Enzymes (Second Edition)
.pdfEnzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis.
Robert A. Copeland Copyright 2000 by Wiley-VCH, Inc.
ISBNs: 0-471-35929-7 (Hardback); 0-471-22063-9 (Electronic)
ENZYMES
SECOND EDITION
ENZYMES
A Practical Introduction to Structure, Mechanism, and Data Analysis
SECOND EDITION
Robert A. Copeland
A JOHN WILEY & SONS, INC., PUBLICATION
New York / Chichester / Weinheim / Brisbane / Singapore / Toronto
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ISBN 0-471-22063-9
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To Clyde Worthen
for teaching me all the important lessons: arigato sensei.
And to Theodore (Doc) Janner for stoking the fire.
CONTENTS
Preface |
xi |
Acknowledgments |
xiii |
Preface to the First Edition |
xv |
1 A Brief History of Enzymology |
1 |
1.1Enzymes in Antiquity / 2
1.2Early Enzymology / 3
1.3The Development of Mechanistic Enzymology / 4
1.4Studies of Enzyme Structure / 5
1.5Enzymology Today / 7
1.6Summary / 8
References and Further Reading / 10
2 Chemical Bonds and Reactions in Biochemistry |
11 |
2.1Atomic and Molecular Orbitals / 11
2.2Thermodynamics of Chemical Reactions / 23
2.3Acid—Base Chemistry / 29
2.4Noncovalent Interactions in Reversible Binding / 32
2.5Rates of Chemical Reactions / 35
2.6Summary / 41
References and Further Reading / 41
3 Structural Components of Enzymes |
42 |
3.1The Amino Acids / 42
3.2The Peptide Bond / 53
3.3Amino Acid Sequence or Primary Structure / 55
3.4Secondary Structure / 57
3.5Tertiary Structure / 62
vii
viiiCONTENTS
3.6Subunits and Quaternary Structure / 65
3.7Cofactors in Enzymes / 68
3.8Summary / 71
References and Further Reading / 74
4 Protein‒Ligand Binding Equilibria |
76 |
4.1The Equilibrium Dissociation Constant, K / 76
4.2The Kinetic Approach to Equilibrium / 78
4.3Binding Measurements at Equilibrium / 80
4.4Graphic Analysis of Equilibrium Ligand Binding Data / 88
4.5Equilibrium Binding with Ligand Depletion (Tight Binding Interactions) / 94
4.6Competition Among Ligands for a Common Binding Site / 95
4.7Experimental Methods for Measuring Ligand Binding / 96
4.8Summary / 107
References and Further Reading / 108
5 Kinetics of Single-Substrate Enzyme Reactions |
109 |
5.1The Time Course of Enzymatic Reactions / 109
5.2Effects of Substrate Concentration on Velocity / 111
5.3The Rapid Equilibrium Model of Enzyme Kinetics / 113
5.4The Steady State Model of Enzyme Kinetics / 115
5.5The Significance of k and K / 120
5.6Experimental Measurement of k and K / 124
5.7Other Linear Transformations of Enzyme Kinetic Data / 133
5.8Measurements at Low Substrate Concentrations / 136
5.9Deviations from Hyperbolic Kinetics / 137
5.10Transient State Kinetic Measurements / 141
5.11Summary / 145
References and Further Reading / 145
6 Chemical Mechanisms in Enzyme Catalysis |
146 |
6.1Substrate—Active Site Complementarity / 147
6.2Rate Enhancement Through Transition State Stabilization / 151
6.3Chemical Mechanisms for Transition State Stabilization / 154
6.4The Serine Proteases: An Illustrative Example / 178
6.5Enzymatic Reaction Nomenclature / 184
CONTENTS ix
6.6 Summary / 186
References and Further Reading / 186
7 Experimental Measures of Enzyme Activity |
188 |
7.1Initial Velocity Measurements / 188
7.2Detection Methods / 204
7.3Separation Methods in Enzyme Assays / 223
7.4Factors Affecting the Velocity of Enzymatic Reactions / 238
7.5Reporting Enzyme Activity Data / 257
7.6Enzyme Stability / 258
7.7Summary / 263
References and Further Reading / 263
8 Reversible Inhibitors |
266 |
8.1Equilibrium Treatment of Reversible Inhibition / 268
8.2Modes of Reversible Inhibition / 270
8.3Graphic Determination of Inhibitor Type / 273
8.4Dose—Response Curves of Enzyme Inhibition / 282
8.5Mutually Exclusive Binding of Two Inhibitors / 287
8.6Structure—Activity Relationships and Inhibitor Design / 291
8.6 Summary / 303
References and Further Reading / 303
9 Tight Binding Inhibitors |
305 |
9.1Identifying Tight Binding Inhibition / 305
9.2Distinguishing Inhibitor Type for Tight Binding Inhibitors / 307
9.3Determining K for Tight Binding Inhibitors / 310
9.4Use of Tight Binding Inhibitors to Determine Active Enzyme Concentration / 313
9.5Summary / 315
References and Further Reading / 316
10 Time-Dependent Inhibition |
318 |
10.1Progress Curves for Slow Binding Inhibitors / 321
10.2Distinguishing Between Slow Binding Schemes / 325
10.3Distinguishing Between Modes of Inhibitor Interaction with Enzyme / 330
xCONTENTS
10.4Determining Reversibility / 332
10.5Examples of Slow Binding Enzyme Inhibitors / 334
10.6Summary / 348
References and Further Reading / 349
11 Enzyme Reactions with Multiple Substrates |
350 |
11.1Reaction Nomenclature / 350
11.2Bi Bi Reaction Mechanisms / 352
11.3Distinguishing Between Random and Compulsory Ordered Mechanisms by Inhibition Pattern / 357
11.4Isotope Exchange Studies for Distinguishing Reaction Mechanisms / 360
11.5Using the King—Altman Method to Determine Velocity Equations / 362
11.6Summary / 364
References and Further Reading / 366
12 Cooperativity in Enzyme Catalysis |
367 |
12.1Historic Examples of Cooperativity and Allostery in Proteins / 368
12.2Models of Allosteric Behavior / 373
12.3Effects of Cooperativity on Velocity Curves / 379
12.4Sigmoidal Kinetics for Nonallosteric Enzymes / 382
12.5Summary / 383
References and Further Reading / 384
Appendix I. Suppliers of Reagents and Equipment for |
|
Enzyme Studies |
385 |
Appendix II. Useful Computer Software and Web Sites |
|
for Enzyme Studies |
387 |
Index |
391 |
PREFACE
In the four years since the first edition of Enzymes was published, I have been delighted to learn of the wide acceptance of the book throughout the biochemical community, and particularly in the pharmaceutical community. During this time a number of colleagues have contacted me to express their views on the value of the text, and importantly to make suggestions for improvements to the content and presentation of some concepts. I have used the first edition as a teaching supplement for a course in which I lecture at the University of Pennsylvania School of Medicine. From my lecture experiences and from conversations with students, I have developed some new ideas for how to better explain some of the concepts in the text and have identified areas that deserve expanded coverage. Finally, while the first edition has become popular with students and industrial scientists, some of my academic colleagues have suggested a need for a more in-depth treatment of chemical mechanisms in enzymology.
In this second edition I have refined and expanded the coverage of many of the concepts in the text. To help the reader better understand some of the interactions between enzymes and their substrates and inhibitors, a new chapter on protein—ligand binding equilibria has been added (Chapter 4). The chapters on chemical mechanisms in enzyme catalysis (Chapter 6) and on experimental measures of enzyme activity (Chapter 7) have been expanded significantly. The discussions of enzyme inhibitors and multiple substrate reactions (Chapters 8 through 11) have been refined, and in some cases alternative treatments have been presented. In all of this, however, I have tried to maintain the introductory nature of the book. There are many excellent advanced texts on catalysis, enzyme mechanisms, and enzyme kinetics, but the level at which these are generally written is often intimidating to the beginner. Hence, as stated in the preface to the first edition, this book is intended to serve as a mechanism for those new to the field of enzymology to develop a reasonable understanding of the science and experimental methods, allowing them to competently begin laboratory studies with enzymes. I have continued to rely on extensive citations to more advanced texts and primary literature as a means for the interested reader to go beyond the treatments offered here and delve more deeply into specific areas of enzymology.
xi
xii PREFACE
In developing this second edition I have had fruitful conversations and advice from a number of colleagues. In particular, I wish to thank Andy Stern, Ross Stein, Trevor Penning, Bill Pitts, John Blanchard, Dennis Murphy, and the members of the Chemical Enzymology Department at the DuPont Pharmaceuticals Company. As always, the love and support of my family has been most important in making this work possible.
R A. C
Wilmington, Delaware