As a prosthetic group ( Intra enzymatic carrier) Many enzyme molecules contain a special chemical compound other than the protien part. e.g. Haem. Flain, a metal atom etc. The enzyme molecule contains identical subunits (1:1 molecular ratio between prosthetic group and sub units). The prosthetic group is regarded as the catalytic centre of the enzyme. In most cases, a prosthetic group acts by removing some part of a substrate molecule (the donor) and transferring it to a second substrats (the acceptor) that combines with the same enzyme molecule, thus acting as a intramolecular carrier. 2. As Inter enzymatic carrier This involves combination of the carrier with an enzyme, transfer of a part of the substrate molecule to the carrier, migration of the loaded carrier from the enzyme to another enzyme.

110   Enzymes

3. By subunit aggregation In many cases, enzyme molecules consist of several subunits, which are inactive singly but active when assembled into the complete enzyme molecule e.g. Phosphate compounds (especially nucleotides). 4. By altering the shape of the enzyme molecule An enzyme molecule is some cases, may not be in a catalytically active configuration without the cofactor. But the combination of the cofactor in some part of enzyme (other than active site) may-change the shape of the molecule and active centre is formed for catalytic action. Such configuration changer is known to be the cofactor which will not take part in catalytic processes. 5. As stabilizers Some enzymes are inherently unstable and become rapidly inactive. These unstable enzymes are stabilized by combination with substance such as a cofactor by various mechanisms. 6. As templates Enzymes like nucleic acid polymerase, which have to synthesize long, polymeric chain in which the units must occur in a definite sequence, require a molecule in which the sequence is embodied, usually a performed nucleic acid chain. The new chain is built up step by step by the enzyme on the preformed chain, which therefore acts as a template without which the enzyme will not work. 7. As primers The function of polymerizing enzymes is to lengthen chains, but usually they cannot start new chains, even when a template is provided. They require a short length of preformed chain as a prime and can then add fresh units to its end sequentially .The difference between a primer and a template is that new units are added to the end of the primer by covalent link, but to the side of the template by a non-covalent link. Since the template remains intact at the end of the synthesis, it is considered to be a cofactor.

Enzyme Cofactors

 111

8. As intermediates In rare case, enzymes may use up a molecule of the cofactor in the reaction and at the same time produce another molecule of the cofactor. e.g. In the case of phosphomutase, the substrate is converted into cofactor and cofactors simutaneously get converted into product.

cofactors actIng as carrIers redox carriers A very important group of cofactors consists of substances which are reduced by one substance and oxidized by another (They act as carriers of reducing equivalents from one molecule to another). E1 AH2 + C CH2 + B

E2

A + CH2 C + BH2

Where, C is the carrier E1 and E2 are enzymes H is the reducing equivalents (The reducing equivalents are transferred as hydrogen atom or electrons. Hence the term “hydrogen carrier and electron carrier”). 1. NAD and NADP These two coenzymes, nicotinamide – adenine dinucleotide (NAD) and NAD phosphate (NADP) are closely related. The thermostable coenzyme NAD was identified by Harden and Young in 1904 and highly purified form was obtained by Van Euler et al., and Warburg and Christian in 1936, NADP was discovered by Warburg and Christian in 1931 as the coenzyme of glucose–6 phosphate dehydrogenase. These enzymes can be reversibly reduce, either by a chemical reducing agent such as dithionite (slowly) or much more rapidly by dehydrogenases. The reduced form is the same in both cases. When the coenzymes are oxidized, the UV absorption spectrum undergoes changes. The oxidized form shows only a band at 260nm due to

112   Enzymes

the puyrine and pyrimidine rings, but the reduced form shows an addition band at 340nm, which is due to quinone bond structure of the reduced nicotinamide ring. 2. Flavins and Flavoproteins The first flavoprotein enzyme was discovered by Warburg and Christian from yeast in 1932 and was resolved into a yellow flavin group (now called riboflavin phosphate or flavin mononucleotide). About 80 flavoprotein enzymes are now known. A few of these have riboflavin-5 Phosphate (Flavin mononucleotide, FMN) as prosthetic group, but the majority contain flavin-adenine dinucleotide (FAD). The flavins bind to a number of different specific proteins (apoenzymes) to give flavorpotein enzymes (generally one molecule of flavin is bound by each enzyme subunit). In other cases they are bound as definite prosthetic group or sometimes the flavin may dissociate from the protein to some extent e.g. D-aminoacid oxidase. The catalysis of redox reactions by flavoproteins is to due to the alternative oxidation and reduction of their flavin group. But FAD differs from NAD in that it completes its redox cycle while it is attached to one and the same enzyme protein, while NAD is reduced by one enzyme and oxidized by another. NAD is an interenzyme redox carrier and FAD is an intraenzyme redox carrier. The fluorescent property of flavin is very different form those of free flavin. The free flavins are strongly fluorescent in the oxidized form and non-fluorescent when reduced. Many flavaproteins do not fluorescence at all when oxidized and a very few show only a weak fluorescence. 3. Glutathione This is known to be widely distributed sulphur containing peptide, which was discovered by Hopkins in 1921. The final structure was established by Harington and Mead as s–L-glutamyl-L-cysteinylglycine by synthesis. Reduced glutathione is oxidized to disulphide by mild oxidizing agents e.g. Iodine or ferricyanide. It is also oxidized by molecular oxygen under suitable conditions in the presence of traces of catalytic metals and by cytochrome C. It is oxidized enzymatically by dehydro

Enzyme Cofactors

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ascorbate in the presence of glutathione dehydrogenase. Powerful reducing agents are needed to reduce the oxidized form back. e.g. NADH or NADHP in the presence of glutathione reductase. Since glutathione undergoes enzymatic oxidation and reduction, it acts as a biological hydrogen carrier. 4. Cytochromes The name “cytochrome” was given by Keilin in 1925 to a group of intacellular haemoproteins, which in the reduced form show a marked absoption spectrum in the visible region. Four banded visible absorption was originally observed by MacMunn in 1886. The cytochromes fall into four groups, differing in the nature of the haem prosthetic group. The four haem types a, b, c and d are characterized by the side chains of their porphyrins. Cytochrome a: The haem group contains a formyl side chain Cytochrome b: Protohaem has prosthetic group, not covalently bound to the proteins. Cytochrome c: Has covalent linkages between the haem side chains and the protein. Cytochrome d: The haem group contains a dihydro-porphyrin (chlorin). The absorption spectrum of a typical single cytochrome in the reduced form shows three main bands in the visible regions in the decreasing order wavelength (α, β and λ-bands). By far the strongest is the λ band, α band and β band . The cytochrome a, shows little or no β Band. On oxidation, the α and β bands of the reduced form disappear and are replaced by a diffuse absorption and λ Band remains and shifts towards UV region.

phosphate carriers The second largest class consists of the enzymes which transfer phosphate groups by way of the phosphate carrier, which transfer substituted phosphate group including nucleotidyl group. 1. ADP The commonest phosphate carrier is ADP which takes up a phosphate group to become ATP and give it up again to an appropriate acceptor.

114   Enzymes

Of the 132 enzymes concerned with transfer of phosphate group, not less than 114 are specific for ADP and they are named as “kinases”. The bond formed in converting ADP into ATP is a pyrophospate link, which has a higher free energy of hydrolysis than an ordinary sugar phosphate link and therefore called as “ high energy bond”. ATP bond energy is the driving force for many biological processes such as protein synthesis. Therefore, ATP may be regarded as an energy carrier as well as phosphate carrier. 2. Phosphomutase coenzymes It differs from all others, in that the coenzyme is converted into a product and an equivalent amount of substrate, converted into coenzyme by the same transfer of a single phosphate residue. In all cases, if the phosphate is transferred from position a to position b, the coenzyme is the a, b-bisphosphate. In one case, an a, b-bisphosphate is convertede into a, b, c-bisphosphate, the coenzyme being b-monophosphate.

co2 carrier Biotin is known as an essential vitamin and the enzyme reaction involves incorporation or transfer of CO2. The mode of action of biotin was studied by Lynen et al., using methyl crotonoyl-CoA carboxylase. During the reaction, CO2 was taken up to form the 1-Ncarboxy derivative and a methyl derivative after methylation and enzyme hydrolysis of the enzyme-biotin-CO2 complex was isolated.

Amino Group Carriers Pyridoxal phosphate This forms the prosthetic group of large number of enzymes which catalyze reactions of several kinds involving amino acids, apart from the formation or hydrolysis of peptide bonds. In many cases, the transamination reactions act as an amino carrier. The cofactor was first purified by Gale and Epps, and Braunstein and Kritzman in 1943 as coenzymes of lysine decarboxylase and aspartate pyruvate transaminase, respectively. The structure was estabilshed by Baddiley and Mathias in 1952.

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 115

The combination of pyridoxal phosphate (called PLP) with apoenzyme is rather a complex process and in some case, they form tetramer with aggregation of the enzyme subunits. In another case, PLP forms a Schiff’s base with a lysine side chain in the protein, especially the aldehyde, phosphate and nitrogen, bind to groups in the protein. The affinity varies greatly from one enzyme to another. The Schiff’s base undergoes tautomerism and the fate of the compound depends on the nature of the enzyme protein to which the PLP is attached and on the group R.

Acyl Group Carriers The study of intermediary metabolism of fats have emphasized the importance of acyl-group transfer, particularly by carrier containing thiol groups with which they form thioesters. These energy rich compounds share their energy with phosphate rich compounds (during biological energy transfer). Coenzyme A Lipmann discovered the coenzyme A from microorganisms in 1947. The structrure of coenzyme A is 3-phospho-ADP-Pantoyl-B-alanylcysteamine, which has similarity to an adenine dinucleotide in which the second nucleoside is replaced by pantetheine. (that is pantothenylcysteamine). Coenzyme A is colorless substance having an absorption band in the UV at 257 NM due to the adenine residue. Coenzyme A acts as an acyl carrier in the transfer of acyl group from one molecule to another. In some cases, the acyl group transferred to the coenzyme A is removed from the substrate by a lyase type reaction leaving a double bond (of the 31 lyases group, 16 has coenzyme A as acyl acceptor).

Carrier of One Carbon Group In the synthesis and breakdown of certain amino acids and purines, single carbon atoms are transferred in the form of hydroxymethyl, formyl and formimino groups as well as methyl groups. Tetra hydrofolate Folic acid derivative is important in one carbon transfer reaction. According to Blakley, the active carrier is not folic acid itself, but

116   Enzymes

the pteridine nucleus is reduced to a tetrahydro form. This reduction occurs enzymatically in crude enzyme preparations. Tetrahydrofolate is a colourless compound and has a characterstic absorption sepctrum in the UV. It is highly autoxidizable by O2 and gets converted into dihydrofolate and oxidized enzymatically by NADP+. These act as a carrier of methyl, hydroxymethyl, formyl or formimino groups. Tetrahydrofolate differs from other carriers in that the group transported may undergo a change while attached to the carrier, so that the group which is given up to the acceptor is not identical with that which was taken up in the first place. It may be accepted as formyl group and bound on to a hydroxy methyl group.

Sulphate Carrier Adenosine 3' – 5 Bisphosphate This catalyzes the transfer of sulphate groups. These reactions involve the sulphate carrier adenosine 3' – 5 bisphosphate which takes up a sulphate group to form 3' phosphaodenly sulphate. Phosphoadenyly-sulphate can also be formed from sulphate by way of adenylyl sulphate. The transferase transfers an AMP group to sulphate from ATP leaving phyrophosphate. In either cases, adenylysulphate is formed and this is in turn phosphorylated by another ATP molecule with kinase to form 3'-phosphoadenyl sulphate.

Aldehyde Carrier Thiamine diphosphate This was first recognised as cofactor by Lohmann and Schuster in 1937 from yeast. The chemical nature showed it to be the prosthetic group of pyruvate decarboxlase. In addition to the decarboxylase system, this is involved in the pyruvate oxidase system and forms acetoin. In all the reactions involving pyruvate, decarboxylation produces and active acetaldehyde residue which becomes attached to the cofactor.

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The point of attachment is the reactive C atom between the S and N of thiazole ring. The enzyme first causes the attachment and undergoes rapid decarboxylation giving hydroxyethyl-thiamine diphosphate. The fate of this compound depends on the enzyme involved and the decarboxylase brings about the direct breakdown to free acetaldehyde. e.g. The pyruvate dehydrogenase in a oxidase system brings its oxidation by lipoate to an acetyl derivative and transfer of acetyl group to the reducing lipoate.

QuestIons 1. Define enzyme cofactors. 2. Write a brief account on the mode of action of enzyme cofactors. 3. Write a short note on redox carrier. 4. Explain the mode of action of flavin and flavoprotein. 5. What are the different types of cytochromes and their functions. 6. Write short notes on suphate carriers. 7. Give an example for CO2 carrier. 8. Explain the mode of action of coenzyme A. 9. Write down the function of tetrahydrofolate as carbon carrier. 10. Describe the action of thiamine diphosphate as aldehyde carrier.

CHAPTER

11 Enzyme Biology

IntroductIon Living matter dependent upon a number of unstable substances which is necessary for break down or get replenished by synthesis for continuous supply of energy keeps the system in a steady state. Even for the protein (stable substance) breakdown under intracellular conditions autolytic enzymes, need energy for their replenishments. If the energy supply is cut off, the whole system runs down by breakdown of the ustatble essential substances and by the predominance of catabolic enzyme processes leading to autolysis of the cell. A living cell is certainly a “bag of enzymes” in a homogeneous solution (a system of many enzymes). Certain enzymes are specifically situated in particular intracellular structures and enzyme system. A typical cell is bounded by a cell membrane, which is permeable only to certain substances and determines the shape of the cell but takes no part in the cell metabolism. The cell contents are divided into cytoplasm and nucleus. The cytoplasm is not homogeneous, but contain particulate structures of various kinds, such as mitochondria, lysosomes, peroxisomes, ribosomes, chloroplasts, golgi apparatus, microtubules, centrosomes, microfibrils, basal granules of cilia or flagellae. Phagocytic inclusions, fat droplets and granules of various metabolic products such as glycogen, starch, sulphur, etc. The structure of the nucleus is very different in dividing and non-dividing cells. In the non-dividing cell, it is surrounded by a double membrane with numerous pores, usually arranged in a regular

120   Enzymes

pattern and apparently closed by a very thin diaphragm or with other material. The nuclear membrane completely disappears during cell division and reforms a round each daughter nuclei.

Intracelluar localIzatIon of enzyMes The intracellular localization can be studied by two methods. 1. Micro histochemical method 2. Separation of particulate fractions from disintegrated cells. e.g. By different centrifugation The subcellular components differ in density and so centrifugation through a density gradient identifies the organelles.

HIstocHeMIcal MetHods This method is regarded as a development from the staining techniques of classical histology, since they depend up on the liberation of a staining substance in tissue sections as a result of the enzyme action, followed by microscopic examination of the stained tissue. The developed histochemical technique makes use of specific antibodies for enzymes. These antibodies are labelled with fluorescent groups for visualization by optical methods or with ferritin for detection by electron microscopy or with peroxidase for subsequent secondary histochemical assay with benzidene. The localization of the conjugated antibody indicates the site of enzyme.

separatIon of partIculate fractIon The intracellular localization of enzymes can be obtained by the preparation of particular cell structures by centrifugation. The cell suspension is subjected to high shear, which disrupts the cell membrance without destroying the integrity of the nucleus or cytoplasmic particles. The various structural components of the homogenate, sediment in the centrifuge at different rates according to the differences in their size. By repeatedly resuspending the centrifuged pellets a fairly homogeneous fraction can be obtained. In order to obtain undamaged preparations, homogenization is made in a special media. e.g. Hypertonic 0.88 M sucrose.

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In the heterogenetic materials for example “Mitochondrial fraction”, De Duve and others developed the use of density gradient centrifugation to improve resolution. For this purpose, gradients of high molecular weight polysaccharides are used. By these methods, mitochondria, lysosomes, peroxisomes were differentiated as organelles containing different enzymes. The nuclear fraction sediments at low centrifugal speed with hypotonic media. 1. Mitochondrial Enzymes

Mitrochondria was first described by Richard Altmann in 1890 who called them as “Bioblast”. The present name “mitochondrion” was coined by Benda in 1897. They are present in all eukaryotic aerobic cells and can be identified by their staining affinity with Janus green B (This has a specific reaction with respiratory enzymes.) Mitochondria are rich in various types of enzymes with variety of functions. As many as 70 enzymes and coenzymes are present in them, which are engaged in metabolic functions such as Oxidative phosphorylation, Electron transport chain reactions, Krebs cycle, Oxidation of fatty acids, Ion transport, etc. The following table list the major enzymes and their location in mitochondria. Table 11.1

Location Outer membrance

Intermembrane space

Major Enzymes in Mitochondria

Enzymes NADH – Cytochrome C oxidoreductase Monoamine oxidase Cytochrome b acyl-coenzyme A synthetase Kynurenine hydroxylase Fatty acid CoA ligase Creatine kinase Adenylate kinase Nucleoside diphoshokinase

122   Enzymes

Inner membrance

Matrix

Cytochrome b, c, c1, a, a3 Succinate dehydrogenase Pyruvate oxidase Carnitine acyltransferase ATP – synthesizing enzymes Cirtate synthetase Aconitase Isocitrate dehydrogenase Fumarase Malate dehydrogenese Glutamate dehydrogenase Aspartate aminotransferase Pyrauvate dehydrogenase complex Enzymes of protein and nucleic acid synthesis α-ketoacid dehyrogenases

The mitochondrial complexes associated with the inner membrane have the following composition: Complex I : NADH-coenzyme Q reductase complex, FMN iron sulphide ubiquinone, lipids. This is the largest electron transport complex having NADH-Q reductase activity. It contains FMN (Flavin mononucleotide) as prosthetic group and six ironsulphur centres. It is situated in the inner membrane and functions in translocating H+ ions across the matrix side to the cytosol side. The complex also has the ability to transfer electrons from NADH to cytochrome Q. Complex II : Succinate-Coenzyme Q reductase complex, FAD, iron, chytochrome b, lipids. This complex is composed of two polypetide chains and contains FAD+ and three iron-sulphur centres. The enzyme succinate dehydrogenase is the principal protein and its binding site is on the matrix side. This complex does not participate in translocating hydrogen ions.

Enzyme Biology

Complex III :

Complex IV :

 123

Coenzyme Q-cytochrome reductase C complex. cytochrome b, cytochrome C, iron, ubiquinone, lipids. This complex contains 6 to 8 subunits as well as cytochromes b and c and iron-sulphur protein. In this system, reduced cytochrome Q is used as the substrate which transfers its electrons to cytochrome C. Cytochrome-C oxidase system, cytochromes a and a3, copper and iron. This is large complex of about 6 to 7 subunits consisting of a number of polypeptide chains. Its main components are cytochrome a a3, and two copper atoms. It is a terminal oxidase system, which transfers electrons to oxygen where hydrogen ions are also available to form water.

2. Enzymes of Endoplasmic Reticulum

Before the advent of electron microscope, it was thought that the plasma membrane is tightly stretched over the eukaryotic cell. But now, it is well known that the plasma membrane either folds itself outward to form microvilli or it invatginates to form vesicles leading right into the depth of the cell. The endomembrane system ramifying the cytoplasmic matrix of the cell is called endoplasmic reticulum (ER). The ER forms compartments in the cytoplasm and owing to its richness in enzymes, it is involved in secretion, synthesis, modification and transport of compounds. The membrances of ER contain enzymes for biosynthesis of cholesterol, which in turn is responsible for the synthesis of hormones and bile acids. The cytosolic β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) is converted by the microsomal enzyme HMG-CoA reductase into muvalonic acid, through a series of enzymes-catalyzed steps. Mevalonic acid is converted to farnesyl pyrophospate which is a substrate for squalene synthesis by membrane-bound enzymes. Bile acids are also synthesized by ER enzymes. Cholestrol is the precursor of estradiol, aldosterone and adrenocorticoids. Biosynthesis of these hormones is medicated through the enzymes located in the membrane of ER. The distribution and the location of enzymes of ER is listed in Table 11.2.

124   Enzymes

Table 11.2 Enzymes of Endoplasmic Reticulum

Enzyme Nucleoside diphosphatase Acetanilide hydrolyzing esterase Gluecose-6-phosphatase β-Glucoronidase Cytochrome P-450 ATPase Cytochrome b3 GDP-manosyl transferase NADH-Cytochrome 5-nucleotidase Nucleoside pyrophospatase

Location Lumen Lumen Lumen Lumen Lumen and cytoplasmic surface Cytoplasmic surface Cytoplasmic surface Cytoplasmic surface Cytoplasmic surface Cytoplasmic surface Cytoplasmic surface

3. Enzymes of Lysosomes

Among the various theories on the origin of lysosomes the most possible one is that primary lysosomes originate from the maturing face of the glogi complex. De Duve and co-workers came across dark-statining bodies rich in a number of enzymes (hydrolytic) which are able to degrade various cellular materials and named it as lysosomes (because of their lytic nature). About 40 lysosomal enzymes have been identified. The enzymes are enclosed by a membrane and are liberated on the substrate on rupturing of the membrane. The majority of enzymes show their catalytic activity in acidic pH. The following table emphasizes the different enzymes present in lysosomes.

Enzyme Biology

Table 11.3

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Major Enzymes in Lysosomes

Class of enzymes Enzymes Hydrolases acting on peptide Acrosin bonds Renin Collagenase Neutral proteinase Kininogen activator Plasminogen activator Diphenyl aminopeptidase Cathepsin A, B1 and B2, C, D and E Dipeptidase Acid carboxypeptidase Glutamate carboxypeptidase Carboxypeptidase A, B and C Hydrolases acting on C–N Amino acid napthylamidase bonds other than peptide Ceramidase bonds Aspartyl glucosylaminase Benzoyl arginine napthylamidase Dipeptide naphthylamide Hydrolases acting on Lysozyme glycosidic bonds Dextranase α-Glucosidase β-Glucosidase α-Galactosidase α-Mannosidase β-Mannosidase α-Acetylglucosaminidase Hyaluronidase β-Xylosidase β-Glucuronidase L-Iduronidase

126   Enzymes

Hydrolases acting on acid anhdride bonds Hydrolases acting on P–N bonds Hydrolases acting on S–N bonds Hydrolases acting on ester bonds

Pyrophosphatase Acylphosphatase Phosphoamidase Hepatic sulphamidases Esterase Lipase Phospholipase A1 and A2 Arylesterase Phosphoprotein phosphatase Phosphodiesterase Deoxyribonuclease II Ribonuclease II Arylsuphatase A and B Cholesterol esterase Arylsulphatase A and B Peroxidase

Oxidoreductases acting H2O2 as acceptor Transferases transferring PO4 Ribonuclease groups 4. Microbody Enzymes

Microbodies were first discovered in the cells of liver and other organs and later in protozoa, yeast and many cell types of higher plants. The microbodies exist in two forms. The first type is rich in peroxidase and catalase, D-amino acid oxidase and urate oxidase and are known as peroxisomes and the other type of microbodies are of plant cell origin, which are rich in enzymes of glyoxylate cycles called glyoxysomes. The enzymes of micorbodies are generally oxidation enzymes that act on limited substrate in a degradative manner. These enzymes appear in both peroxisomes and glyoxysomes which are catalases and variety of hydrogen peroxide producing flavin oxidases.

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Enzymes common to both types of microbodies (peroxisomes and glyoxysomes) are listed Table 11.4 Table 11.4 Enzymes of Microbodies

S.No 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Enzymes Aconitase Isocitrate lyases Glycolate oxidase Catalase Malate dehydrogenase Gluatamate oxaloacetate aminotransferase Malate synthetase Uricase Altlantoinase Amino acid oxidase Glyoxylate aminotransferase Thioase Fatty acyl CoA Synthetase Xanthine dehydrogenase Urate oxidase NADP-Isocitrate dehydrogenase Glumate-oxaloacetate aminotransferase Glycerophosphate dehydrogenase

QuestIons What are the two methods of intracellular localization of enzymes? List out the major enzymes present in mitohondria. What are the major functions of mitochondria? Explain briefly about the different mitochondrial complexes associated with the inner membrane of mitochondria. 5. Illustrate the various locations of enzymes in endoplasmic reticulum. 1. 2. 3. 4.

128   Enzymes

6. Explain the various classes of enzymes of lysosomes with examples. 7. What are microbodies and list out the enzyumes present in them.

CHAPTER

12 Enzyme Engineering

defInItIon Improvement in the activity and usefullness of an existing enzyme or creation of new enzyme activity by making suitable changes in amino acid sequence is called as enzyme engineering. If this technique is applied for protein molecules, then it is termed as protein engineering. Enzyme engineering utilizes recombinant DNA technology. DNA technology is used to transfer gene encoding useful enzymes from dangerous, unapproved, slow growing or low-producing microorganisms into safe, fast growing and highproducing microorganisms. The level of production of an enzyme may also be increased by this method. E.g. The E.coli gene encoding penicillin-G-oxidase was integrated in pBR322 and the recombinant plasmid was introduced into E.coli. Upto 50 copies per cell of the plasmids were produced, 50-fold increase was achieved.

oBJectIVes The objectives of enzyme engineering are: 1. Improvement of kinetic properties. 2. Elimination of allosteric regulation. 3. Enhanced substrate and reaction specificity. 4. Increase of thermostability. 5. Alteration in optimal pH. 6. Suitability for use in organic solvents. 7. Increase / decrease of optimal temperature.

130 

Enzymes

Other than these objectives, enzyme engineering is adopted to produce an enzyme, which may be useful for industrial or other applications.

prIncIple of enzyMe engIneerIng Since enzymes are proteins they may be determined by its aminoacid sequence, that is, its primary structure, so that any change in properties is always reflected in its primary structure. It allows an explanation of the changes in structure and function on the basis of the changes in amino acid sequence.

procedure In enzyMe engIneerIng Step 1: Isolation of the concerned enzyme and determination of its structure and properties: Both amino acid sequence and 3D structure of the enzyme are determined. They can be obtained by x-ray diffraction, magnetic resonance (NMR) etc. Step 2: The data obtained from Step 1 are analyzed with the known database. Molecular modelling is performed to determine the desired improvement in function/structure of the enzyme. Step 3: This step involves the construction of genes. Specified changes in the base sequence are introduced at a specific site of genes by site-directed mutagenesis, that is, via PCR. Step 4: If one of the appropriate gene is constructed, it is introduced and expressed in a suitable host, for example, E.coli. Step 5: The isolated recombinant or mutant enzyme is purified and used for the determination of its structure and properties.

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Isolation and cloning of the concerned gene (site-directed mutagenesis) Expression of the modified gene in a suitable host. e.g. E.coli Analysis of structure, molecular modeling, prediction of the desired amino acid sequence.

Isolation and purificantion of enzyme, determination of structure and function.

DATA BASE

Fig 12.1 Steps Involved in Enzyme Engineering

eXaMple This technique is extensively used for studies on the relationships between amino acid sequences, structures and functions of various enzymes. Many studies have also focused on modifications of industrial enzymes to enhance their usefulness and generated valuable data on sequence-structure-function relationships. An example of enzyme engineering can be illustrated with an enzyme from B. amyloliquefaciens, used in detergents. It was desired to enhance its stability at higher temperature, pH and oxidant (bleach) strength, in order to improve its activity in detergents. The P1 cleft of this enzyme holds the amino acid residue on C-terminal side of the targeted peptide bond. P1 cleft enhances the specificity of the enzyme for specific peptide bonds. So, substitution is unusually non-specific in activity. Non-specific inactivition. Substitution is inactivated by oxygen produced by bleaches (The activation is due to oxidation of the methionine residue at position 222 (Met-222)to its sulphoxide) Met-222 residue was replaced by serine or alanine.The lactate dehydrogenase from Bacillus stearothermophilus was used for enzyme engineering. When glutamine residue at position 102 (Gln-102) was replaced by arginine, the engineered enzyme became specific to malate.

132 

Enzymes

proteIn engIneerIng Only about 20 of the many thousands of enzymes that have been studied and characterized biochemically account for over 90% of the enzymes that are currently being used industrially. Table 12.1 Industrial Enzymes and their Commercial Uses

Enzymes α-amylase Aminoacylase Bromelain Catalase Cellulase Ficin Gluco amylase Glucose isomerase Glucose oxidase Invertase Lactase Lipase Papain Pectinase Protease Rennet

Industrial Use Beer making, alcohol production Preparation of L-amino acids Meat tenderizer, juice-clarification Antioxidant in prepared foods Alcohol and glucose production Meat tenderizer, juice-clarification Beer making, alcohol production Manufacture of high fructose syrups Antioxidant in prepared foods Sucrose inversion Whey utilization, lactose hydrolysis Cheese making, preparation of flavourings Meat tenderizer, juice-clarification Clarifying fruit juices, alcohol production Detergent and alcohol production Cheese making

A major reason for not using more enzymes in industrial processes is that an enzyme actively evolved under natural conditions, usually is not well suited for a highly specialized invitro function. Most enzymes are easily denatured by the exposure to conditions such as high temperature, presence of organic solvents, etc that are used in many industrial processes. However, with the availability of directed mutagenesis and gene cloning, these constraints are no longer significant.

Addition of Disulphide Bond The thermostability of a protein can be increased by creating a molecule that will not readily unfold at elevated temperatures. These

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thermostable enzymes are often resistant to denaturation by organic solvents and non-physiological conditions such as extremes of pH. The addition of disulphide bonds can significantly increase the stability of a protein. For example, in one study, six variants of the enzyme T4 lysozyme were constructed by oligonucleotide-directed mutagenesis. To introduce internal disulphide bond (Table 12.2), two, four or six amino acid residues were changed to cysteine, thereby generating one, two or three disulphide bonds, respectively. Table 12.2 Properties of T4 Lysozyme and Six Engineered Variants Enzyme

Wt Pwt A B C D E F

3

9

21

Ile Ile Cys Ile Ile Cys Ile Cys

Ile Ile Ile Cys Ile Cys Cys Cys

Thr Thr Thr Thr Cys Thr Cys Cys

Amino acid Positions No Relative Tm 54 97 142 164 of Activity (ºC) –S–S– % Cys Cys Thr Leu 0 100 41.9 Thr Ala Thr Leu 0 100 41.9 Thr Cys Thr Leu 1 96 46.7 Thr Ala Thr Cys 1 106 48.3 Thr Ala Cys Leu 1 0 52.9 Thr Cys Thr Cys 2 95 57.6 Thr Ala Cys Cys 2 0 58.9 Thr Cys Cys Cys 3 0 65.5

Wt = Wild type T4 lysozyme Pwt = Pseudo-type enzyme A-F = Six engineered cysteine variants S-S = Disulphide bonds Tm = Melting temperature (a measure of thermo stability) The newly introduced cysteines created disulphide bonds between positions 3 and 97, 9 and 164, 21 and 142 of the enzymes (the number denotes the amino acid position in the polypeptide, starting from the N-terminus). Following mutagenesis, each mutated gene was identified and expressed in E.Coli. The engineered enzymes were purified and the enzymatic activity and thermostability of each were determined.

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Enzymes

The thermostability of a protein is often defined as the temperature at which the overall structure of the protein is 50% denatured. (the state of denaturation can be assessed by monitoring the circular dichorism of the protein in solution). The wild type (native) form of the enzyme T4 lysozyme has two free cysteine residues neither of which is involved in a disulphide bond, In the so called pseudo-wild type enzyme, these cysteine residues were changed by oligonucleotide-directed mutagenesis to Thr and Ala without altering either the activity or thermostability of the enzyme. Consequently, the pseudo-wild type sequence provided a standard for comparing variants with potentially thermostabilizing disulphide bonds and also prevented spurious disulphide bonding between the mutant cysteine residues and the naturally occurring ones. The constructed lysozyme derivatives had 1-3 disulphide bonds. The result of this experiment indicates that the thermal stability of the enzyme increases as a result of the presence of disulphide bonds (more stable than wild-type or pseudo wild type).

changing asparagine to other aminoacids When proteins are exposed to high temperatures, asparagine and glutamine residues may undergo deamidation, a reaction that releases ammonia. With the loss of the amide moiety, these amino acids become aspartic acid and glutamic acid, respectively. Localized changes in the folding of the peptide chain may ensure and lead to a loss of activity. For example, a study on changing some asparagine residues in Sacchromyces cerevisiae (yeast) enzyme, triose phosphate isomerase, was examined. (Table 12.3) This enzyme consists of two identical subunits. Each subunit has two asparagine residues that may contribute to the thermosensitivity of the enzyme because they are located at the junction of the subunit interface.

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By means of oligonucleotide-directed mutagenesis, the asparagine residues at positions 14 and 78 were targeted for change. Converting either of these residues to threonine or isoleucine enhanced the thermostability of the enzyme, whereas, as predicted, changing one of the asparagine residue to asparatic acid reduced thermostability. When both asparagine residues were changed to asparatic acid residues, the resulting enzyme was unstable even at ambient temperature and had low enzymatic activity. The engineered proteins were also listed for their sensitivity to proteolytic digestion. A positive correlation was observed between thermostability and resistance to proteolysis. Table 12.3 Stability at 1000c of Yeast Enzyme, Triose Phosphate Isomerase and its Engineered Derivatives.

Enzyme Wild type Variant A Variant B Variant C Variant D

Amino Acid 14 Asn Asn Asn Thr Asp

Position 78 Asn Thr Ile Ile Asn

Half-Life (Min) or enzyme inactivation 13 17 16 25 11

Reduction of Number of Free Sulphydryl Residues Occasionally, an expressed foreign protein is less active than expected. Protein engineering can be used to increase the activity. For example, when the cDNA for human β-interferon (IFN-β) was cloned and expressed in E.coli, the protein product showed only a disappointing 10% of the antiviral activity of the authentic, glycosylated form. Although, reasonable amounts of IFN-β were synthesized, most of it were found to exist as dimers and higher oligomers, which were inactive. Analysis of the DNA sequence of the IFN-β gene revealed that the encoded protein has three cysteine residues, so one or more of these residues could be involved in the intermolecular disulphide bonding that produce the dimer and oligomers in E.coli but not in human cells. It was reasoned that conversion of one or more of the cysteine

136 

Enzymes

codons into serine codons might result in an IFN-β derivatives that would not form oligomers. Cys 138

Cys 29

Cys 98

Cys 1

Fig 12.2

Cys 31

(a) Known IFN α

Cys 141

Cys 17

Fig 12.2 (b) Deduced IFN β

Alignment of the aminoacid sequences of these two protein molecules indicated that Cys-31 and Cys-141 in IFN-β are located in the positions similar to those of Cys-29 and Cys-138 in1FNα. Because Cys-29 and Cys-138 in IFN-α are involved in the formation of an intramolecular disulfide bond, it seemed reasonable to assume that Cys-17 of IFN-β was not involved in intramolecular disulphide bonding. This deduction proved to be correct. No multimeric complexes were formed when a Ser-17 variant of IFN-β was expressed in E.coli. Moreover, the ser-17 IFN-β had a specific activity similar to that of authentic native IFN-β and more stable long-term storage than the native form.

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Increase of Enzymatic Activity In addition to stabilizing an enzyme by directed mutagenesis, it may be feasible to modify its catalytic function. For this, detailed information about the geometry of the active site of a well-characterized enzyme is required to alter enzymatic activity in a meaningful way. For example, the enzyme tyrosyl-tRNA synthetase from Bacillus Stearothermophillus has been modified with respect to substrate binding. Tyrosyl tRNA synthetase catalyzes the aminoacylation of a transfer RNA that specifically accepts tyrosine (that is, tRNA tyr) in a two-step process. Step 1: Tyr + ATP → Tyr – A + PPi Step 2: Tyr – A + tRNA Tyr → Tyr – tRNA + AMP In the first step, tyrosine (Tyr) is activated by ATP to yield the enzyme-bound tyrosyl adenylate (Tyr–A) with the concomitant formation of pyrophosphate (PPi). In the second step, the tyrosyl adenylate is hydrolyzed by the free 3, hydroxyl of the incoming tRNA molecule, so the tyrosine moiety becomes attached to the tRNA and AMP is released. Both of these reactions take place while the substrates are bound to tyrosyl-tRNA synthetase. e.g. The three-dimensional structure of tyrosyl-tRNA synthetase from B. stearothermophilus had already been determined and the active site has been mapped. With the aid of computer graphics, predictions were made about the effects of changing one or more amino acid residues of the active site on the interaction of the enzyme with the substrates. Experiments were conducted and the gene for tyrosyl-tRNA synthetase was specifically modified by oligonucleotide-directed mutagenesis. A threonine residue at position 51 (Thr-51) was replaced by either an alanine or a proline residue. In the native enzyme, the hydroxyl group of Thr-51 forms a long hydrogen bond with the ring oxygen of the ribose moiety of tyrosine adenylate. The resultant enzyme variants were characterized by determining their kinetic constants.

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For the Ala-51 variant, the binding affinity (Km) of the enzyme for ATP increased approximately two-fold without any significant change in the catalytic rate constant (Kcat). The enzyme that contained a proline in position 51 is bound to ATP 100-fold more tightly than the native enzyme. The catalytic efficiency (Kcat / Km ) of the aminoacylation reaction was increased with both of the variants. Table 12.4

Enzyme Thr-51 Ala-51 Pro-51

Aminoacylation Activity of Native (Thr-51) and Modified (Ala-51 And Pro-51) Tyrosyl-trna Synthetases.

Kcat (S-¹) 4.7 4.0 1.8

Km (mM) a Kcat / Km (S-¹ M-¹) 2.5 1.2 0.019

1,860 3,200 95,800

a = The unit of Km, the binding constant of the enzyme for ATP in millimoles / litre (mM) Kcat = The catalytic rate constant, in reciprocal seconds (S-¹).

Modification of Enzyme Specificity It has focused on modifying native properties of specialized enzymes (it is conceivable that an enzyme could be redesigned to produce an entirely unique catalytic entity). For example,the gene for the relatively non-specific enzyme staphylococcal nuclease was, specifically mutated. Then its protein product was covalently coupled to a short single-stranded oligonucleotide to create a sequence specific for single-stranded DNA nuclease. Staphylococcal nuclease is a single polypeptide of 149 amino acids. It cleaves the phosphodiester bonds of single-stranded RNA and of both single and double-stranded DNA at A,U or A,T-rich regions to generate 5, phosphate and 3, hydroxyl termini. The Lys-116 of Staphylococcal nuclease was changed to Cys116 by oligonucleotide that had been synthesized to contain a 3, sulphydryl group which was added to the modified enzyme to establish a mixed disulphide bond in which the oligo-nucleotide was covalently linked to the enzyme through Cys-116.

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However, staphylococcal nuclease does not have any other cysteine residues. There are three possible disulphide linkages that could be formed in the reaction mixture. 1. An enzyme dimer. 2. An oligonucleotide dimer. 3. An enzyme-oligonucleotide mixed disulphide. Gel-filtration chromatography was used to separate the three reaction products on the basis of size. When enzyme-oligonucleotide complexes were added to a mixture of single-stranded DNAs, only the DNA molecules that had segments complementary to the oligonucleotide attached to the enzyme are bound. After binding, the enzyme specifically cleaves the single-stranded DNA substrate adjacent to the region of base pairing.

Lys

Staphylococal nuclease

Directed mutagenesis SH Cys Single-stranded oligonucleotide SH

S S

Cys

Fig 12.3 Modification of Enzyme Specificity in Staphylococal nuclease

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secretIon of cloned proteIns The production of commercial products by genetically engineered microorganisms requires a partnership with two kinds of experts: 1. Molecular biologists, for isolating, characterizing, modifying and creating effectively expressed genes. 2. Biochemical engineers, to ensure that these microorganisms can be grown in large quantities under optimal conditions to give a product. A good growth of aerobic microorganisms is well achieved within a standard flask of 200 ml which is well mixed by a 300 watt motor. If this system is directly scaled up, a 10,000 liter container will require a mixer of 15MW motor which will be equivalent to a large house. There are parameters which has to be regulated to get the maximum yield, such as (pH, temperature, rate and nature of mixing of growing cells, O2 demand etc,. Apart from these parameters there are technical considerations such as 1. Design of the reactor. 2. Adequate sterility. 3. Accurate and continuous online monitoring of reactor.

large scale fermentation Large-scale fermentation includes the following typical procedure: Step 1: Formulation and sterilization of the growth medium. Step 2: Cells grown in stock culture (5–10 ml) Step 3: Grown in shake flask (200–1000 ml) Step 4: Transferred to seed fermenter (10–100 L) Step 5: Transferred to production fermenter (>100 L) Step 6: Harvesting

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Centrifugation or filtration Intracellular

Cells

Disruption

Product

n

Extracellular

Cell-free culture medium

Kill cells

Discard

Extraction

Kill residual cells

Product purification

Discard

Fig 12.4 Flow chart of Extraction Proceduse

Principles of Microbial Growth Microorganisms can be grown by 1. Batch culture The sterile growth medium is inoculated with appropriate micro organisms and fermentation proceeds without any further addition, under optimum conditions and the product is recovered. 2. Fed-batch culture Nutrients are added incrementally at various times during the fermentation reaction, and no growth medium is removed until the end of the process. 3. Continuous culture Fresh growth medium is added continuously during fermentation. Throughout the process, oxygen is supplied, antifoaming agent is supplied, and acid or base is injected into the bioreactor.

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Cells

Conc.

Cells

Conc.

Substrate

Fed substrate Time Fed-batch

Time Batch Cells

Conc. Growth limiting substrate

Time Continuous

Fig 12.5 Schematic Representation of Time Course for Batch, Feet-batch and Continuous Fermentation Batch Fermentation

During this fermentation, the composition of the culture medium, the concentration of micro organisms, internal chemical composition of microorganisms and the amount of target protein or metabolite, all these change as a consequence of the state of cell growth, cellular metabolism and availability of nutrients. There are six phases observed during this fermentation. (Figure 12.6). They are: 5

6

Log of cell numbers

4

3

2

Time

Fig 12.6 Growth Curve for Batch Fermentation

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1. Lag phase 2. Acceleration phase 3. Logarithmic phase or exponential phase 4. Deceleration phase 5. Stationary phase 6. Death phase 1. Lag phase Microbial cells adapt to the new environmental conditions. Cells need time for their metabolic systems to adjust to the new medium. 2. Acceleration phase The growth begins immediately. The rate of cell growth increases until it reaches the log phase growth. 3. Log phase Cell mass undergoes several cell doubling and the specific growth rate of the culture remains constant. It can be represented in a mathematical form: i. The rate of increase of cell biomass with time is dx/dt. ii. The product of specific growth rate is µ iii. The biomass concentration is X. Therefore, dx / dt = µX Similarly, The rate of increase of cell no is dN / dt The product of the specific growth rate is µ The cell no number is N Therefore, dN / dt = µN 4. Decleration phase At this stage the microorganism utilizes the substratum available and maintains its metabolic processes. The growth rate of microorganism will ne at a lower rate. 5. Stationary phase At this phase the growth rate is zero. The population of organization are still metabolically active and produces secondary metabolite which are not produced during the exponential phase.

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6. Death phase In this phase the amount of substratum will be at a very low level where the survival of microorganism is difficult therefore the organism reaches the motality phase. Fed-batch Fermentation

Substrate is added in increments at various times throughout the course of the reaction. The addition prolongs both the log and stationary phases, thereby increasing the biomass and metabolites such as antibiotics. In the stationary phase, the microorganisms produce proteolytic enzymes or proteases, which attach themselves the protein product. This fermentation needs more monitoring and they are used to a lesser extent. Due to the production of proteins from recombinant microorganisms, they are popular. This method is also used with mammalian and insect cells in culture. Merits • Used for production of human therapeutic proteins. • In the absence of fed-batch, the animal cells are not efficient in producing foreign proteins. Continuous Fermentation

In continuous fermentation, a steady state condition where dx / dt = 0 is attained, when the total number of cells and total volume in the bioreactor remain constant, or the loss of cells due to outflow (product removal) is exactly balanced by the gain of the cells by growth (division). The amount of cell biomass is potentially much lower than that of batch fermentation. Merits • Uses smaller fermentors. • After the fermentation process, large-scale equipments are needed for cell harvesting, cell breakage and subsequent downstream processing of proteins. But in continuous fermentation, it needs small equipments.

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•

Continuous fermentation avoids "downtime" that is, batch run, sterilization, repairs etc. Continuous fermentation has less downtime because a single input / dosage can be maintained for a much longer period. • The physiological state of cells during continuous fermentation is more uniform. Continuous fermentation is used for commercial production of single cell protein, antibiotics and organic solvents. Demerits • The duration may be 500–1000 hours, therefore some cells might lose the recombinant plasmid constructs. Therefore, yield may be declined with time. • Maintenance of sterile conditions for long periods is difficult. • The quality of culture medium has no assurance (which varies form batch to batch).

Bioreactors Bioreactors fall under three fundamental classes. They are: 1. Stirred tank reactors (STR): Has an internal mechanical agitation. 2. Bubble column: Introduction of air or gas for agitation (sparging). 3. Airlift reactor: It has internal or external loop. Mixing and circulation of culture in these reactors causes density differences. Motor

Gas outlet

Culture Medium Impeller

Air inlet (a) STR

(b) Bubble column

(c) Internal loop air lift reactor

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Enzymes Gas outlet

Gas liquid seperator Culture medium

Air inlet (d) External loop air lift reactor

Fig 12.7

Types of Bioreactors

Stirred Tank Reactors Merits 1. Highly flexible operating conditions. 2. Commercially available. 3. It provides efficient gas transfer to the growing microbial cells. 4. Extensively used for a variety of microorganisms. 5. STR is usually constructed in stainless steel or glass. Glass is limited to laboratory scale bioreactors (50 L). Bubble column reactors 1. Air is introduced under high pressure near the bottom. 2. Smaller bubbles become larger one as they rise in the reactor. 3. Due to pressure, air tends to cause excessive foaming in the medium. Airlift reactors 1. This can be readily adapted for either pilot plant or largescale fermentation processes. 2. Air is introduced into the bottom of a vertical channel (riser) which reach the space at the top (gas–liquid separator).

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3. The degassed liquid which is denser than gassed liquid descends in a vertical channel (down corner) and flows along the base and reaches the bottom of the riser. 4. The medium is circulated continuously. 5. There are two types of airlift reactors: • Internal loop • External loop (This can be modified according to the requirement). 6. Airlift bioreactors are more efficient than bubble columns for more denser or more viscous suspension of microorganisms. 7. The largest airlift reactor is 1,500,000 L fermentor, built by ICI in England for the production of SCP.

Harvesting of Microbial Cells The steps involved in the purification of a product are: 1. Separation of cells from the culture medium. For large volumes, either high-speed centrifugation or membrane microfiltration is used to separate cells from the growth medium. 2. High-speed semi continuous centrifuge is used to harvest microbial cells. 3. The cell suspension is continuously fed into a running centrifuge. 4. The cells are concentrated within it, while the clarified medium is collected externally in a container. 5. When the centrifuge chamber is fully packed with the cells, the run is stopped and the cells are removed. Membrane Filtration

This is an alternative method of separation of cells from the medium. 1. Dead end filtration • The microbial cells accumulate on the surface of the polymeric membrane filter.

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•

Therefore, the flow rate of spent medium through the membrane decreases rapidly. • The average pore size of membrane is 0.2–0.45 μ. 2. Cross flow filteration Cell Suspension

Cells

Membrane

Cell-free culture medium

Fig 12.8

• • • •

Dead End Filteration

The cell suspension is passed at a high speed across the surface of the membrane. In this only a small fraction of liquid passes in one pass. The remaining cell suspension sweeps out the membrane clean of accumulated cells. So there won’t be decrease in liquid flow through the membrane.

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The average pore size is 0.2 to–0.45 μ

Cells

Cell suspension

Membrane Cell concentration

Cell-free culture medium

Fig 12.9

Cross Flow Filteration

The next step depends on the nature and location of the product. • If the target protein purified is very concentrated then column chromatography is employed. • If the product is of low-molecular weight, then it is subjected to extraction procedure. • If the product is in cellular fraction, the cells must be disrupted (lysed).

Disruption of Microbial Cells The cell wall of micro organisms is composed of different polymers: 1. Gram-positive bacteria consist of thick peptidoglycan of N-acetyl glucosamine and N-acetyl muramic acid, which is cross-linked by oligopeptides. 2. Gram-negative bacteria have an outer membrane, a thin peptidoglycan layer and a cytoplasmic membrane.

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3. Yeast cell has a thick layer of phosphorylated mannans and beta glycan. 4. The lower fungi have multilayered cell wall consisting of alpha-beta-glycan, glycol proteins and chitin. Chemical methods of disruption

It involves treatment with alkali, organic solvents or detergents. i. If the protein product is stable at pH 10.5–12.5, bacterial cell lysis can easily be carried out at low cost. e.g. Recombinant human growth hormone released from E.coli is carried out in NaOH at pH 11. ii. Treatment with organic solvent is inexpensive and used for the isolation of enzymes from yeast. iii. Detergents permeablize the bacterial cells resulting in holes through which the proteins and other molecules are released from the cells. However, detergents are expensive and sometimes, they retain the contaminants even after purification. Biological methods of disruption

Enzymatic lysis Gram-positive bacteria are readily hydrolyzed by the enzyme lysozyme (isolated from egg whites). Gram-negative bacteria are readily hydrolyzed by lysozyme and EDTA. Yeast can be lysed with one or more enzymes such as beta-1,3 glucanase, beta-1,6 glucanase, mannanase, chitinase, etc. This method is highly specific and the conditions are mild. Non-mechanical methods of disruption

The non-mechanical methods include: • Osmotic shock • Repeated cycles of freezing • Thawing

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Mechanical methods of disruption

Sonication, wet milling, high pressure homogenization and impingement are some of the mechanical methods. Mechanical methods are highly efficient. i. Sonicator Generates high pressure sound waves which causes cell disruption by shear and cavitation (production of internal holes). ii. Wet milling Used to disrupt large quantity of cell suspensions. Cell suspension is Pumped into the chamber with high-speed agitator bead mill, which is filled with inert abrasive materials such as small glass beads (4 mm in diameter) and fitted with a central shaft, which has attached blades. iii. High pressure homogenization: Concentrated cells are pumped into a valve assembly under high pressure and then the pressure is rapidly decreased. This causes the cell lysis. iv. Impingement This involves a high velocity stream of suspended cells under pressure that hits either a stationary surface or a second fluid stream of suspended cells. The force created at the point of contact disrupts the cells.

Downstream Processing After cell disruption, the cell debris are removed by 1. Low speed centrifugation 2. High capacity centrifugation 3. Membrane microfiltration The protein product is precipitated with organic solvents (alcohol or acetone) or ammonium sulphate. Likewise, the target protein is enriched 2 or 5-fold. The crude protein mixtures are concentrated and fractionated by crossflow ultrafiltration through membranes of smaller pore size. The required degree of purity depends on usage. Usually, the crude preparation is satisfactory.

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QuestIons 1. Define enzyme engineering. 2. Define protein engineering. 3. Explain the technique used behind enzyme engineering with an example. 4. List out the objectives of enzyme engineering. 5. What are the typical procedure followed in large scale fermentation 6. Discuss about the principles of microbial growth. 7. Explain the various phase of microbial growth. 8. Write an essay on fed batch fermentation. 9. Discuss about continuous fermentation. 10. Write an essay on the types of bioreactors. 11. Write short notes on airlift reactor. 12. What are the methods of harvesting microbial cells? 13. Write short notes on cross flow filteration. 14. Write essay on the disruption of microbial cells.

13

CHAPTER

Immobilization of Enzyme Many enzymes secreted by microorganisms are available on a large scale and there is no effect on their cost, if they are used only once in a process. In addition, many more enzymes are such that they affect the cost and could not be economical, if not reused. Therefore, reuse of enzymes led to the development of immobilized techniques. It involves the conversion of water soluble enzyme protein into a solid form of catalyst by several methods. Thus, immobilization is “The imprisonment of an enzyme in a distinct phase that allows exchange with, but is separated from the bulk phase in which the substrate, effector or inhibitor molecules are dispersed and monitored”(Treven,1980). (Imprisonment refers to the arresting of the enzyme by polymer matrix). The first commercial application of immobilized enzyme technology was realized in 1969 in Japan with the use of Aspergillus orzyae amino acylase for the industrial production of L-amino acids. Consequently, pilot plant processes were introduced for 6-amino pencillanic acid (6APA) product from penicillin G and for glucose to fructose conversion by immobilized glucose isomerase.

adVantages The advantages of enzymes immobilization are: 1. Reuse 2. Continuous use 3. Less labour intensive 4. Saving in capital cost

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5. 6. 7. 8. 9.

Enzymes

Minimum reaction time Less chance of contamination in the products More stability Improved process control High enzyme : substrate ratio.

MetHods of enzyMe IMMoBIlIzatIon There are five different techniques of immobilizing enzymes. They are: 1. Adsorption 2. Covalent bonding 3. Entrapment 4. Copolymerization 5. Encapsulation For the purpose of immobilization of enzymes carriers, that is the support materials such as matrix system, a membrane or a solid surface are used.

adsorption An enzyme may be immobilized by bonding to either the external or internal surface of a carrier or support such as mineral support (aluminium oxide, clay) organic support (starch) and ion-exchange resins. Bonds of low energy are involved, for example ionic interactions, hydrogen bonds. If the enzyme is immobilized externally, the carrier particle size must be very small in order to achieve a appreciable surface of bonding. (The particles may range from 500Ao to about 1mm). There are four procedures for immobilization by adsorption. i. Static process Enzyme is immobilized on the carrier simply by allowing the solution containing the enzyme to contact the carrier without stirring. ii. Dynamic batch process Carrier is placed into the enzyme solution and mixed by stirring, or agitated continuously in a shaker.

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iii. Reactor loading process Carrier is placed into the reactor, then the enzyme solution is transferred to the reactor and carrier is loaded in a dynamic environment by agitating the carrier and enzyme solution. iv. Electro deposition process Carrier is placed proximal to one of the electrodes in an enzyme bath, and current is applied to it. The enzyme migrates to the carrier and gets deposited on the surface.

covalent Bonding Covalent bonding is formed between the chemical group of enzyme and chemical groups on the surface of the carrier. This is utilized under a broad range of pH, ionic strength and other variable conditions.Covalent attachment may be directed to a specific group such as amine, hydroxyl, tyrosyl etc. On the surface of the enzyme. Hydroxyl and amino groups are the main groups of the enzymes with which it forms bonds. Different methods of covalent bonding are: i. Diazoation: Bonding between the amino group of the support such as, aminobenzyl cellulose, amino silanized porous glass, amino derivatives, and a tyrosyl or histidyl group of the enzyme. ii. Formation of peptide bond: Bond formation between the amino or carboxyl group of the support and amino or carboxyl group of the enzyme. iii. Group activation: Use of cyanogen bromide to a support containing glycol group that is, cellulose, sephadex, sepharose, etc. iv. Polyfunctional reagents: Use of a bifunctional or multi functional reagent such as glutaraldehyde, which forms bonding between the amino group of the support and the amino group of the enzyme. The major problem with covalent bonding is that the enzyme may be inactivated by bringing about changes in conformation at the active sites. This problem can be overcome through immobilization in the presence of enzyme’s substrate or a competitive inhibitor or protease. The most common activated polymers such as, cellulose or carbodimide or azide groups are incorporated.

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entrapment Enzymes are physically entraped inside a matrix (support) of a water soluble polymer such as polyacrylamide type gels and naturally derived gels such as, cellulose triacetate, agar, gelatin, carrageenan, alginate etc .The form and nature of the matrix vary, and the pore size of the matrix should be adjusted to prevent the loss of enzyme from the matrix due to excessive diffustion. There is a possibility of leakage of low molecular weight enzymes from the gel. Agars have large pore sizes (> 1, the diffusion rate is limiting, known as the diffusion limited regime. For Da >1) And the system is pseudo-first order. When the system in reaction is limited (Da 15cm diameter). ii Deviations from ideal plug-flow are due to back-mixing within the reactors, the resulting product stream having a distribution of residence times. iii In an extreme case, back-mixing may result in the kinetic behaviour of the reactor approximating to that of the CSTR, and the consequent difficulty in achieving a high degree of conversion. These deviations are caused by channeling, where some substrate passes through the reactor more rapidly, and hold-up, which involves stagnant areas with negligible flow rate. Channels may form in the reactor bed due to excessive pressure drop, irregular packing or uneven application of the substrate stream, causing flow rate differences across the bed. The use of a uniformly sized catalyst in a reactor with an upwardly flowing substrate stream reduces the chance and severity of non-ideal behaviour. iv Fabrication and commissioning costs are high although running cost may be low. v Particulate, colloidal or high viscosity substrate streams tend to block packed bed reactors, and in addition, unless the catalyst is incompressible, channelling or blocking of the flow through the catalyst bed may occur. This latter problem and that of potential gas liberation can be overcome by using a sheet immobilized enzyme rolled up into a spiral and inserted longitudinally into the reactor, or by using skeins of immobilized enzyme fibres. Advantages of PBRs: i Small size ii High productivity even in the presence of product inhibition iv Low void volume v Ease of automation

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Product

SP

Substrate

Fig 13.5 Packed Bed Reactor

The superficial flow velocity through the reactor is equal to the volumetric flow of feed divided by the void cross-sectional area which is the total cross-sectional area times the void fraction ε. The appropriate rate expression used in the tubular reactor material balance is based upon use of effectiveness factors. For example, consider a single reaction S → P with intrinsic rate V = V (S, P) the rate of product formation per unit volume of immobilized biocatalyst pellet at a point in the reactor is: V = η (SS , PS) V (SS, PS) (1) Overall/unit volume of pellet Ss = Substrate concentration at the exterior pellet surface at that position inside the reactor. PS = Product concentration at the exterior pellet surface at that position inside the reactor. η = Effectiveness factor which accounts for intra particle diffusion. V = Rate expression If mass-transfer resistance between the bulk liquid phase and the pellet surface is examined next, a steady-state material balance on substrate over the pellet gives for a spherical catalyst pellet of radius R.

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Rate of substrate diffusion Out of bulk liquid = rate of substrate disappearance By reaction within pellet. 4π R² KS (S–SS) = (4/3) πR³ η (SS, PS) v (SS, PS) (2) ........ With this equation and reaction stoichiometry, SS and PS can be evaluated in terms of the bulk liquid concentrations. Substituting these expressions equation (1) then gives the total rate of substrate disappearance per unit volume of catalyst pellets in terms of the bulk fluid substrate concentration. In writing the material balance on differential slice in the plug-flow packed bed reactor, we must remember that bulk fluid exists only in a fraction ε of this volume and catalyst particles occupy a fraction 1–ε of this volume. The substrate concentration S used in this equation is the concentration per unit fluid volume. Accordingly, the material balance on substrate becomes U dS / dz = –((1–ε) / ε) η (SS, PS) V (SS, PS) (3) ........ Where, the quantities on the right hand side can be evaluated in terms of S, allowing integration of equation (3) for given values of feed substrate and product concentration. The situation is greatly simplified if intraparticle and external mass-transfer resistances are negligible, since these conditions imply η → 1 and SS → S. In such circumstances, the governing mass balances can be integrated analytically.

fluIdIzed-Bed reactors Introduction

These reactors generally behave in a manner intermediate between CSTRs and PBRs. They consist of bed of a immobilized enzymes, which is fluidized by the rapid upwards flow of the substrate stream, alone or in combination with gas or secondary liquid stream either of which may be inert or contain material relevant to the reaction. A gas stream is usually preferred as it does not dilute the product stream.

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Detailed Account On FBRs i. There is a minimum fluidization velocity needed to achieve bed expansion, which depends upon the size, shape, porosity and density of the particles and the density and viscosity of the liquid. ii. This minimum fluidization velocity is generally fairly low _ (about 0.2–1.0 cm / s 1) as most immobilized enzyme particles have densities close to that of the bulk liquid. In this case, the relative bed expansion is proportional to the superficial gas velocity and inversely proportional to the square root of the reactor diameter. iii. Fluidizing the bed requires a large power input, but once fluidized, there is little further energetic input needed to increase the flow rate of the substrate stream through the reactor. iv. At high flow rate and low reactor, diameters almost ideal plugflow characteristics may be achieved. However, the kinetic performance of the FBR normally lies between that of the PBR and the CSTR, as the small fluid linear velocities allowed by most biocatalyst particles causes a degree of back-mixing that is often substantial, although never total. v. The actual design of the FBR will determine whether it behaves in a manner that is closer to that of PBR or CSTR. It can, for example, be made to behave in a manner very similar to that of a PBR, if it is baffled in such a way that substantial backmixing is avoided. FBRs are chosen when these intermediate characteristics are required, for example, where a high conversion if needed by the substrate stream is colloidal or the reaction produces a substantial pH change or heat output. They are particularly useful if the reaction involves the utilization or release of gaseous material. Immobilized Catalyst i. The FBRs are normally used with fairly small immobilized enzyme particles (20–40 μm diameter) inorder to achieve a high catalytic surface area. These particles must be sufficiently dense, relative to the substrate stream, that they are not swept out of the reactor. Less-dense particle must be

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somewhat larger for efficient operation. The particles should be of nearly uniform size otherwise a non-uniform biocatalytic concentration gradient will be formed up the reactor. Disadvantages i. FBRs are usually tapered outwards at the exit to allow for a wide range of flow rates. Very high flow rates are avoided as they cause channeling and catalyst loss. ii. The major disadvantage of development of FBR process is the difficulty in scaling-up these reactors. PBRs allow scaleup factors of greater than 50000 but, because of the markedly different fluidization characteristics of different-sized reactors, FBRs can only be scaled-up by a factor of 10–100 each time. iii. In addition, changes in the flow rate of the substrate stream causes complex changes in the flow pattern within these reactors that may have consequences unexpected, upon the conversion rate. Advantages i. Channelling cannot occur. ii. Colloidal or solid substrate can be used iii. Mixing is improved and diffusion limitation reduced with the result that high biocatalyst loading may be used efficiently and productivity of reactor is increased. A rudimentary model of such fluidized reactors can be developed by assuming that • The biocatalyst particles are uniform in size. • The fluid phase density is a function of substrate concentration. • The liquid phase moves upward through the vessel in plug flow. • Substrate-utilization rates are first-order in biomass concentration but zero-order in substrate concentration. • The catalyst particle Reynolds number, based on the terminal velocity, is small enough to justify Stoke’s law.

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Under these assumptions, the substrate conversion equation is of the form: d (SU) / dZ = –KX (1) (or) u dS / dZ + s dU / dZ = –KX (2) For Stoke’s flow, the concentration of the suspended biomass can be related to the liquid flow velocity in a fluidized bed by X = ρ0 [1– (U/Ut)1/4.65] (3) Where ρ0 = Microbial density on a dry weight basis Ut = Terminal velocity of sphere in Stoke’s flow. Substituting Equation (3) in Equation (2) leaves two unknown, S and U as functions of position Z in the tower. We complete the model by applying the equation. d (UC) / dZ = rfC to total mass (rf=0) to reveal d (ρU) / dZ = 0 (4) Expanding eq.4 and using ρ = ρ (s) gives ρ(S) dU / dZ + (U dρ / dS) dS / dZ = 0 (5) To cast the model in standard form suitable for numerical integration, we may view equation 2 and equation 5 as simultaneous algebraic equations in the unknowns dS / dZ and dU / dZ. Solving this algebraic set, which need not be written out in full, gives dS/dZ and dU/dZ in terms of S and U, a set of two simultaneous differential equations to be integrated with the intial conditions. S(0) = Sf U(0) = Uf = Ff /Af Where, Af is the tower cross-section at the bottom. The effluent substrate concentration Se is S (Z = L) All this is much simplified if we assume that whatever fluid density changes occur will not affect U significantly where in equation (2) integrates directly with the result. Sc = Sf – Kρo [1 – (u / ut)1/4.65] L/U Where, X from equation (3) is inserted and L is the tower height.

Immobilization of Enzymes

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

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Product Out

Substrate In

Fig 13.6 Fluidized Bed Reactor

MeMBrane reactors A membrane reactor is really just a plug-flow reactor that contains an additional cylinder of some porous material within it, like a tube within the shell of a shell and tube heat exchanger. This porous inner cylinder is the membrane that gives the membrane reactor its name. The membrane is a barrier that only allows certain components to pass through it. The selectivity of the membrane is controlled by its pore diameter, which can be of the order of angstroms, or micro porous layers, of the order of microns or macro porous layers. Why use a membrane reactor Membrane reactors combine reaction with separation to increase conversion. One of the products of a given reaction is removed from the reactor through the membrane, forcing the equilibrium of the reaction “to the right”, so that more of that product is produced. Membrane reactors are commonly used in dehydrogenation reactions (E.g. dehydrogenation of ethane), where only one of the products (molecular hydrogen) is small enough to pass through the membrane. This raises the conversion for the reaction, making the process more economical. KINDS OF MEMBRANE REACTORS

i. Inert membrane reactor ii. Catalytic membrane reactor

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Inert membrane reactor Inert membrane reactor allows catalyst pellets to flow with the reactants on the feed side (usually the inside of the membrane). It is known as IMRCF, which stands for Inert Membrane Reactor with Catalyst on the feed side. In this kind of membrane reactor, the membrane does not participate in the reaction directly; it simply acts as a barrier to the reactants and some products. Catalytic Membrane Reactor (CMR) Catalytic membrane reactor has a membrane that has either been coated with or is made of material that contains catalyst, which means that the membrane itself participates in the reaction. Some of the reaction products (those that are small enough) pass through the membrane and exit the reactor on the permeate side. Design Of Membrane Reactors The membrane reactor has two individual compartments separated by a chemically specific membrane. Each compartment can hold a different solution, for instance, one can contain the catalyst and the other, the starting materials/products. Additionally, the membrane itself can contain an immobilized catalyst and the two compartments can hold the reagents and products separately. It can provide benefits in separating products, reagents or catalysts, which can eliminate the need for solvent intensive workup procedures. The usual choice for a membrane reactor is a hollow fibre reactor with a formed module, containing hundereds of thin tubular fibres each having a diameter of about 200μm and a membrane thickness of about 50μm. Membrane reactors may be used in either batch or continuous mode and allow the easy separation of enzyme from the product. They are normally used with soluble enzymes, avoiding the costs and problems associated with other methods of immobilization and some of the diffusion limitations of immobilized enzymes. If the substrate is able to diffuse through the membrane, it may be introduced to either side of the membrane with respect to the enzyme, otherwise it must be within the same compartments as the enzyme, a configuration that imposes a severe restriction on the flow rate through the reactor, if used in continuous mode.

Immobilization of Enzymes

 179

Due to the ease with which membrane reactor systems may be established, they are often used for production on a small scale (gm to kg), especially where a multi-enzyme pathway or coenzyme regeneration is needed. They allow the easy replacement of enzyme in processes involving particularly labile enzymes and can also be used for biphasic reactions. Disadvantages i. Cost of membranes is high. ii. Membranes need to be replaced at regular intervals. iii. The kinetics of membrane reactors are similar to those of the batch STR, in batch mode, or the CSTR, in continuous mode. Deviations from these models occur primarily in configurations where the substrate stream is on the side of the membrane opposite to the enzyme and the reaction is severely limited by its diffusion through the membranes. Advantages i. Membrane reactors achieve efficiencies by combining in one unit a reactor that generates a product with a semipermeable membrane that extracts it. The result is more compact design and a greater conversion. ii. Removal of product increases the residence time for a given volume of reactor and drives equilibrium-limited reactions towards completion. iii. Membrane reactors fundamentally change the pressure decomposition reactions so that the reactions are preferentially performed at high pressure rather than at low pressure. High pressures allow much small reactors and more efficient purification. iv. Membrane reactors can be advantageous also for sequential endothermic and exothermic reactions, by using the product extraction to promote heat transfer. Enhanced heat transfer permits plug flow where CSTR designs would have been necessary otherwise. The net result is small reactors, low capital costs, and often fewer side reactions.

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Enzymes

B

A

B+C

C

Catalytic ceramic membrames

B

A C

Fig 13.7 Membrane Reactor

A mixed feed of A and B enters the membrane reactor. C is produced in the reactor, and B diffuses out through the membrane pores. There are multiple ceramic membranes, but only two are shown in the figure for simplicity.

QuestIons 1. 2. 3. 4. 5. 6. 7. 8. 9.

What is immobilization of enzymes? List out the advantages of immobilization techniques. Give an account on various methods of enzyme immobilization. Explain the working principle of adsorption method with a neat diagram. Define diazoation. Explain briefly about the various types of bonding in immobilization. Write short notes on entrapment techniques with neat diagram. What is encapsulation? Explain. List out the various areas in which enzyme immobilization techniques are adopted.

CHAPTER

14 Biosensors

A biosensor is an analytical device consisting of an immobilized layer of biological material (enzyme, antibody, organelle, hormone, nucleic acids or whole cells), in intimate contact with a transducer that is, a sensor (a physical component), which analyzes the biological signals.

prIncIple The biological material is immobilized on an immobilization support (permeable membrane) in the direct vicinity of a sensor. The substances (analyte) to be measured is passed through the membrane and is made to interact with the immobilized material to yield the product. A product (monitor substrate) may be heat, gas (oxygen), electrons, hydrogen ions or the product of ammonium ions. The product passes to the transducer which converts the product into electrical signal, which is then amplified and then displaced, read out and recorded. Biosensor must have following features: 1. It should be highly specific for the analyte. 2. The reaction used should be independent of factors like stirring, pH, temperature, etc. 3. The response should be linear over a useful range of analyte concentrations. 4. The device should be tiny and biocompatible (when it is to be used for analysis within the body).

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5. The device should be cheap, small and easy to use. 6. It should be durable (should be capable of repeated use).

general features A biosensor has two distinct types of components: 1. Biological (enzyme, antibody, etc.) 2. Physical (transducer, amplifier, etc.) The biological component of biosensor performs two key functions: i. Specifically recognizes the analyte. ii. Interacts with it and produces some physical change and detectable by the transducer. The biological component is suitably immobilized on to the transducer. (Thus, this enzyme immobilized system can be used for more than 10,000 times over a period of several months). The biological component interacts specifically with the analyte to produce a physical change, close to the transducer surface. The changes may be: 1. Heat released or absorbed by the reaction (measured by calorimetric biosensors). 2. Production of an electrical potential due to charged distribution of electrons (potentiometric biosensors). 3. Movement of electrons due to redox reaction (aperometric biosensors). 4. Light produced or absorbed during the reaction (optical biosensors). 5. Change in mass of the biological component as a result of the reaction (acoustic wave biosensor).

Biosensors

Transport

 183

201 Amplifier

Processor

Display

Analyte

BIOLOGICAL COMPONENT

Transducer PHYSICAL COMPONENTS

Fig 14.1

Components of a Biosensor

The transducer detects and measures these changes and converts it into electrical signals. The signal is amplified by an amplifier before it is fed into the microprocessor. Thus, the biosensor converts a chemical information flow into an electrical information flow.

BIOSENSOR

MIXTURE OF DIFFERENT MOLECULES

Output

Detector

Fig 14.2

Transducer

Mechanism of a Biosensor

184 

Enzymes

glucose electrode In 1987, Yellow Springs Instruments Co., USA, developed a biosensor for diagnostic purposes for measuring glucose in blood plasma. This was a hand-held machine, which can measure six components of blood plasma such as glucose, urea, nitrogen, sodium, potassium and chloride. The glucose electrode can be prepared by immobilizing glucose oxidase in polyacrylamide gel around a platinum–oxygen electrode separated, by a Teflon membrane. Potassium chloride solution is placed around the platinum–oxygen electrode. The glucose oxidase is covered by a cellulose acetate membrane. When glucose is brought into the membrane, glucose and oxygen passes through membrane and next to the enzyme layer and results in oxidation–reduction reaction. Conversion of gluconic acid and hydrogen peroxide occurs in the presence of water, oxygen and glucose oxidases. Hydrogen peroxidase brings about a change in the current that is, measurable signal. Electrode records the rate of reactions. The rate of diminition of oxygen concentration is proportional to glucose concentration of the sample. This electrode responds to 10–1 to 10–5 mol dm–3 of glucose concentration in 1 minute. Scientists at Central Electrochemical Research Institute (CECRI), Karaikudi, constructed a biosensor which can be used to measure glucose concentration as low as 0–15 millimoles. Table 14.1

List of Some Biological Materials and Detection Devices Used In Biosensors

Biological Materials 1. Enzymes 2. Nucleic acids

Detection Device (or Transdecers) Potentiometric electrodes i. Amperometric electrodes ii. Amperometric electrodes

Examples Enzyme electrode for urea (based on urease) Enzyme electrode for organophosphorus pesticides (using acetyl cholinesterases) Glucose biosensor (based on Glucose oxidase)

Biosensors

3. Antibodies 4. Lectins

5. Organs 6. Cells 7. Tissue slices

 185

Wave guides (optical biosensors) Grating couplers Acoustic wave sensors (optical biosensors) Conductometric sensors Thermometric sensors.

types of BIosensors The biosensors are of different types namely, 1. Calorimetric biosensors 2. Potentiometric biosensors 3. Amperometric biosensors 4. Optical biosensors 5. Acoustic wave biosensors 6. Whole cell biosensors.

calorimetric Biosensors Calorimetric biosensors measure a change in temperature of the solution containing the analyte. During this process, heat is liberated (exothermic). The analyte solution is passed through a small (1 ml) packed bed column containing immobilized enzyme. The temperature is recorded during the entry into the column and its exit from the column. This type can be used for turbid or even strongly coloured solution. The sensitiving can be increased by using two or more enzymes. This type has one disadvantage, where the sample is maintained at 0.01oC (constant temperature). e.g. glucose oxidase for determination of glucose.

amperometric Biosensors The electrons function due to the potential developed between two electrodes, and its magnitude is proportional to the substrate concentration. This type employs the Clark oxygen electrode, which

186 

Enzymes

determines the reduction of O2 concentration. Which can be rectified by using mediators. e.g. Ferrocenes (transfers the electrons). Electrode

Product Εnzyme

θ θ

Redox mediator

Substinite

θ Fig 14.3 Amperometric (Mediated Biosensor)

optical Biosensors These biosensors measure both catalytic and affinity reactions. They measure a change in fluorescence or in absorbance caused by the products generated by catalytic reactions. A most promising biosensor involves luminescence technology, where luciferase used for detection of bacteria, are lysed to release ATP and the luciferase in the presence of O2 produce light which is measured by the biosensor. Bioaffinity sensors are developed recently and it measures the concentration of the determinants that is, substrates based on equilibrium, binding and shows high degree of selectivity. A receptor is radiolabelled and allowed to bind with a determinant analogue immobilized onto the surface of a transducer. When concentrations of a determinant are increased, the labelled receptor forms an intimately bound complex with the determinant. Finally, radiolabelled receptor– determinant complex is removed from the immobilized determinant analogue.

Biosensors Transducer

 187

Determinant analogue Labelled receptor Determinant

Fig 14.4

Bioaffinity Sensor

Acoustic Wave Biosensor The surface is coated with antibodies which bind to the complementary antigen present in the sample solution. This leads to increased mass which reduces their vibrational frequency. This change is used to determine the amount of antigen present in the sample solution.

potentiometric Biosensors These biosensors adopt ion-selective electrodes, which convert the biological reaction into electronic signal. A glass pH electrode is coated with a gas selective membrane (for CO2, NH3, or H2S) or solid state electrodes. During the process H+ is generated or used up and it is measured by the biosensor. E.g. Electrode based on urease. The reaction is as follows. CO ( NH2) + 2H2O + H+ 2NH4 + + HCO3–

Whole Cell Biosensors (Microbial Biosensors) In this type, the whole cell of microorganism or their organelles are used. Immobilized Azotobacter vinelandii coupled with ammonia electrode shows sensitivity between 10-5 and 8 x 10 mol dm-3..

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Table 14.2 Microbial Biosensors Containing Oxygen Electrode

Microorganism Brevibacterium lactofermentum Bacillus subtills Clostridium butyricum Methylomonas flagellate Trichosporon brassicae Trichosporon brassicae Psuedomonas fluorescens

Substrate for Sensoring Assimilate sugars

Response Time (Min) 1–10

Mutagen screening Formate

90–100 20

Methane

1

Acetate

6–10

Ethanol

10

Glucose

15–20

Microbial biosensors have many advantages for a wide range of substrates. One important difference results from the nature of substrate oxidation in the cells, which provides reducing intermediates from several degradative stages and provide a bigger electrochemical signal than single enzyme reaction. e.g. Indirect glucose sensor, the oxidation of glucose to gluconolactone yields two electrons per molecule using glucose oxidase. D-Gluconolactone + 2H+ + 2e– β-D-glucose In contrast, the oxidation of glucose in a whole cell may be represented as, 6CO2 + 24H+ + 24e– C6H12O6 + 6H2 O 1. When O2 probe is used, it is the respiratory activity which is generally measured. 2. Electroactive metabolites such as proton, CO2, hydrogen, formic acid and reduced cofactor could be measured by the electrode.

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 189

applIcatIons of BIosensors 1. 2. 3. 4. 5. 6. 7. 8. 9.

10.

11.

12.

Medicine: For the diagnosis of infectious and non-infectious diseases. Agriculture: For the detection of plant nutrients, fertilizers, pesticides. Environment: For pollution monitoring–BOD, COD, NH3, NO3, SO3. Food industry: To sense freshness, odour, taste, and to find out toxic substances. Industries: For the detection of acids, alcohols, phenols. Defence: To detect mustard gas, nerve gas. Mining: To detect toxic compounds and gases. Sports: To determine fatigue by measuring levels of lactic acid, ammonia in perspiration. Glucose level: Glucose level as low as 0.15 milimoles can be measured using glucose oxidase enzyme. The device induces a current flow that is proportional to glucose concentrations. Estimation of hormones: Graves disease result due to less amount of thyroxine hormone leading to physical and mental retardation in children. In this device, hormone sensing molecule is used in the biolayer. Penicillin concentration: Biosensor is used to measure penicillin concentration. The biosensor consists of a silicon wafer pH sensitive layer on which the pencillinase enzyme is immobilized. Then the probe is immersed in an aqueous solution containing millimolar penicillin. The penicillin forms a pencilloate ion and a proton, generating a current that is proportional to the degree of penicillin concentration. Detection of freshness: Biosensor is used to measure freshness of fish or meat where ATPase, amino oxidase, putrescine oxidase are used. This sensor measures nitrate by a colour change in pH indicator, which is detected by the transducer.

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Enzymes

13. Immunosensors: Nowadays, immunosensors are more common and commercialized. Surface Plasmon Resonance (SPR) biosensor is used to measure optical changes. With this biosensor protein interactions can be followed in real time without the requirement of any labelling technique. 14. Portable biosensor: It can measure as low as 5 ng of cocaine in urine sample in just 45 seconds. This sensor is cheaper when compared to RIA (1,50,000). When urine is passed through the column, cocaine if present displaces fluorescent molecules which come in the elute. To detect this, American navy spends $10 million on screening alone. Table 14.3 Enzyme Biosensors with Industrial Applications

Target Trans Response Stability Range substance -ducer Time (Days) Alcohol (alochol 30 120 5–103 mg/dm3 O2 oxidase) Sucrose (invertase 360 14 O2 2 × 103 M muta-rotase) Glucose (glucose – 21 0.5–4% W/V O2 oxidase) Cholestrol Pt 120 30 3 × 10–5 M 2 × 103 Lactate Pt 200–600 7 –2 × 10–4 M

QuestIons 1. 2. 3. 4. 5. 6. 7.

What is a biosensor? Explain the principle of the biosensor. Write a short on the significant features of a biosensor. What are the different physical changes occurring in a biosensor? Write a short note on glucose electrode. What are the different types of biosensors? Write short notes on calorimetric biosensor with an example.

Biosensors

 191

8. Explain the working principle of amperometric biosensor. 9. What are optical biosensors? Explain them with a suitable example. 10. Write short notes on acoustic biosensor, potentiometric biosensor and whole cell biosensor. 11. List out the advantages of microbial biosensors. 12. Write a note on the various applications of biosensors.

CHAPTER

15 Clinical Enzymology

Enzymes are present in high concentration in blood. Enzymes that perform physiological functions such as lipoprotein lipase in circulation and enzymes of blood coagulation, are termed as functional enzymes. Enzymes that are present in low concentration in plasma and have no physiological functions are called as nonfunctional enzymes. Measurement of plasma levels of non-functional enzymes is employed in clinical diagnosis. Enzymes may be assayed by determining the catalytic activity or by determining the protein antigens.

aMylase α-amylase hydrolyzes the α-1,4 linkages in starch and glycogen to yield maltase. The optimum pH is 6.9. Amylase is found in saliva and pancreatic juice at higher concentration, and skeletal muscle, adipose tissue, gonads, fallopian tubes, colostrums, tears, milk and urine have low concentration of amylase.

Clinical Significance Increased activity of plasma amylase is recorded in certain body conditions. They are 1. Severe diabetic ketoacidosis. 2.

Salivary gland disorders such as mumps, Salivary calculi.

3.

Ruptured ectopic pregnancy.

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4. Acute oliguric renal failure. 5. Acute pancreatitis (exceeds 2000 U/L). 6. In duodenal ulcer, the ampulla is involved in the inflammatory process thus blocking secretion of pancreatic juice into duodenum. 7. 8. 9.

Sphincter of oddi: It Results due to contraction of pancreatic duct sphincter (morphine administration). Macroamylasaemia results due to increase in plasma amylase with decrease in urinary excretion. Due to increased plasma amylase activity, cerebral trauma, shock and pneumonia may occur.

transaMInases These enzymes are involved in the transfer of amino groups (aminotransferases or transaminases). The important transaminases are aspartate transaminase (AST) and alanine transaminase (ALT). Heart, skeletal muscles, liver, kidney and erythrocytes have high concentration of aspartate transaminase. AST is higher in neonates than adults and alanine transaminase is present in the liver.

Clinical Significance 1.

During cardiac arrest, the activities of both AST and ALT are increased (10–100 times ULN). 2. Myocardial infarction: Plasma AST level increases following myocardial infarction. The activity is more at 6–8 hours after the onset and reaches its peak between 24–48 hours and attains normal level by 4–6 days. 3. Liver disease: Both AST and ALT are sensitive markers (10–100 times U/L) of hepatocellular injury in which ALT is a more specific indicator. The values of transaminases reaches a peak when the illness is maximum. In case of inflammation, ALT activities are higher than AST.

Clinical Enzymology

4. 5.

 195

In carcinoma of the liver, both AST and ALT seems to be elevated (but AST levels are higher than ALT). Plasma AST is elevated in severe haemolysis of erythrocyte origin.

ALKALINE PHOSPHATASE (ALP) A group of functionally related enzymes that hydrolyze organic phosphates at a pH range between 9–10.5 constitute the alkaline phosphatase enzyme (ALP). The enzyme is activated by divalent cations such as Mg2+, Co2+, Mn2+ and inhibited by phosphate, borate, oxalate and cyanide. This enzyme is present in all tissues and high concentration of this enzyme is seen in osteoblasts of bone, hepatobiliary tract, intestinal epithelium, kidney and placenta. Plasma ALP activity is higher in males than in females and more during childhood. High fat meal can increase the intestinal plasma ALP activity.

Clinical Significance Cholestasis: A high plasma ALP (>180 µ/L) is associated with an increase in bilirubin, which is the indicator of cholestasis. 2. An increase in plasma ALP without any association of bilirubin is seen in primary and secondary liver malignancy and early cirrhosis. 3. Bone disease: i Very high values (>500 µ/L) are seen in Paget’s disease. ii In osteomalacia and rickets, the increase in enzyme activity reverts to normal after vitamin D therapy. iii Levels of ALP is slightly elevated in bone cancer. 4. Cretinism and hypophosphatasis: Low level of ALP activity and the bone growth is arrested. 1.

ACID PHOSPHATASE (ACP) Acid phosphatase are a group of enzymes that catalyze the hydrolysis of organic phosphates at acidic pH.

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Enzymes

The prostate is the richest source of ACPs and the other organs are liver, spleen, semen, eythrocytes, platelets, bone marrow, milk and urine. High concentration of ACP in semen is the indicator in the investigation of rape. The optimum pH is 5–6 and this enzyme is inhibited by formaldehyde and therefore it is called formol labile ACP and it is also inhibited by tartrate ions, so they are called as tartrate labile or prostatic ACP.

Clinical Significance Increased levels of non-prostatic ACP are seen in Paget’s disease of the bone.

LACTATE DEHYDROGENASE (LD) Lactate dehydrogenase catalyzes the oxidation of L-lactate to Pyruvate with NAD+ as hydrogen acceptor at an optimum pH range of 8.8–9.8. This enzyme is inhibited by mercuric ions and p-chloromercuribenzoate, borate and oxamate. LD is widely distributed in heart, skeletal muscle, liver, kidney, brain and erythrocytes. Plasma LD is predominant when derived from erythrocytes and platelets during their turnover.

Clinical Significance 1.

2.

3. 4. 5.

Myocardial infarction: The enzyme activity rises within 12–24 hours after the onset of chest pain and reaches its peak by 48–72 hours and remains elevated for 7–12 days. Haematological disorders: About 20 times U/L concentration of LD is seen in blood diseases such as megaloblastic anaemias, acute leukaemias and lymphomas. A marked increase of LD (10 times) is seen in toxic hepatitis with jaundice. Malignancies: Elevated LD level is seen in Hodgkin’s disease, abdominal and lung cancer. Excretion of LD in urine is seen in kidney disorder.

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 197

CREATINE KINASE (CK) Creatine kinase catalyzes the reversible phosphorylation of creatine by ATP at an optimum pH of 9 and the reversible reaction occurs at pH 6.7. The enzyme is activated by Mg2+ and inhibited by Ca2+, Zn2+, Cu2+, Mn2+, citrate, fluoride, urate, cystine and thyroxine. The enzyme is present in cardiac, skeletal and smooth muscles. The brain, which is a non-muscular tissue contains high level of CK. The neonates and children have higher CK activities than adults. Men show greater activity than women.

Clinical Significance 1. The CK is very useful in the diagnosis of myocardial infarction and an increase in level is seen within 2–5 hours after the onset of chest pain. 2. Deuchenne muscular dystrophy: This is a sex-linked muscular disorder which has high levels of CK during the early clinical stage. 3. Plasma CK activity is increased in hypothyroidism. 4. Reye’s syndrome: Plasma CK activity is increased upto 70fold. This occurs in childhood, which is characterized by brain swelling and dysfunction of the liver. 5. Plasma CK levels are elevated in lymphomas, leukaemias, sarcomas and tumors of the prostate. So, this enzyme acts as a turnover marker.

GAMMA-GLUTAMYL TRANSFERASE (GGT) This enzyme catalyzes the transfer of a gamma glutamyl group from peptides to acceptors. GGT is found in kidney, liver, pancreas and prostate.

Clinical Significance 1. 2.

Liver disease: Extremely high concentration of GGT is an indicator of chronic cholestasis and liver malignancy. Plasma GGT is increased by ingestion of alcohol and drugs such as phenobarbitone, phenytonic, and antidepressants.

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cHolInesterases These enzymes catalyzes the hydrolysis of choline esters. The enzyme is inhibited by alkaline and organic insecticides such as diisopropyl fluorophosphate.

Clinical Significance This enzyme acts as an indicator of insecticide poisoning. High levels are seen in nephritic syndrome, thyrotoxicosis, brain diseases and obese diabetes.

QuestIons 1. Define functional and non-functional enzymes. 2. Write down the clinical significance of the enzyme amylase. 3. Define the term macroamylasaemia. 4. Explain the significance of AST and ALT. 5. Explain the role of AST and ALT in liver disease. 6. Expand ALP and write a short note on it. 7. Define the term cholestasis. 8. Write short notes on ACP. 9. Illustrate the clinical significance of ACP. 10. Explain haematological disorder of LD. 11. Write a short note on creatine kinase. 12. Define Reye’s syndrome. 13. Explain the significance of GGT. 14. Write short notes on cholinesterases.

CHAPTER

16 Applications of Enzymes

applIcatIons In MedIcIne Assay of Plasma Enzymes Assays of some of the enzymes present in blood plasma or serum is carried out routinely in most clinical laboratories and these play an important role in diagnosis (plasma is the fluid from unclotted blood). A few plasma enzyme have a clearly defined role in that particular location (for example, in coagulation). For each enzyme present in plasma, a balance is normally set up between its rate of arrival by leakage from cells and its rate of removal by catabolism or excretion. Therefore, for each plasma enzyme, there is a normal concentration range and hence, a normal range of activity, which can be determined. If the cells of a particular tissue are affected by a disease in such a way that many of them no longer have intact membranes, then their content will leak out into the blood stream at an increased rate and the enzyme associated with those cells will be found in the plasma in elevated levels. 1. Lactate dehydrogenase (LDH) LDH can be assayed since it catalyzes reactions involving NAD/ NADH as coenzymes. LDH is found in most cells of human body such as liver, skeletal muscles etc. The total activity of LDH is the sum of activities of five isoenzymes such as M4, M3H, M2H2, MH3 and H4.. M4 is present in liver and skeletal muscles and H4 in heart muscles. If plasma is subjected to electrophoresis (on cellulose acetate strips)

200 

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at pH 8.6, the LDH isoenzymes may be located by means of specific strain (a mixture of lactate, NAD +and a chromogen). LDH catalyzes the first step of the reaction. Separation is achieved by ion-exchange (QAE-sephadex) chromatography. Immunological reagents may be used to precipitate all isoenzymes containing M-subunits leaving only H4 in solution. If the total LDH activity is found to be higher than normal, there are several ways of establishing whether it is due to excess H4 (in case of heart disease, hematological disease) or due to excess M4.(in case of skeletal muscle or liver disease). 2. Aspartate transaminase (AST) AST was formerly known as glutamate oxaloacetate transaminase (GOT), which is widely distributed throughout the body and carry out a general reaction given below: oxaloacetate + glutamate Aspartate + 2-oxoglutarate This may be assayed by coupling the product, oxaloacetate to an indicator, catalyzed by malate dehydrogenase in which NAD+ / NADH act as coenzymes. Plasma activities of AST indicates a severe damage to the cells of heart (as in myocardial infarction ) or liver( in viral hepatitis or toxic liver necrosis). 3. Alanine transaminase (ALT) ALT was formerly known as glutamate pyruvate transaminase (GPT) It catalyzes the reaction given below: pyruvate + glutamate. Alanine + 2-oxoglutarate This enzyme is predominant and found in liver cells. Hence, a markedly raised plasma activity indicates a severe liver disease (viral hepatitis or toxic liver necrosis). 4. Alkaline phosphatase These are a group of isoenzymes which catalyze the hydrolysis of phosphates at alkaline pH. They are found in bone, liver, kidney, intestinal wall, lactating mammary gland and placenta. These enzymes can be separated by electrophoresis at pH 8.6 with calcium alpha naphthyl phosphate mixed with a diazonium salt. Because of its osteoblastic origin (bone), it is useful in the detection of bone diseases. The activity of this enzyme is raised in bone diseases (osteomalacia, rickets, Paget’s disease etc.)

Applications of Enzymes

 201

5. Digestive enzymes Digestive enzymes, trypsin, triacylglycerol, lipase and alphaamylase are produced in the pancreas. Their increased amount in plasma causes acute pancreatitis. Alpha-amylase utilizes starch as substrate and produces reducing sugars as products. Alpha-amylase is very small enough to pass through the glomerulus of the kidney to be excreted in urine. In some cases alpha amylases form complexes with other proteins called macro-amylasaemia which causes plasma alpha amylase activity to increase. 6. Acid phosphatase This isoenzyme is found in large amounts in the prostate gland, liver, red cells, platelets and bone. This enzyme is used in the diagnosis of prostatic carcinoma. They catalyze the same reaction as alkaline phosphatase but at lower pH value. 7. Cholinesterase This is another enzyme which is usually assayed in plasma. The enzyme catalyzes the hydrolysis of choline esters, such as acetylcholine. Acetic acid + Choline. Acetylcholine + H2O Acetyl cholinesterase is found in nerve tissues and red blood cells. This acts on the substrate, acetyl beta-methyl choline. Patients with low plasma activities of cholinesterase experience severe breathing difficulties for several hours after treatment with choline (muscle relaxant).

Therapeutic Uses Microbial enzymes have a lot of importance in medicine. According to one estimate, microbial enzymes will be 60% of the total $1 billion diagnostic enzyme market. Microbial enzymes find their applications in therapy as well, as in testing kits. 1. Methotrexate (MTX) is widely used as an anticancer drug damaging dihydrofolate reductase enzyme. This enzyme is required by cancer cells. MTX is not selective in action and affects normal cells. Since different patients break down the drug at different rates, it is important to monitor the level of MTX in the body. This can be done by in-vitro testing by the effect

202 

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of MTX on dihydrofolate reductase enzyme from the blood sample. Here dihydrofolate reductase enzyme, obtained from Lactobacillus casei is used. 2. In most cases, certain diseases are treated by administering the appropriate enzyme. E.g. Virilization of a disease is developed due to loss of an hydroxylase enzyme from adrenal cortex. (Addition of hydroxyl group (–OH) on 21-carbon of ring structure of steroid hormone). E.g. Treatment of leukaemia (a disease in which leukaemic cells require exogenous asparagines for its growth) could be done by administering asparaginase of bacterial origin (obtained from Erwinia). 3. Gaucher’s disease is one of the hereditary lipid storage diseases affecting the nervous system of children where they lack the enzyme which breaks down lipids (glucocerebroside). This compound starts accumulating in spleen, liver, and bones, resulting in swelling and damage of nerves and leads to death in children before the age of two. Gaucher’s disease is prevented by injecting a right enzyme. Nowadays, this enzyme is purified from human urine or plasma. The enzyme streptokinase is isolated from Streptococcus sp. These enzymes are efficient in breaking down the blood clots in vein and prevent heart attacks or strokes. In 1982, US Food and Drug administration department approved this enzyme for treating heart attacks. Human superoxide dismutase (HSOD) is an enzyme is capable of neutralizing free radical oxygen, known to produce cell and tissue damage. This enzyme prevents oxidative damage and the resultant loss of organ function (this damage is expected during heart surgery, heart attacks and organ transplants). HOSD is used to treat such patients. 4. Immobilized enzymes may be components of artificial kidney machines, which are used to remove urea and other waste products from the body where kidney disease prevents this from being done by natural processes. Urea enters the machine from the blood by dialysis (termed haemodialysis) and is converted to CO2 and NH4 by immobilized urease, toxic NH4+ is then either trapped on ion exchange resins

Applications of Enzymes

 203

or incorporated into glutamate by the action of immobilized glutamate dehydrogenase linked to alcohol dehydrogenase to ensure coenzyme recycling before the fluid is returned to the blood stream. 5. Enzymes are of great value in the pharma industry for the conversion of naturally occurring Penicillin G (Benzyl pencillin) to 6-amino penicillanic acid (6-APA), which is catalyzed by bacterial pencillin amidase (cleaves the side chain of the substrate at mild alkaline pH). S CH2CONH

Benzene

O

N

CH3 CH2CO2H + H2N

CH3 CHO2H

Pencicilling G

S

CH3 CH3

O

N

CO2H

6-amino penicillanic acid

Commercial synthesis of 6-APA is carried out by the use of E.coli, immobilized in polyacrylamide gel and the enzyme is entrapped in fibres of cellulose acetate. 6-APA is more advantageous because, new side chain can be attached to give a variety of semisynthetic pencillins for example, Ampicillin (α-methyl benzyl pencillins), a broad spectrum antibiotic. Bacterial strains enzymes may be employed to catalyze forward and backward reactions. For e.g. Mutant of Kluyvera citrophila for 6-APA production and Pseudomonas melanogenum for ampicillin synthesis.

applIcatIon In Industry In Food Industry In the food industry, the activity of certain enzymes may be determined before and after pasteurization and sterilization procedures, to check whether these have been properly carried out. E.g. Alkaline phosphatase and invertase present in milk are inactivated within the same temperature range as required for pasteurization, so the activities of these enzymes at the end of the process give an indication of its effectiveness. Similarly, the degree of bacterial contamination of foodstuffs can be estimated by the assay of microbial enzymes, not normally present in food.

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Milk contains only small amounts of reductases and can may be easily assayed. They catalyze the reduction of methylene blue to colourless leuco-methylene blue under anaerobic conditions. A test strip (Bactostrip) incorporating 2,3,4-triphenyl tetrazolium salts provides a convenient way of testing for the presence of bacteria. A red formazan dye is produced as a result of the action of reductase. Enzyme assay may also be used to determine whether stored plant products are suitable for use as foodstuff. For e.g. Alpha amylase is present in relatively low amounts in stored wheat seeds. However, if sprouting (germination of the stored crop) takes place, there is increased production of alpha-amylase and some proteolytic enzymes. The freshness of meat may be determined by the use of monoamine oxidase to detect amines formed during degradation. Enzyme Immuno Assay (EIA) is used to detect food alteration. Papain (from papaya) is used as a meat tenderizer. South Americans traditionally wrapped their meat in leaves of papaya. Papain (and other proteases) is used in the brewing industry to prevent chill hazes, caused by precipitation of complexes of protein and tannin at low temperatures.

Baking of Bread In the baking of bread, wheat flour (mainly starch and protein) is mixed with yeast and water. Starch consists of D-glucose units linked by α-1,4 glycosidic bonds, with α-1, 6 bonds at branching points. The enzymes α-amylase and β-amylase present in the flour, cleave some of the α-1,4 bonds and results in glucose, maltose (a disaccharide), which is further metabolized by yeast to CO2 which distends the protein framework of the dough and ready for baking. (wheat flour has low α-amylase content; therefore, it may be supplemented with malt flour or fungal α-amylase (Aspergillus oryzae). Proteases from Aspergillus oryzae may be added for breakdown of wheat protein (gluten). The advantages of using this is. The mixing time is shortened enabling a smoother and more uniform dough. Sacchromyces carlsbergensis (yeast) is used in the baking and brewing industries because they contain enzymes for alcoholic

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fermentation. They metabolize hexose sugars to produce pyruvate and the end product is ethanol and CO2, under anaerobic conditions. CH3COCOOH Pyruvic acid

Pyruvate decarboxylse Mg2+ .TPP

Alcohol decarboxylse

CH3CHO

CH3CH2OH + CO2

Acetaldehyde NADH

Ethanol NAD+

Enzyme assay is also used for research into such process as the browning of plant products, which poses problems during the conversion of fruits and vegetables into drinks and preserves. The browning process involves in (involves in cyanide resistant due to uptake of oxygen), which oxidizes phenols to quinones thus indicating the sequence of reactions resulting in the formation of dark melanines. Many industrial processes utilize microorganisms and enzymatic analysis may be employed to ensure that the concentrations of vital ingredients in the growth media are within the required limits. E.g. Glycerol kinase is obtained commercially from Candida mycoderma and it is essential that the glycerol content of the growth medium is high enough throughout, to induce glycerol kinase synthesis by the organism. (A suitable example may be oxidation of glycerol by glycerol dehydrogenase to dihydroxyacetone in the presence of NAD).

Enzymes and Recombinant DNA Technology DNA technology make use of a variety of enzymes, for example, restriction endonucleases (from bacteria) and DNA ligases (from bacteria and bacteriophages) to insert extra genes into cells with the help of vectors such as plasmids. Plasmids are small, circular, cytoplasmic molecules of DNA, acting as extra chromosomal genes in bacteria (sometimes). They may also get attach themselves to chromosomes known as episomes. Plasmids are not essential for the functioning of particular organism, but confers additional properties on it (E.g. resistance to certain antibiotics).

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Example: 1. The plasmid pBR322 is cleaved at a single specific site by the EcoRI restriction enzyme. 2. This makes staggered cut in the double helix, leaving complementary single stranded ends. 3. The cut plasmid is spliced by means of DNA ligase. 4. The new circular plasmid so formed is taken up to a small degree by E.coli since the plasmid contains genes resistance to antibiotics ampicillin and tetracycline. 5. In this way, synthetic prokaryotic and eukaryotic genes are incorporated into E.coli and other bacteria. 6. The bacterium thus grown in culture, will replicate together with the vector. 7. This production of identical copies is termed cloning.

(a) Into a plasmid + DNA ligase

(b) Cut made by restriction endonuclease

(c) Insertion of gene fragment

Fig 16.1

DNA construction rDNA

λ Phage Another group of vectors are variants of a bacterial virus known as λ phage (about 45kb that is, 45,000 basepairs long). The middle third has no role in infectious process, which can be replaced by another piece of DNA of same length (by restriction endonuclease and ligase enzyme) without affecting the ability of phage to infect bacteria and reproduce in abundance.

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In Dairy Industry Calf rennet is being used in dairy industry. In recent years it has been replaced by microbial rennet (E.g Mucor proteases.) They are acid aspartate proteases. They slightly differ from calf rennets.

Cheese Making This involves the conversion of milk protein casein to paracasein by limited hydrolysis by chymosin (rennin). Chymosin, an aspartic acid protease, causes the clothing of milk, a process which involves cleavage of a single peptide bond in K-casein between the C-terminal peptide. The release of the C-terminal peptide is followed by Ca induceed aggregation of the modified micelles to form a gel. The fungal aspartate from Mucorpusillus, Rhizomucor miehei and Cryphonectria parasiticas can be substitute for chymosin, Recombinant DNA chymosin in which the chymosin gene has been cloned into expression system such as oxyzae and Kluyveromyces lactis has been used in the manufacture of certain cheese separated from the whey after which the clot is allowed to mature under controlled conditions (in the presence of rennin) to form cheese. Lactase produced by Bacillus stearothermophilus is used for hydrolysis of lactose in whey or milk. Lipase is used for flavour development in special cheeses.

In Starch Industry Glucose isomerase is an important enzyme used commercially in conversion of glucose to fructose (via.) isomerization. The mixture is sweeter than glucose and as sweet as sucrose.

In Detergent Industry Enzymes have been used in detergents since the 1960s. The use of enzymes in laundry detergents and automatic dish washing machines provides consumers with well proven benefits—both in the washing process and in the wider environment.

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Enzymes can reduce the environmental load of detergent products due to the following reasons: 1. Save energy by enabling a lower wash temperature. 2. Partly replace other, often less desirable, chemicals in detergents. 3. Are biodegradable, leaving no harmful residues. 4. Have no negative environmental impact on sewage treatment processes. 5. Do not present a risk to aquatic life. Proteases Proteases are the most widely used enzymes in the

detergent industry. They remove protein stains such as grass, blood, egg and human sweat. These organic stains have a tendency to adhere strongly to textile fibres. The proteins act as glues, preventing the water borne detergent systems from removing some of the other components of soiling, such as pigments and street dirt. The inefficiency of non-enzymatic detergents at removing proteins can result in permanent stains due to oxidation and denaturation caused by bleaching and drying. Blood, for example, will leave a rust-coloured spot unless it is removed before bleaching. Proteases hydrolyze proteins and break them down into more soluble polypeptides or free amino acids. As a result of the combined effect of surfactants and enzymes, stubborn stains can be removed from fibres. Lipases Though enzymes can easily digest protein stains, oily

and fatty stains have always been troublesome to remove. The trend towards lower washing temperatures has made the removal of grease spots an even bigger problem. This applies particularly to materials made up of a blend of cotton and polyester. The lipase is capable of removing fatty stains such as fats, butter, salad oil, sauces and the tough stains on collars and cuffs. Amylases Amylases are used to remove residues of starch-based

foods like potatoes, spaghetti, custards, gravies and chocolates. These enzymes can be used in laundry detergents as well as in dishwashing detergents.

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Cellulases The development of detergent enzymes has mainly focused on enzymes capable of removing stains. However, a cellulase enzyme has properties enabling it to modify the structure of cellulose fibre on cotton and cotton blends. When it is added to a detergent, it results into the following effects: Colour brightening When garments made of cotton or cotton

blends have been washed several times, they tend to get a “fluffy” look and the colours become dull. This effect is due to the formation of microfibrils that become partly detached from the main fibres. The light falling on the garment is reflected back to a greater extent giving the impression that the colour is duller. These fibrils, however, can be degraded by the cellulase enzyme, restoring a smooth surface to the fibre and restoring the garment to its original colour. Softening The enzyme also has a significant softening effect on the fabric, probably due to the removal of the microfibrils. Soil removal Some dirt particles are trapped in the network of

microfibrils and are released when the microfibrils are removed by the cellulase enzyme.

In Leather Industry Alkaline proteases (0.1 – 1% w/w) are used to remove hair from hides. This method is more safer than the use of sodium sulphide. The dehaired are processed or bated further by using pancreatic enzymes for softness in appearance. (Bating is for soft leather clothing.)

Wool Industry Papain (protease) enzyme is used to the scales in wool fibres (wool fibres have lapping scales pointing towards the tip which lead to shrinkage). Addition of papain gives wool a silky appearance and adds value. This is not adopted nowadays due to high cost of papain.

Production Syrups 1. Glucose syrup Glucose syrup is produced from liquefied strarch of 8 – 12 DE (dextrose equivalent).

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DE =

Amount of reducing sugar expressed as glucose × 100 Total amount of carbohydrate

Liquified starch is obtained by heating to 105o C for 5 minutes. A 30 – 40% granular starch slurry (of 20 – 80 ppm Ca++) is obtained to which alpha-amylase has been added to about 0.5–0.6 kg/ton. The starch becomes gelatinized due to heat and then the temperature is lowered to 90 – 100oC and incubated for 1–2 hours. The enzyme alphaamylase digests the starch to produce soluble dextrin molecules. This process is called liquefaction. The liquefied starch (a small quantitiy) is spray-dried in foods and used as bulking agents. Starch slurry (30 _ 40%, pH 6.5, Ca ++ 40 ppm )

Gelatinized starch

Liquefied starch (18 _ 22 DE)

Glucose syrup (99 DE)

Maltose syrup (44 DE)

Fig 16.2 Production of Glucose and Maltose Syrups

Alpha-amylase is obtained from B. amyloliquefaciens and B. licheniformis. The debranching enzyme, pullulanase is obtained from B. acidopullulyticus (added to digest branched oligosaccharides) Glucoamylase is obtained from Aspergillus niger. The purification may be done by using activated charcoal and ion exchange resins. The syrup consists of 95 – 07 % glucose, 1 – 2% maltose and 0.5 – 2% isomaltose.

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2. Maltose syrup High conversion syrups are used to produced hard candy, in soft drinks, brewing and baking. It has 60 – 70 DE and it consists of 30 – 37 % maltose and 35 – 43% glucose, 10% maltotriose, 15% other oligosaccharides.

In Sugar Industry Invert sugar, a mixture of glucose and fructose can be produced from sucrose by action of yeast β-fructofuranosidase (invertase). Invert sugar is widely used in jam making. +H2O Glucose + Fructose. Sucrose 70% Invertase is added to sucrose (w/v) syrup at 50o C pH 4.5. This hydrolyses sucrose to glucose and fructose. Glucose + Fructose. Sucrose +H2O The resulting syrup is called “Invert syrup” is sweeter and less crystallize than pure sucrose syrup. Xylene is added to prevent microbial growth. The enzyme and xylene are removed by refining and evaporation. Invertase enzyme is used in soft or liquid confectioneries and then coated with chocolates. After few days / weeks the sucrose gets hydrolyzed and produces a soft centre in chocolates. In the sugar industries, the presence of dextran and raffinases produced in cane / sugar beet inhibits crystallization of sucrose. Therefore, dextranase enzyme (from Penicillium sp.,) degrade the dextran and raffinase is hydrolyzed to galactose and sucrose by a fungal raffinase.

In Brewing Industry Germinated barley seeds are used as raw material in brewing. The reserve starch is broken down by amylase. (The grains may be roasted to prevent further growth and to add flavour). The soluble material present is extracted by water (wort). Yeast is added to produce ethanol by alcoholic fermentation of glucose and maltose. Bacterial α-amylase (Bacillus subtilis) is even more heat stable than wheat α-amylase and is used extensively in brewing industries.

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Wort is prepared from unmalted barely (and maize). To it, fungal exo-1, 4-α-glucosidase, known as amylo-glucosidase or glucamylase (from Aspergillus niger) is added to cleave α-1,6 and α-1,4 glycosidic bonds so as to increase the yield of glucose and maltose from starch. In the production of glucose from starch, bacterial α-amylase may be used followed by fungal amyloglucosidase. (cleaving of glycosidic bonds). Glucose may also be obtained from cellulose containing waste products by treatment with cellulose (Aspergillus niger). The clarification of cider, wines and fruit juices (eg. apple) can be done using fungal pectinases. The pectins of fruits and vegetables play an important role in jam making (gel formation), however, they cause fruit drinks to be cloudy by preventing the flocculation of suspended particles. Pectinases are a group of enzymes including polygalacturonases (breaks the main chains of pectins) and pectinesterases (hydrolyze methyl esters). Their action releases the trapped particles and allow them to flocculate.

BIotecHnologIcal applIcatIons of enzyMes Although enzymes have been used in certain industrial processes for centuries, their precise role, or even their identity was not known over most of this period. They were often utilized as components of intact cells. E.g. Yeast in the baking and brewing industries, or as extracts containing a mixture of many enzymes. Malt, containing amylase and proteolytic enzymes. The first enzyme to be made commercially available in a partially purified form (by Hansen, 1874) was the acid protease, rennin (chymosin), a crude preparation obtained from the fourth stomach of young calves used to curdle milk in cheese production. Recombinant DNA technology can be used to increase the yield of enzymes already produced by microorganisms (E.g. Beta galactosidase and aspartate carbamoyl transferase by E.coli) or to produce completely different enzymes, including ones normally synthesized by eukaryotic cells. These techniques may even be used to produced enzymes of modified or synthesized genes. This is termed as protein engineering. Once a suitable strain of suitable microorganism

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has been found (or produced), a culture may be grown to produce a large quantity of the enzymes (loosely termed fermentation). In the same way, animal or plant cells could be cultivated to enable largescale extractions of their enzymes to be performed on commercial basis. Some enzymes for example, Aspergillus sp proteases may be obtained from semi-solid culture of low water content. A high degree of aeration at the surface apparently favours production of these enzymes. However, the vast majority are produced in aseptic conditions. In general, in a single batch fermentation procedure, the number of cells present may increase for about a day (termed the growth phase) until a limiting value is reached, but enzyme production is likely to continue at a significant level for several days after this. The culture medium must contain the essential nutrients needed by the microorganisms, together with any inducer necessary for the synthesis of the required enzyme. Alternatively, it may be possible by growing cells under conditions where the substrate of the enzyme is present in only limited amounts throughout. To isolate and culture a mutant strain which does not require induction, the synthesis of many catabolic enzyme is repressed if the cells are grown rapidly on readily utilizable carbohydrate or protein. This catabolic repression can be overcome if the rate of utilization of the carbon source is controlled, by feeding small amounts into the system at regular intervals, which is slowly hydrolyzed to yield the nutrient. Similarly, feedback repression of biosynthetic enzymes may occur in the presence of metabolic end products, so the build up of these should be avoided if the synthesis of such an enzyme is required. The problems of repression may also be overcome by the use of mutants.

analytIcal uses Use of enzymes for analytical purposes is very important. The endpoint reaction and kinetic analysis are possible by using enzymes. End point reaction refers to total conversion of substrate into products in the presence of enzymes in a few minutes. Kinetic analysis involves the rate of reaction and substrate / product concentration. E.g. alkaline phosphatase, beta-galactosidase, beta-lactamase. Enzymes are used in the analysis of antibodies immunoglobin, etc.

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Every enzyme catalyze a reaction, which is both substrate specific and product specific. These enzymes are used to estimate a specific substances, at very low concentration, in the presence of other chemically similar substances. Advantages 1. The enzyme-based methods of analysis are both specific and sensitive. 2. Enzyme-catalysed reactions make them suitable for analytical applications under relatively mild conditions, for example,

at near neutral pH and at room temperature. To minimize wastage, the use of immobilized enzymes are increasingly available and they can be recovered intact at the end of the procedure. The enzymes in this form will be more stable than in free solutions. Enzyme-based analytical procedures may be designed to determine the concentrations of substrates, coenzymes, activators and inhibitors.

Principles of Enzymatic Analysis 1. End-point methods 2. Kinetic methods 3. Immunoassay methods End-Point Methods End-point methods of enzymatic analysis are

also called total change equilibrium methods. The nature of the reaction and the conditions should be such that the position of equilibrium is very much in favour of product formation that is, the reaction goes very close to completion in which almost all the substrate is being converted to product. At equilibrium, the concentrations of activators and inhibitors do not affect the situation. Analysis should be performed under conditions where the concentration of the added enzyme is high, to ensure rapid progress towards equilibrium, while the concentration of the substrate under investigation should be low enough for the reaction to be first-order with respect to this substrate.

Applications of Enzymes

Vmax

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Zero-order reaction

V

First-order reaction [So]

Fig 16.3 Graph showing the order of reaction

The amount of sample used for analysis, therefore, should be in excess, so that they do not limit the reaction or change its first order characteristics (including co-substrate as NAD+). No products are initially present, so the concentrations of all products (including co-products such as NADH) at equilibrium will be dependent solely on the intial concentrations that can be measured (E.g. NADH by absorbance at 340 nm). If the reaction has gone to completion, then the final product concentration will be a measure of the intial substrate concentration. For any first order (or pseudo first-order) reaction S → P, the rate of reaction at time t is given by, – d[s] / dt = K[S] Where, [S] is the concentration of substrate at time t, K is a constant. By integration, T = (1 / K) loge([S0] / [S]) = (2.303 / K) log10 ([S0] / [S]) where, [S0] is the concentration of substrate at zero time. For an enzyme-catalyzed reaction S → P, which goes to completion in the absence of any appreciable back reaction, a steady state may be assumed to exist throughout.

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Therefore, the rate of reaction at time t, is given by – d[S] / dt = (Vmax [S]) / ([S] + Km) At very low substrate concentrations, [S] Vmax / Km

This ensures that the B being formed from A is quickly converted to C. If the coupled procedure is satisfactory, the final concentration of C (or of a coproduct of the indicator reaction) is a measure of the initial concentration of A. The main advantage of end-point methods of analysis is that they do not require constant attention. (A procedure may be set up and left to come to equilibrium). 2. Kinetic Methods Most procedures for enzymatic analysis involve steady-state kinetic methods.

The initial velocity of the reaction is determined at fixed temperature and pH and used to calculate the concentration of the substance being investigated. This procedure is used for the analysis of enzymes (that is, enzyme assay), substrate, coenzyme, activators and inhibitors. The initial velocity is directly proportional to the enzyme concentration if all other relevant factors are kept fixed and nonlimiting. It is arranged that all substrates which influence the rate of reaction are present in fixed and non-limiting concentrations, with the exception of the one being analyzed, whose concentration may thus be determined from the observed reaction rate. The relationship between substrate concentration and initial velocity is apparent from the Michaelis – Menten equation. V0 = Vmax [S0] / [S0] + Km = K2 [E0] [S0] / [S0] + Km Therefore, at constant [Eo], in the absence of inhibitors and at fixed concentrations of any activators and additional substrates, there is a hyperbolic relationship between V0 and [S0].

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Vmax

Vo

Vmax / 2

Km

[S]

Fig 16.4 Relationship between V0 and [S]

At low substrate concentrations there is a linear relationship between V0 and [S0] since if [S0]