ENZYMES Biochemistry, Biotechnology and Clinical Chemistry

ENZYMES Biochemistry, Biotechnology and Clinical Chemistry
 
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: pdf
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

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