Ebook Enzymes - Biochemistry, biotechnology and clinical chemistry (2/E): Part 1

ENZYMES:
Biochemistry, Biotechnology and Clinical Chemistry

Second Edition

"Talking of education, people have now a-days" (said he) "got a
strange opinion that every thing should be taught by lectures. Now, I
cannot see that lectures can do so much good as reading the books
from which the lectures are taken. I know nothing that can be best
taught by lectures, except where experiments are to be shewn. You
may teach chymestry by lectures - You might teach making of shoes
by lectures!"
James Boswell: Life ofSamuel Johnson, 1766


ABOUT THE AUTHORS
Trevor Palmer was born in South Yorkshire and graduated from Cambridge
University in 1966 with an honours degree in biochemistry, being influenced
by (amongst others) Peter Sykes in organic chemistry and Malcolm Dixon in
enzymology. He then worked as a clinical biochemist at the Queen Elizabeth
Hospital for Children, linked to the Institute of Child Health, University of
London, obtaining a PhD for research into inherited disorders. From this
emerged the two main interests of his subsequent career, enzymology and
evolution, the latter stimulating a further interest in the long-term effects of
natural catastrophes. He moved to Nottingham Trent University (then Trent
Polytechnic) in 1974, initially as a lecturer in biochemistry, before becoming
Head of Department of Life Sciences (1987), Dean of the Faculty of Science
and Mathematics (1992), Senior Dean of the University (1998) and Pro
Vice-Chancellor for Academic Development (2002), returning to
predominantly academic activity as Emeritus Professor in 2006. His books

include Understanding Enzymes (1981), Principles of Enzymology for
Technological Applications (1993), Controversy - Catastrophism and
Evolution (1999) and Perilous Planet Earth (2003). His wife, Jan, teaches
psychology and sociology (and is currently a part-time PhD student at
Leicester University). Their son, James, is carrying out postdoctoral studies
as a Leverhulme Fellow at Nottingham University and their daughter,
Caroline, is researching for a PhD at Sheffield University.
Philip L. Bonner went to school in Coventry, West Midlands, before
graduating from the University of Sussex in 1978 with an honours degree in
biochemistry. He then worked as a research assistant at Glaxo plc on
Merseyside before leaving to take up a Research Assistant/Demonstrator
post at Trent Polytechnic, where he obtained a PhD for research concerning
enzymes associated with seed germination. Several postdoctoral
appointments followed, at Bristol, Lancaster and Central Lancashire
Universities, working on a variety of topics including relaxin, aspartate
kinase and phospholipase C, before he was appointed as Senior Lecturer at
Nottingham Trent University in 1991. There, he has maintained his research
interests in enzymology and analytical biochemistry, working on the role of
transglutaminase in plant/animal tissue and methods to isolate and
characterise post-translationally-modified MHC peptides. His first singleauthor book, on protein purification, was published in 2007. His wife, Liz, is
a manager of an occupational therapist team in Nottingham and their
daughter, Francesca, is at junior school.


ENZYMES:
Biochemistry, Biotechnology and Clinical Chemistry
Second Edition

Trevor Palmer, BA, PhD, CBiol, FIBiol, FIBMS, FHEA
Emeritus Professor in Life Sciences

Nottingham Trent University
Philip L. Bonner, BSc, PhD
Senior Lecturer in Biochemistry
Nottingham Trent University

WP
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For:
Caroline, Francesca, James, Jan and Liz
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First edition published by Horwood Publishing Limited, 2001
Second edition published by Horwood Publishing Limited, 2007
Reprinted by Woodhead Publishing Limited, 2011
© T. Palmer and P.L. Bonner, 2007
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Printed by Lightning Source


Table of Contents
Authors' preface ............................................................................. xiv
Part 1 : Structure and function ofenzymes

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


vi

Table of Contents
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


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vii

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


viii

Table of Contents

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 ....................................... 174
10.1.3 Enzyme modification by chemical procedures affecting

amino acid side chains ................................................ 175


Table of Contents

ix

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 B 12 ......................................................... 218
Summary of Chapter 11 .............................................................. 220
Further reading ......................................................................... 220

12 The binding of ligands to proteins
12.1 Introduction ...................................................................... 222


x

Table of Contents
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


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xi

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 enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. .. 293
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


xii

Table of Contents

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


Table of Contents

Xlll

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 ........................................................... 377
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


Authors' Preface

This 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.
Although the book is intended mainly for students taking first degree courses
which have a substantial biochemistry component, large portions may be of value to
students on comparable courses in biological sciences, biomedical sciences or
forensic sciences, and even to ones emolled on, in one direction, foundation
programmes, or, in the other, MSc or other advanced courses who are approaching
the subject of enzymology for the first time (or the first time in many years).
No previous knowledge of biochemistry, and little of chemistry, is assumed.
Most scientific terms are defined and placed in context when they are first
introduced. Enzymology inevitably involves a certain amount of elementary
mathematics, and some of the equations which are derived may appear somewhat
complicated at first sight; however, once the initial biochemical assumptions have
been understood, the derivations usually follow on the basis of simple logic, without
involving any difficult mathematical manipulations. Numerical and other problems
(with answers) are included, to test and reinforce the student's grasp of certain
points. These problems generally use hypothetical data, although the results are
often based on findings reported in the biochemical literature.

If the size of the book is to be kept reasonable, some things of value have to be
left out. The chief aim of this particular book is to help the student understand the
concepts involved in enzymology, and the historical context in which they were
worked out. It is not a reference book for practising enzymologists, so no
comprehensive tables of data or long, finely-detailed accounts are included. Instead,
an attempt has been made to give a perspective of each topic, and examples are
quoted where appropriate. Credit has been given wherever possible to those
responsible for the development of the subject, but many names deserving of
mention have been excluded for reasons of space.


Authors' Preface

xv

Individual scientific papers have not, in general, been referred to, but at the end of
each chapter is a list of relevant books and articles, to provide context and an up-to
date viewpoint, from which references to the original papers may usually be
obtained.
As with any book at this level, certain topics have been presented in a simplified
(possibly even over-simplified) form. However, a considerable effort has been made
to avoid giving a distorted account of any topic. It is hoped that this book can
provide a foundation for those wishing to pursue more advances studies, and that
nothing learned from it will have to be 'unlearned' later. There are good reasons for
thinking that this is a realistic hope.
For this second edition of Enzymes, we have revised and updated the first edition,
reducing coverage of techniques whose use is declining to make room for discussion
of topics of greater current and future interest, e.g. expanded bed chromatography,
affinity precipitation, immobilized metal affinity chromatography, hydroxyapatite
chromatography, hydrophobic charge induction chromatography, lectin affmity

chromatography, covalent chromatography, membrane technology, capillary
electrophoresis, absorbance fluorescence and lumimetric methods, high-throughput
screening methods, 6-His tag and fusion protein technology, mass spectrometry and
the use of protein arrays. A completely new section has been added on the use of
enzymatic analysis in forensic science, and the final chapter of the first edition has
been split into two to allow greater discussion of the rapidly-expanding areas of
genomics, bioinformatics and proteomics. Elsewhere, coverage of protein structure,
synthesis and function and mechanisms of enzyme activity has been revised to take
into account recent developments (e.g. concerning the mechanism of action of
lysozyme).
Acknowledgements. In the preparation of this new edition, we are grateful for
the help of many people, including Lesley Atherton, Mark Crowley, Nick Howard,
Elaine James, Caroline Palmer, Jan Palmer and Karen Roberts. However, any errors
of fact or interpretation which may have crept into the book are entirely our own
responsibility, and we would be grateful if we could be informed about them.
Finally, we would like to acknowledge the helpful cooperation of the staff of
Horwood Publishing and, in particular, to express our gratitude to, and admiration
for, the distinguished scientific publisher, the late Ellis Horwood, without whom this
book would never have come into being.
Trevor Palmer and Philip Bonner, 2007



1
An Introduction to Enzymes
1.1 WHAT ARE ENZYMES?
Enzymes are biological catalysts. They increase the rate of chemical reactions taking
place within living cells without themselves suffering any overall change. The
reactants of enzyme-catalysed reactions are termed substrates. Each enzyme is
quite specific in character, acting on a particular substrate or substrates to produce a

particular product or products.
All enzymes are proteins. However, without the presence of a non-protein
component called a cofactor, many enzyme proteins lack catalytic activity. When
this is the case, the inactive protein component of an enzyme is termed the
apoenzyme, and the active enzyme, including cofactor, the holoenzyme. The
cofactor may be an organic molecule, when it is known as a coenzyme, or it may be
a metal ion. Some enzymes bind cofactors more tightly than others. When a cofactor
is bound so tightly that it is difficult to remove without damaging the enzyme, it is
sometimes called a prosthetic group.
To summarize diagrammatically:
COENZYME
INACTIVE PROTEIN+ COFACTOR
METAL ION
ENZYME
ACTIVE PROTEIN

<

<

As we shall see later, both the protein and cofactor components may be directly
involved in the catalytic processes taking place.

1.2 A BRIEF HISTORY OF ENZYMES
Until the nineteenth century, it was considered that processes such as the souring of
milk and the fermentation of sugar to alcohol could only take place through the
action of a livitJg organism. In 1833, the active agent breaking down the sugar was
partially isolated and given the name diastase (now known as amylase).

Sec 1.3]

The naming and classification of enzymes

3

A little later, a substance which digested dietary protein was extracted from
gastric juice and called pepsin. These and other active preparations were given the
general name ferments. Justus von Liebig recognized that these ferments could be
non-living materials obtained from living cells, but Louis Pasteur and others still
maintained that ferments must contain living material.
While this dispute continued, the term ferment was gradually replaced by the
name enzyme. This was first proposed by Wilhelm Kfthne in 1878, and comes from
the Greek, enzume (i:v?;vµq), meaning 'in yeast'. Appropriately, it was in yeast that a
factor was discovered which settled the argument in favour of the inanimate theory
of catalysis: brothers Eduard and Hans Buchner showed, in 1897, that sugar
fermentation could take place when a yeast cell extract was added even though no
living cells were present.
In 1926, James Sumner crystallized urease from jack-bean extracts and, in the
next few years, many other enzymes were purified and crystallized. Once pure
enzymes were available, their structure and properties could be determined, and the
findings form the material for most of this book.
Today, enzymes still form a major subject for academic research. They are
investigated in hospitals as an aid to diagnosis and, because of their specificity of
action, are of great value as analytical reagents. Enzymes are still widely used in
industry, continuing and extending many processes which have been used since the
dawn of history.
1.3 THE NAMING AND CLASSIFICATION OF ENZYMES
1.3.1 Why classify enzymes?
There is a long tradition of giving enzymes names ending in '-ase'. The only major

exceptions to this are the proteolytic enzymes, i.e. ones involved in the breakdown
of proteins, whose names usually end with '-in', e.g. trypsin.
The names of enzymes usually indicate the substrate involved. Thus, lactase
catalyses the hydrolysis of the disaccharide lactose to its component
monosaccharides, glucose and galactose:
(1.1)
lactose

glucose

galactose

The name lactase is a contraction of the clumsy, but more precise, lactosase. The
former is used because it sounds better but it introduces a possible trap for the
unwary because it could easily suggest an enzyme acting on the substrate lactate.
There is nothing in the name of this enzyme or many others to indicate the type of
reaction being catalysed. Fumarase, for example, by analogy with lactase might be
supposed to catalyse a hydrolytic reaction, but, in fact, it hydrates fumarate to form
malate:
-02C.CH=CH.co2 + H20
fumarate

-0 2C.CHOH.CH2C02
malate

(1.2)


An Introduction to Enzymes

4

[Ch. 1

The names of other enzymes, e.g. transcarboxylase, indicate the nature of the
reaction without specifying the substrates (which in the case of transcarboxylase are
methylmalonyl-CoA and pyruvate). Some names, such as catalase, indicate neither
the substrate nor the reaction (catalase mediates the decomposition of hydrogen
peroxide).
Needless to say, whenever a new enzyme has been characterized, great care has
usually been taken not to give it exactly the same name as an enzyme catalysing a
different reaction. Also, the names of many enzymes make clear the substrate and
the nature of the reaction being catalysed. For example, there is little ambiguity
about the reaction catalysed by malate dehydrogenase. This enzyme mediates the
removal of hydrogen from malate to produce oxaloacetate:
-0 2 C.C.CH 2 .C0.2 + NADH + H+ (1.3)

II

0

oxaloacetate

However, malate dehydrogenase, like many other enzymes, has been known by
more than one name.
So, because of the lack of consistency in the nomenclature, it became apparent as
the list of known enzymes rapidly grew that there was a need for a systematic way
of naming and classifying enzymes. A commission was appointed by the
International Union of Biochemistry (later re-named the International Union of
Biochemistry and Molecular Biology, IUBMB), and its report, published in 1964,

forms the basis of the currently accepted system. Revised editions of the report were
published in 1972, 1978, 1984 and 1992. An electronic version is now maintained
by the IUBMB on an accessible web-site, and this is updated on a regular basis.

1.3.2 The Enzyme Commission's system of classification
The Enzyme Commission divided enzymes into six main classes, on the basis of the
total reaction catalysed. Each enzyme was assigned a code number, consisting of
four elements, separated by dots. The first digit shows to which of the main classes
the enzyme belongs, as follows:

First digit Enzyme class
Oxidoreductases

2

Transferases

3

Hydro lases

Type of reaction catalysed
Oxidation/Reduction reactions
Transfer of an atom or group between two molecules
(excluding reactions in other classes)
Hydrolysis reactions

4

Lyases

Removal of a group from substrate
(not by hydrolysis)

5

Isomerases

Isomerization reactions

6

Ligases

The synthetic joining of two molecules, coupled with
the breakdown of the pyrophosphate bond in a
nucleoside triphosphate


Sec. 1.3]

The naming and classification of enzymes

5

The second and third digits in the code further describe the kind of reaction being
catalysed. There is no general rule, because the meanings of these digits are defmed
separately for each of the main classes. Some examples are given later in this
chapter. Note that, for convenience, and in line with normal practice, some
structures are written in a slightly simplified form in the lists provided. So, for

example, in the case of the acyl group, which is transferred in reactions catalysed by
E.C. 2.3 enzymes, it should be understood that the structure written -COR
represents:

-C-R

II

0
Enzymes catalysing very similar but non-identical reactions, e.g. the hydrolysis of
different carboxylic acid esters, will have the same first three digits in their code.
The fourth digit distinguishes between them by defining the actual substrate, e.g. the
actual carboxylic acid ester being hydrolysed.
However, it should be noted that isoenzymes, that is to say, different enzymes
catalysing identical reactions, will have the same four figure classification. There
are, for example, five different isoenzymes of lactate dehydrogenase within the
human body and these will have an identical code. The classification, therefore,
provides only the basis for a unique identification of an enzyme. The particular
isoenzyme and its source still have to be specified.
It should also be noted that all reactions catalysed by enzymes are reversible to
some degree and the classification which would be given to the enzyme for the
catalysis of the forward reaction would not be the same as that for the reverse
reaction. The classification used is that of the most important direction from the
biochemical point of view, or according to some convention defined by the
Commission. For example, for oxidation/reduction involving the interconversion of
NADH and NAD+ (see section 11.5.2) the classification is usually based on the
direction where NAD+ is the electron acceptor rather than that where NADH is the
electron donor.
Some problems are given at the end of this chapter to help the student become
familiar with this system of classification.

1.3.3 The Enzyme Commission's recommendations on nomenclature
The Commission assigned to each enzyme a systematic name in addition to its
existing trivial name. This systematic name includes the name of the substrate or
substrates in full and a word ending in '-ase' indicating the nature of the process
catalysed. This word is either one of the six main classes of enzymes or a
subdivision of one of them. When a reaction involves two types of overall change,
e.g. oxidation and decarboxylation, the second function is indicated in brackets, e.g.
oxidoreductase (decarboxylating). Examples are given below.
The systematic name and the Enzyme Commission (E.C.) classification number
unambiguously describe the reaction catalysed by an enzyme and should always be
included in a report of an investigation of an enzyme, together with the source of
enzyme, e.g. rat liver mitochondria.


An Introduction to Enzymes

6

[Ch. 1

However, these names are likely to be long and unwieldy. Trivial names may,
therefore, be used in a communication, once they have been introduced and defined
in terms of the systematic name and E.C. number. Trivial names are also inevitably
used in everyday situations in the laboratory. The Enzyme Commission made
recommendations as to which trivial names were acceptable, altering those which
were considered vague or misleading. Thus, 'fumarase', mentioned above, was
considered unsatisfactory and was replaced by 'fumarate hydratase'.

1.3.4 The six main classes of enzymes
Main Class 1: Oxidoreductases

These enzymes catalyse the transfer of H atoms, 0 atoms or electrons from one
substrate to another. The second digit in the code number of oxidoreductases
indicates the donor of the reducing equivalents (hydrogen or electrons) involved in
the reaction. For example:
Second digit
1
2
3
4
5
6

Hydrogen or electron donor
alcohol (>CHOH)
aldehyde or ketone (>C=O)
--CH.CHprimary amine (>CHNH2 or >CHNH3"')
secondary amine (>CHNH-)
NADH or NADPH (only when some other redox catalyst
is the acceptor)

The third digit refers to the hydrogen or electron acceptor, as follows:

Third digit
1
2
3

99

Hydrogen or electron acceptor

NAD+ or NADP+
Fe3+ (e.g. cytochrome)
02
An otherwise unclassified acceptor

Trivial names of oxidoreductases include oxidases (transfer of H to 0 2) and
dehydrogenases (transfer ofH to an acceptor other than 0 2). These often indicate the
identity of the donor and/or acceptor. Here are some examples:
(S)-lactate: NAD+ oxidoreductase
dehydrogenase, catalyses the reaction:
CH3.CH.co2 +NAD+

I

OH
(S)-lactate

(E.C.

1.1.1.27),

trivial

CH3 c.co-2 + NADH + H+

·11

name

lactate

(1.4)

0
pyruvate

Note that it is the alcohol group oflactate, rather than the carboxyl group, which is
involved in the reaction and this is indicated in the classification.


Sec. 1.3]

The naming and classification of enzymes

7

Isocitrate: NAD+ oxidoreductase (decarboxylating) (E.C. 1.1.1.41), trivial name
isocitrate dehydrogenase, catalyses:
-02C.CH2.CH.c.co2 + NADH + H+ + C02

II

0

(1.5)

2-oxoglutarate

isocitrate

D-amino acid: oxygen oxidoreductase (deaminating) (E.C. 1.4.3.3), trivial name Damino acid oxidase, catalyses:

D-amino acid

oxoacid

Note that this enzyme is less specific than most and will act on any D-amino acid.
Main Class 2: Trans/erases
These catalyse reactions of the type:

AX+ B

BX + A

but specifically exclude oxidoreductase and hydrolase reactions. In general, the
Enzyme Commission recommends that the names of transferases should end 'Xtransferase', where Xis the group transferred, although a name ending 'trans-X-ase'
is an acceptable alternative. The second digit in the classification describes the type
of group transferred. For example:
Second digit
1
2
3

4
7

Group transferred
I -carbon group
aldehyde or ketone group (>C=O)
acyl group (--COR)

glycosyl (carbohydrate group)
phosphate group

In general, the third digit further describes the group transferred. Thus:
E.C. 2.1.1 enzyme are methyltransferases (transfer --CH3), whereas
E.C. 2.1.2 enzymes are hydroxymethyltransferases (transfer --CH20H) and
E.C. 2.1.3 enzymes are carboxyl transferases (transfer --COOR)
or carbamoyl transferases (transfer --CONH2).
Similarly,
E.C. 2.4.1 enzymes are hexosyltransferases (transfer hexose units), and
E.C. 2.4.2 enzymes are pentosyltransferases (transfer pentose units).


[Ch. 1

An Introduction to Enzymes

8

The exception to this general rule for transferases is where there is transfer of
phosphate groups: these cannot be described further, so there is opportunity to
indicate the acceptor.
E.C. 2.7.l enzymes are phosphotransferases with an alcohol group as acceptor,
E.C. 2.7.2 enzymes are phosphotransferases with a carboxyl group as acceptor,
E.C. 2.7.3 enzymes are phosphotransferases with a nitrogenous group as acceptor.
Phosphotransferases usually have a trivial name ending in '-kinase'. Some
examples oftransferases are:
(S)-2-methyl-3-oxopropanoyl-CoA: pyruvate carboxyltransferase (E.C. 2.1.3.1)
(trivial name: methylmalonyl-CoA carboxyltransferase, formerly transcarboxylase)
which catalyses the transfer of a carboxyl group from methylmalonyl-CoA to

pyruvate:
CH3.CH.COSC0A + CH3CO.co2 ~ CH3_CH2.COSC0A + ?H2.CO.co2 (1.7)

I

co2

co2

methylmalonyl-CoA

pyruvate

propionyl-CoA

oxaloacetate

ATP: D-hexose-6-phosphotransferase (E.C. 2.7.l.1) (trivial name: hexokinase)
which catalyses:
C5 H9 0 5 .CH2 0H + ATP ~ C5 H 9 0 5 .CH 2 0Poi- + ADP
D-hexose

D-hexose-6-phosphate

This enzyme will transfer phosphate to a variety ofD-hexoses.
Main Class 3: Hydrolases
These enzymes catalyse hydrolytic reactions of the form:
A-X + H20 ~ X-OH + HA
They are classified according to the type of bond hydrolysed. For example:
Second digit

1
2

4
5

Bond hydrolysed
ester
glycosidic (linking carbohydrate units)
peptide (-CONH-) (see chapter 2)
C-N bonds other than peptides

The third digit further describes the type of bond hydrolysed. Thus:
E.C. 3.1.1 enzymes are carboxylic ester (-COO-) hydrolases,
E.C. 3.1.2 enzymes are thiol ester (-COS-) hydrolases,
E.C. 3.1.3 enzymes are phosphoric monoester ( -0 - Poi-) hydrolases,
E.C. 3.1.4 enzymes are phosphoric diester ( -O-P02 -0-) hydrolases.

(1.8)


Sec. 1.3

The naming and classification of enzymes

9

For example, orthophosphoric monoester phosphohydrolase (E.C. 3.1.3.1)
(alkaline phosphatase) catalyses:
R-0-POj- + H 20

R-OH + HO-Poj-

organic phosphate

(1.9)

inorganic phosphate

Alkaline phosphatases are relatively non-specific, and act on a variety of
substrates at alkaline pH.
The trivial names of hydrolases are recommended to be the only ones to consist
simply of the name of the substrate plus '-ase'.
Main Class 4: Lyases
These enzymes catalyse the non-hydrolytic removal of groups from substrates, often
leaving double bonds.
The second digit in the classification indicates the bond broken, for example:
Second digit
1
2

Bond broken
C-C

3
4

C-N
C-S

C-0

The third digit refers to the type of group removed. Thus, for the C-C lyases:
Group removed
carboxyl group (i.e. C02)
aldehyde group (-CH=O)
ketoacid group ( -co.coz-)

Third digit
1
2
3

For example, L-histidine carboxy-lyase (E.C. 4.1.1.22) (trivial name: histidine
decarboxylase, catalyses:
C3 N 2 H 3 .CH 2 CH.NH; ~ C3 N 2 H 3 .CH 2 .CH 2 .NH; + C0 2

I
co2

histidine

(1.10)

histamine

(Note the importance of the hyphen and the extra 'y' in the systematic name,
because carboxy-lyase and carboxylase do not mean the same thing: carboxylase
simply refers to the involvement of C02 in a reaction without being specific.)
Also classified as lyases are enzymes catalysing reactions whose biochemically

important direction is the reverse of the above, i.e. addition across double bonds.
These may have the trivial name synthase or, if water is added across the double
bond, hydratase, as discussed earlier in the example of fumarate hydratase
(fumarase), the systematic name of this particular enzyme being (S)-malate hydrolyase (E.C. 4.2.1.2).