| Author: |
Trevor Palmer & Philip L. Bonner
|
| Publisher: | Woodhead Publishing Limited |
| ISBN No: | 978-1-904275-27-5 |
| Release at: | 2007 |
| Pages: | 431 |
| Edition: |
Second Edition
|
| File Size: | 33 MB |
| File Type: | |
| Language: | English |
Description of ENZYMES Biochemistry, Biotechnology and Clinical Chemistry
This ENZYMES Biochemistry, Biotechnology and Clinical Chemistry book was written, as all textbooks should be, with the requirements of the student firmly in mind. It is intended to provide an informative introduction to Enzymology, and to give a balanced, reasonably-detailed, account of all the various theoretical and applied aspects of the subject which are likely to be included in an honours degree course.
Furthermore, some of the later chapters may serve as a bridge to more advanced texts for students wishing to proceed further in this area of biochemistry.
Content of ENZYMES Biochemistry, Biotechnology and Clinical Chemistry
Part 1: Structure and function of enzymes
1 An introduction to enzymes
1.1 What are enzymes? 2
1.2 A brief history of enzymes 2
1.3 The naming and classification of enzymes. 3
1.3 .1 Why classify enzymes? 3
1.3 .2 The Enzyme Commission's system of classification 4
1.3.3 The Enzyme Commission's recommendations on
nomenclature 5
1.3.4 The six main classes of enzymes . 6
Summary of Chapter 1 11
Further reading 11
Problems 11
2 The structure of proteins
2.1 Introduction 14
2.2 Amino acids, the building blocks of proteins 15
2.2.1 Structure and classification of amino acids 15
2.2.2 Stereochemistry of amino acids. 17
2.3 The basis of protein structure 18
2.3.1 Levels of protein structure. 18
2.3.2 Bonds involved in the maintenance of protein structure 19
2.4 The determination of primary structure. 21
2.4.1 The isolation of each polypeptide chain. 21
2.4.2 Determination of the amino acid composition of
each polypeptide chain 24
2.4.3 Determination of the amino acid sequence of each polypeptide chain 26
2.4.4 Determination of the positions of disulphide bridges 29
2.4.5 Some results of experimental investigation of primary structure .29
2.4.6 Indirect determination of primary structure 30
2.5 The determination of protein structure by X-ray crystallography .30
2.5.1 The principles of X-ray crystallography . 30
2.5.2 Some results of X-ray crystallography 35
2.6 The investigation of protein structure in solution .40
Summary of Chapter 2 42
Further reading 42
Problems. 43
3 The biosynthesis and properties of proteins
3 .1 The biosynthesis of proteins .44
3.1.1 The central dogma of molecular genetics .44
3.1.2 The double-helix structure of DNA .46
3.1.3 The translation of genetic information into protein structure. .48
3.1.4 Modification of protein structure after translation 51
3.1.5 Control of protein synthesis .52
3.1.6 Sequence determination .55
3.2 The properties of proteins 57
3.2.1 Chemical properties of proteins. .57
3.2.2 Acid-base properties of proteins. 58
3.2.3 The solubility of globular proteins 62
Summary of Chapter 3 64
Further reading 64
Problems 65
4 Specificity of enzyme action
4.1 Types of specificity 67
4.2 The active site 68
4.3 The Fischer 'lock-and-key' hypothesis 70
4.4 The Koshland 'induced-fit' hypothesis 70
4.5 Hypotheses involving strain or transition-state stabilization 72
4.6 Further comments on specificity 73
Summary of Chapter 4 74
Further reading . 75
5 Monomeric and oligomeric enzymes
5.1 Monomeric enzymes . 76
5.1.1 Introduction 76
5.1.2 The serine proteases 76
5.1.3 Some other monomeric enzymes 78
5.2 Oligomeric enzymes . 79
5.2.1 Introduction 79
5.2.2 Lactate dehydrogenase . 79
5.2.3 Lactose synthase . 81
5.2.4 Tryptophan synthase . 81
5.2.5 The pyruvate dehydrogenase multienzyme complex 82
Summary of Chapter 5 83
Further reading 83
Part 2 : Kinetic and chemical mechanisms of enzyme-catalysed reactions
6 An introduction to bioenergetics, catalysis and kinetics
6.1 Some concepts ofbioenergetics 85
6.1.1 The first and second laws of thermodynamics . 85
6.1.2 Enthalpy, entropy and free energy 85
6.1.3 Free energy and chemical reactions . 86
6.1.4 Standard free energy 87
6.1.5 Bioenergetics and the living cell . 88
6.2 Factors affecting the rates of chemical reactions 89
6.2.1 The collision theory 89
6.2.2 Activation energy and the transition-state theory . 89
6.2.3 Catalysis . 92
6.3 Kinetics ofuncatalysed chemical reactions . 93
6.3.l The Law of Mass Action and the order ofreaction 93
6.3.2 The use of initial velocity 95
6.4 Kinetics of enzyme-catalysed reactions: an historical introduction . 96
6.5 Methods used for investigating the kinetics of enzyme-catalysed
reactions . 98
6.5.1 Initial velocity studies 98
6.5.2 Rapid-reaction techniques 100
6.6 The nature of enzyme catalysis 100
Summary of Chapter 6 102
Further reading . 102
Problems . 102
7 Kinetics of single-substrate enzyme-catalysed reactions
7 .1 The relationship between initial velocity and substrate
concentration . 105
7.1.1 The Henri and Michaelis-Menten equations 105
7.1.2 The Briggs-Haldane modification of the Michaelis-Menten
equation 107
7.1.3 The significance of the Michaelis-Menten equation . 109
7 .1.4 The Lineweaver-Burk plot 111
7.1.5 The Eadie-Hofstee and Hanes plots 112
7.1.6 The Eisenthal and Comish-Bowden plot 114
7.1.7 The Haldane relationship for reversible reactions 115
7 .2 Rapid-reaction kinetics 116
7.2.1 Pre-steady-state kinetics . 116
7.2.2 Relaxation kinetics 120
7.3 The King and Altman procedure . 121
Sunimary of Chapter 7 . 124
Further reading . 124
Problems 125
8 Enzyme inhibition
8.1 Introduction 126
8.2 Reversible inhibition . 126
8.2.1 Competitive inhibition 126
8.2.2 Uncompetitive inhibition 133
8.2.3 Non-competitive inhibition 136
8.2.4 Mixed inhibition 140
8.2.5 Partial inhibition . 143
8.2.6 Substrate inhibition 144
8.2.7 Allosteric inhibition 146
8.3 Irreversible inhibition 147
Summary of Chapter 8 149
Further reading 150
Problems 150
9 Kinetics of multi-substrate enzyme-catalysed reactions
9.1 Examples of possible mechanisms . 153
9.1.1 Introduction 153
9 .1.2 Ping-pong bi-bi mechanism 153
9.1.3 Random-order mechanism 154
9.1.4 Compulsory-order mechanism . 154
9.2 Steady-state kinetics . 155
9.2.1 The general rate equation of Alberty 155
9.2.2 Plots for mechanisms which follow the general rate
equation . 157
9.2.3 The general rate equation of Dalziel 158
9.2.4 Rate constants and the constants of Alberty and Dalziel 158
9 .3 Investigation of reaction mechanisms using steady-state methods 160
9.3.1 The use of primary plots 160
9 .3 .2 The use of inhibitors which compete with substrates
for binding sites 161
9.4 Investigation of reaction mechanisms using non-steady-state
methods 165
9.4.1 Isotope exchange at equilibrium 165
9.4.2 Rapid-reaction studies 167
Summary of Chapter 9 . 168
Further reading . 168
Problems 168
10 The investigation of active site structure
10.1 The identification of binding sites and catalytic sites 173
10.1.l Trapping the enzyme-substrate complex 173
10.1.2 The use of substrate analogues 17 4
10.1.3 Enzyme modification by chemical procedures affecting amino acid side chains . 175
10.1.4 Enzyme modification by treatment with proteases 179
10.1.5 Enzyme modification by site-directed mutagenesis 179
10.1.6 The effect of changing pH 180
10.2 The investigation of the three-dimensional structures of
active sites . 185
Summary of Chapter 10 187
Further reading 187
Problem 188
11 The chemical nature of enzyme catalysis
11.1 An introduction to reaction mechanisms in organic chemistry 189
11.2 Mechanisms of catalysis 191
11.2.1 Acid-base catalysis 191
11.2.2 Electrostatic catalysis 192
11.2.3 Covalent catalysis . 192
11.2.4 Enzyme catalysis 193
11.3 Mechanisms of reactions catalysed by enzymes without cofactors 194
11.3.1 Introduction 194
11.3.2 Chymotrypsin 194
11.3.3 Ribonuclease 195
11.3.4 Lysozyme 196
11.3.5 Triose phosphate isomerase 199
11.4 Metal-activated enzymes and metalloenzymes 200
11.4.1 Introduction 200
11.4.2 Activation by alkali metal cations (Na+ and K+) 200
11.4.3 Activation by alkaline earth metal cations
(Ca2+ and Mg2l 201
11.4.4 Activation by transition metal cations (Cu, Zn, Mo, Fe and Co cations) 202
11.5 The involvement of coenzymes in enzyme-catalysed reactions 204
11.5.1 Introduction 204
11.5.2 Nicotinamide nucleotides (NAD+ and NADPl 205
11.5.3 Flavin nucleotides (FMN and FAD) 207
11.5.4 Adenosine phosphates (ATP, ADP and AMP) 210
11.5.5 Coenzyme A (CoA.SH) . 211
11.5.6 Thiamine pyrophosphate (TPP) . 212
11.5. 7 Pyridoxal phosphate 214
11.5.8 Biotin 216
11.5.9 Tetrahydrofolate 217
11.5.10 Coenzyme B12 . 218
Summary of Chapter 11 220
Further reading . 220
12 The binding of ligands to proteins
12.1 Introduction 222
12.2 The binding of a ligand to a protein having a single ligand-binding
site 222
12.3 Cooperativity 223
12.4 Positive homotropic cooperativity and the Hill equation 224
12.5 The Adair equation for the binding of a ligand to a protein
having two binding sites for that ligand 227
12.5.1 General considerations 227
12.5.2 Where there is no interaction between the binding sites 228
12.5.3 Where there is positive homotropic cooperativity 230
12.5.4 Where there is negative homotropic cooperativity 230
12.6 The Adair equation for the binding of a ligand to a protein
having three binding sites for that ligand 231
12. 7 The Adair equation for the binding of a ligand to a protein
having four binding sites for that ligand 232
12.8 Investigation of cooperative effects 232
12.8.1 Measurement of the relationship between Y and (S] 232
12.8.2 Measurement of the relationship between v0 and [So] 233
12.8.3 The Scatchard plot and equilibrium dialysis techniques 233
12.9 The binding ofoxygen to haemoglobin . 236
SummaryofChapter 12 . 237
Further reading . 237
Problems 238
13 Sigmoidal kinetics and allosteric enzymes
13 .1 Introduction 23 9
13 .2 The Monod-Wyman-Changeux (MWC) model 239
13.2.1 The MWC equation 239
13.2.2 How the MWC model accounts for cooperative effects . 242
13.2.3 The MWC model and allosteric regulation 242
13.2.4 The MWC model and the Hill equation 244
13.3 The Koshland-Nemethy-Filmer (KNF) model 245
13.3.1 The KNF model for a dimeric protein . 245
13.3.2 The KNF model for any oligomeric enzyme . 247
13 .3 .3 The KNF model and allosteric regulation . 248
13 .4 Differentiation between models for cooperative binding
in proteins 248
13.5 Sigmoidal kinetics in the absence of cooperative binding 249
13.5.1 Ligand-binding evidence versus kinetic evidence 249
13.5.2 The Ferdinand mechanism 250
13.5.3 The Rabin and mnemonical mechanisms 250
Summary of Chapter 13 251
Further reading 251
Problems 252
14 The significance of sigmoidal behaviour
14.1 The physiological importance of cooperative oxygen-binding
by haemoglobin 255
14.2 Allosteric enzymes and metabolic regulation 257
14.2.1 Introduction 257
14.2.2 Characteristics of steady-state metabolic pathways 258
14.2.3 Regulation of steady-state metabolic pathways by
control of enzyme activity 260
14.2.4 Allosteric enzymes and the amplification of metabolic
regulation . 262
14.2.5 Other mechanisms of metabolic regulation 263
14.2.6 Some examples of allosteric enzymes involved in
metabolic regulation 268
Summary of Chapter 14 271
Further reading 272
Part 3: Application of enzymology
15 Investigation of enzymes in biological preparations
15 .1 Choice of preparation for the investigation of enzyme
characteristics . 274
15.2 Enzyme assay 276
15.2.l Introduction 276
15 .2.2 Enzyme assay by kinetic determination of catalytic activity . 277
15.2.3 Coupled kinetic assays 280
15.2.4 Radioimmunoassay (RIA) of enzymes . 282
15 .3 Investigation of sub-cellular compartmentation of enzymes 284
15. 3 .1 Enzyme histochemistry 2 84
15 .3 .2 The use of centrifugation . 286
15.3.3 Some results of the investigation of enzyme
compartmentation 289
Summary of Chapter 15 291
Further reading 291
Problem 292
16 Extraction and purification of enzymes
16.1 Extraction of enzymes293
16.1.1 Introduction 293
16.1.2 The extraction of soluble enzymes . . . 293
16.1.3 The extraction of membrane-bound enzymes . 294
16.1.4 The nature of the extraction medium . 297
16.2 Purification of enzymes . 298
16.2.1 Preliminary purification procedures 298
16.2.2 Further purification procedures 299
16.2.3 Criteria of purity . 307
16.3 Determination of molecular weights of enzymes .311
Summary of Chapter 16 312
Furtherreading 313
Problem .314
17 Enzymes as analytical reagents
17 .1 The value of enzymes as analytical reagents . 315
17 .2 Principles of enzymatic analysis 316
17.2.l End-point methods 316
17.2.2 Kinetic methods 319
17.2.3 Immunoassay methods . 323
17.3 Handling enzymes and coenzymes . 324
Summary of Chapter 17 .326
Further reading .326
Problems . .327
18 Instrumental techniques available for use in enzymatic analysis
18.1 Principles of the available detection techniques .328
18.1.1 Introduction 328
18.1.2 Manometry 328
18.1.3 Spectrophotometry 329
18.1.4 Spectrofluorimetry 330
18.1.5 Electrochemical methods 331
18.1.6 Enthalpimetry . 334
18.1. 7 Radiochemical methods . 334
18.1.8 Dry-reagent techniques 335
18.2 Automation in enzymatic analysis 336
18.2.1 Introduction 336
18.2.2 Fixed-time methods 33 7
18.2.3 Fixed-concentration methods 339
18.2.4 Methods involving continuous monitoring 340
18.3 High-throughput assays (HTA) 341
Summary of Chapter 18 . 342
Further reading 342
19 Applications of enzymatic analysis in medicine, forensic science and industry
19.1 Applications in medicine 343
19 .1.1 Assay of plasma enzymes 343
19 .1.2 Enzymes and inborn errors of metabolism 348
19.1.3 Enzymes as reagents in clinical chemistry 352
19.2 Applications in forensic science 352
19.3 Applications in industry 353
Summary of Chapter 19 355
Further reading 355
20 Biotechnological applications of enzymes
20.1 Large-scale production and purification of enzymes 356
20.1.1 Production of enzymes on an industrial scale 356
20.1.2 Large-scale purification of enzymes 359
20.1.3 Synthesis of artificial enzymes . 360
20.2 Immobilized enzymes 361
20.2.1 Preparation of immobilized enzymes 361
20.2.2 Properties of immobilized enzymes 366
20.2.3 Applications of immobilized enzymes: general principles .368
20.3 Enzyme utilization in industry 369
20.3.1 Introduction 369
20.3.2 Applications in food and drink industries . 370
20.3.3 Applications in other industries . 373
Summary of chapter 20 . 374
Further reading 374
Problems 376
21 Genomics, proteomics and bioinformatics
21.1 Enzymes and recombinant DNA technology 377
21.1.1 Introduction 3 77
21.1.2 Applications 378
21.2 Proteomics 387
21.2.1 The application of mass spectrometry to the investigation
of the proteome 387
21.2.2 Proteomics research 389
21.3 Enzymes and bioinformatics . 390
21.3.l Introduction 390
21.3.2 Systems biology and microarrays 392
Summary of Chapter 21 393
Further reading 393
Problems 396
Answers to problems 397
Abbreviations 403
Index 405
1 An introduction to enzymes
1.1 What are enzymes? 2
1.2 A brief history of enzymes 2
1.3 The naming and classification of enzymes. 3
1.3 .1 Why classify enzymes? 3
1.3 .2 The Enzyme Commission's system of classification 4
1.3.3 The Enzyme Commission's recommendations on
nomenclature 5
1.3.4 The six main classes of enzymes . 6
Summary of Chapter 1 11
Further reading 11
Problems 11
2 The structure of proteins
2.1 Introduction 14
2.2 Amino acids, the building blocks of proteins 15
2.2.1 Structure and classification of amino acids 15
2.2.2 Stereochemistry of amino acids. 17
2.3 The basis of protein structure 18
2.3.1 Levels of protein structure. 18
2.3.2 Bonds involved in the maintenance of protein structure 19
2.4 The determination of primary structure. 21
2.4.1 The isolation of each polypeptide chain. 21
2.4.2 Determination of the amino acid composition of
each polypeptide chain 24
2.4.3 Determination of the amino acid sequence of each polypeptide chain 26
2.4.4 Determination of the positions of disulphide bridges 29
2.4.5 Some results of experimental investigation of primary structure .29
2.4.6 Indirect determination of primary structure 30
2.5 The determination of protein structure by X-ray crystallography .30
2.5.1 The principles of X-ray crystallography . 30
2.5.2 Some results of X-ray crystallography 35
2.6 The investigation of protein structure in solution .40
Summary of Chapter 2 42
Further reading 42
Problems. 43
3 The biosynthesis and properties of proteins
3 .1 The biosynthesis of proteins .44
3.1.1 The central dogma of molecular genetics .44
3.1.2 The double-helix structure of DNA .46
3.1.3 The translation of genetic information into protein structure. .48
3.1.4 Modification of protein structure after translation 51
3.1.5 Control of protein synthesis .52
3.1.6 Sequence determination .55
3.2 The properties of proteins 57
3.2.1 Chemical properties of proteins. .57
3.2.2 Acid-base properties of proteins. 58
3.2.3 The solubility of globular proteins 62
Summary of Chapter 3 64
Further reading 64
Problems 65
4 Specificity of enzyme action
4.1 Types of specificity 67
4.2 The active site 68
4.3 The Fischer 'lock-and-key' hypothesis 70
4.4 The Koshland 'induced-fit' hypothesis 70
4.5 Hypotheses involving strain or transition-state stabilization 72
4.6 Further comments on specificity 73
Summary of Chapter 4 74
Further reading . 75
5 Monomeric and oligomeric enzymes
5.1 Monomeric enzymes . 76
5.1.1 Introduction 76
5.1.2 The serine proteases 76
5.1.3 Some other monomeric enzymes 78
5.2 Oligomeric enzymes . 79
5.2.1 Introduction 79
5.2.2 Lactate dehydrogenase . 79
5.2.3 Lactose synthase . 81
5.2.4 Tryptophan synthase . 81
5.2.5 The pyruvate dehydrogenase multienzyme complex 82
Summary of Chapter 5 83
Further reading 83
Part 2 : Kinetic and chemical mechanisms of enzyme-catalysed reactions
6 An introduction to bioenergetics, catalysis and kinetics
6.1 Some concepts ofbioenergetics 85
6.1.1 The first and second laws of thermodynamics . 85
6.1.2 Enthalpy, entropy and free energy 85
6.1.3 Free energy and chemical reactions . 86
6.1.4 Standard free energy 87
6.1.5 Bioenergetics and the living cell . 88
6.2 Factors affecting the rates of chemical reactions 89
6.2.1 The collision theory 89
6.2.2 Activation energy and the transition-state theory . 89
6.2.3 Catalysis . 92
6.3 Kinetics ofuncatalysed chemical reactions . 93
6.3.l The Law of Mass Action and the order ofreaction 93
6.3.2 The use of initial velocity 95
6.4 Kinetics of enzyme-catalysed reactions: an historical introduction . 96
6.5 Methods used for investigating the kinetics of enzyme-catalysed
reactions . 98
6.5.1 Initial velocity studies 98
6.5.2 Rapid-reaction techniques 100
6.6 The nature of enzyme catalysis 100
Summary of Chapter 6 102
Further reading . 102
Problems . 102
7 Kinetics of single-substrate enzyme-catalysed reactions
7 .1 The relationship between initial velocity and substrate
concentration . 105
7.1.1 The Henri and Michaelis-Menten equations 105
7.1.2 The Briggs-Haldane modification of the Michaelis-Menten
equation 107
7.1.3 The significance of the Michaelis-Menten equation . 109
7 .1.4 The Lineweaver-Burk plot 111
7.1.5 The Eadie-Hofstee and Hanes plots 112
7.1.6 The Eisenthal and Comish-Bowden plot 114
7.1.7 The Haldane relationship for reversible reactions 115
7 .2 Rapid-reaction kinetics 116
7.2.1 Pre-steady-state kinetics . 116
7.2.2 Relaxation kinetics 120
7.3 The King and Altman procedure . 121
Sunimary of Chapter 7 . 124
Further reading . 124
Problems 125
8 Enzyme inhibition
8.1 Introduction 126
8.2 Reversible inhibition . 126
8.2.1 Competitive inhibition 126
8.2.2 Uncompetitive inhibition 133
8.2.3 Non-competitive inhibition 136
8.2.4 Mixed inhibition 140
8.2.5 Partial inhibition . 143
8.2.6 Substrate inhibition 144
8.2.7 Allosteric inhibition 146
8.3 Irreversible inhibition 147
Summary of Chapter 8 149
Further reading 150
Problems 150
9 Kinetics of multi-substrate enzyme-catalysed reactions
9.1 Examples of possible mechanisms . 153
9.1.1 Introduction 153
9 .1.2 Ping-pong bi-bi mechanism 153
9.1.3 Random-order mechanism 154
9.1.4 Compulsory-order mechanism . 154
9.2 Steady-state kinetics . 155
9.2.1 The general rate equation of Alberty 155
9.2.2 Plots for mechanisms which follow the general rate
equation . 157
9.2.3 The general rate equation of Dalziel 158
9.2.4 Rate constants and the constants of Alberty and Dalziel 158
9 .3 Investigation of reaction mechanisms using steady-state methods 160
9.3.1 The use of primary plots 160
9 .3 .2 The use of inhibitors which compete with substrates
for binding sites 161
9.4 Investigation of reaction mechanisms using non-steady-state
methods 165
9.4.1 Isotope exchange at equilibrium 165
9.4.2 Rapid-reaction studies 167
Summary of Chapter 9 . 168
Further reading . 168
Problems 168
10 The investigation of active site structure
10.1 The identification of binding sites and catalytic sites 173
10.1.l Trapping the enzyme-substrate complex 173
10.1.2 The use of substrate analogues 17 4
10.1.3 Enzyme modification by chemical procedures affecting amino acid side chains . 175
10.1.4 Enzyme modification by treatment with proteases 179
10.1.5 Enzyme modification by site-directed mutagenesis 179
10.1.6 The effect of changing pH 180
10.2 The investigation of the three-dimensional structures of
active sites . 185
Summary of Chapter 10 187
Further reading 187
Problem 188
11 The chemical nature of enzyme catalysis
11.1 An introduction to reaction mechanisms in organic chemistry 189
11.2 Mechanisms of catalysis 191
11.2.1 Acid-base catalysis 191
11.2.2 Electrostatic catalysis 192
11.2.3 Covalent catalysis . 192
11.2.4 Enzyme catalysis 193
11.3 Mechanisms of reactions catalysed by enzymes without cofactors 194
11.3.1 Introduction 194
11.3.2 Chymotrypsin 194
11.3.3 Ribonuclease 195
11.3.4 Lysozyme 196
11.3.5 Triose phosphate isomerase 199
11.4 Metal-activated enzymes and metalloenzymes 200
11.4.1 Introduction 200
11.4.2 Activation by alkali metal cations (Na+ and K+) 200
11.4.3 Activation by alkaline earth metal cations
(Ca2+ and Mg2l 201
11.4.4 Activation by transition metal cations (Cu, Zn, Mo, Fe and Co cations) 202
11.5 The involvement of coenzymes in enzyme-catalysed reactions 204
11.5.1 Introduction 204
11.5.2 Nicotinamide nucleotides (NAD+ and NADPl 205
11.5.3 Flavin nucleotides (FMN and FAD) 207
11.5.4 Adenosine phosphates (ATP, ADP and AMP) 210
11.5.5 Coenzyme A (CoA.SH) . 211
11.5.6 Thiamine pyrophosphate (TPP) . 212
11.5. 7 Pyridoxal phosphate 214
11.5.8 Biotin 216
11.5.9 Tetrahydrofolate 217
11.5.10 Coenzyme B12 . 218
Summary of Chapter 11 220
Further reading . 220
12 The binding of ligands to proteins
12.1 Introduction 222
12.2 The binding of a ligand to a protein having a single ligand-binding
site 222
12.3 Cooperativity 223
12.4 Positive homotropic cooperativity and the Hill equation 224
12.5 The Adair equation for the binding of a ligand to a protein
having two binding sites for that ligand 227
12.5.1 General considerations 227
12.5.2 Where there is no interaction between the binding sites 228
12.5.3 Where there is positive homotropic cooperativity 230
12.5.4 Where there is negative homotropic cooperativity 230
12.6 The Adair equation for the binding of a ligand to a protein
having three binding sites for that ligand 231
12. 7 The Adair equation for the binding of a ligand to a protein
having four binding sites for that ligand 232
12.8 Investigation of cooperative effects 232
12.8.1 Measurement of the relationship between Y and (S] 232
12.8.2 Measurement of the relationship between v0 and [So] 233
12.8.3 The Scatchard plot and equilibrium dialysis techniques 233
12.9 The binding ofoxygen to haemoglobin . 236
SummaryofChapter 12 . 237
Further reading . 237
Problems 238
13 Sigmoidal kinetics and allosteric enzymes
13 .1 Introduction 23 9
13 .2 The Monod-Wyman-Changeux (MWC) model 239
13.2.1 The MWC equation 239
13.2.2 How the MWC model accounts for cooperative effects . 242
13.2.3 The MWC model and allosteric regulation 242
13.2.4 The MWC model and the Hill equation 244
13.3 The Koshland-Nemethy-Filmer (KNF) model 245
13.3.1 The KNF model for a dimeric protein . 245
13.3.2 The KNF model for any oligomeric enzyme . 247
13 .3 .3 The KNF model and allosteric regulation . 248
13 .4 Differentiation between models for cooperative binding
in proteins 248
13.5 Sigmoidal kinetics in the absence of cooperative binding 249
13.5.1 Ligand-binding evidence versus kinetic evidence 249
13.5.2 The Ferdinand mechanism 250
13.5.3 The Rabin and mnemonical mechanisms 250
Summary of Chapter 13 251
Further reading 251
Problems 252
14 The significance of sigmoidal behaviour
14.1 The physiological importance of cooperative oxygen-binding
by haemoglobin 255
14.2 Allosteric enzymes and metabolic regulation 257
14.2.1 Introduction 257
14.2.2 Characteristics of steady-state metabolic pathways 258
14.2.3 Regulation of steady-state metabolic pathways by
control of enzyme activity 260
14.2.4 Allosteric enzymes and the amplification of metabolic
regulation . 262
14.2.5 Other mechanisms of metabolic regulation 263
14.2.6 Some examples of allosteric enzymes involved in
metabolic regulation 268
Summary of Chapter 14 271
Further reading 272
Part 3: Application of enzymology
15 Investigation of enzymes in biological preparations
15 .1 Choice of preparation for the investigation of enzyme
characteristics . 274
15.2 Enzyme assay 276
15.2.l Introduction 276
15 .2.2 Enzyme assay by kinetic determination of catalytic activity . 277
15.2.3 Coupled kinetic assays 280
15.2.4 Radioimmunoassay (RIA) of enzymes . 282
15 .3 Investigation of sub-cellular compartmentation of enzymes 284
15. 3 .1 Enzyme histochemistry 2 84
15 .3 .2 The use of centrifugation . 286
15.3.3 Some results of the investigation of enzyme
compartmentation 289
Summary of Chapter 15 291
Further reading 291
Problem 292
16 Extraction and purification of enzymes
16.1 Extraction of enzymes293
16.1.1 Introduction 293
16.1.2 The extraction of soluble enzymes . . . 293
16.1.3 The extraction of membrane-bound enzymes . 294
16.1.4 The nature of the extraction medium . 297
16.2 Purification of enzymes . 298
16.2.1 Preliminary purification procedures 298
16.2.2 Further purification procedures 299
16.2.3 Criteria of purity . 307
16.3 Determination of molecular weights of enzymes .311
Summary of Chapter 16 312
Furtherreading 313
Problem .314
17 Enzymes as analytical reagents
17 .1 The value of enzymes as analytical reagents . 315
17 .2 Principles of enzymatic analysis 316
17.2.l End-point methods 316
17.2.2 Kinetic methods 319
17.2.3 Immunoassay methods . 323
17.3 Handling enzymes and coenzymes . 324
Summary of Chapter 17 .326
Further reading .326
Problems . .327
18 Instrumental techniques available for use in enzymatic analysis
18.1 Principles of the available detection techniques .328
18.1.1 Introduction 328
18.1.2 Manometry 328
18.1.3 Spectrophotometry 329
18.1.4 Spectrofluorimetry 330
18.1.5 Electrochemical methods 331
18.1.6 Enthalpimetry . 334
18.1. 7 Radiochemical methods . 334
18.1.8 Dry-reagent techniques 335
18.2 Automation in enzymatic analysis 336
18.2.1 Introduction 336
18.2.2 Fixed-time methods 33 7
18.2.3 Fixed-concentration methods 339
18.2.4 Methods involving continuous monitoring 340
18.3 High-throughput assays (HTA) 341
Summary of Chapter 18 . 342
Further reading 342
19 Applications of enzymatic analysis in medicine, forensic science and industry
19.1 Applications in medicine 343
19 .1.1 Assay of plasma enzymes 343
19 .1.2 Enzymes and inborn errors of metabolism 348
19.1.3 Enzymes as reagents in clinical chemistry 352
19.2 Applications in forensic science 352
19.3 Applications in industry 353
Summary of Chapter 19 355
Further reading 355
20 Biotechnological applications of enzymes
20.1 Large-scale production and purification of enzymes 356
20.1.1 Production of enzymes on an industrial scale 356
20.1.2 Large-scale purification of enzymes 359
20.1.3 Synthesis of artificial enzymes . 360
20.2 Immobilized enzymes 361
20.2.1 Preparation of immobilized enzymes 361
20.2.2 Properties of immobilized enzymes 366
20.2.3 Applications of immobilized enzymes: general principles .368
20.3 Enzyme utilization in industry 369
20.3.1 Introduction 369
20.3.2 Applications in food and drink industries . 370
20.3.3 Applications in other industries . 373
Summary of chapter 20 . 374
Further reading 374
Problems 376
21 Genomics, proteomics and bioinformatics
21.1 Enzymes and recombinant DNA technology 377
21.1.1 Introduction 3 77
21.1.2 Applications 378
21.2 Proteomics 387
21.2.1 The application of mass spectrometry to the investigation
of the proteome 387
21.2.2 Proteomics research 389
21.3 Enzymes and bioinformatics . 390
21.3.l Introduction 390
21.3.2 Systems biology and microarrays 392
Summary of Chapter 21 393
Further reading 393
Problems 396
Answers to problems 397
Abbreviations 403
Index 405
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