Kinetics of Enzyme Action: Essential Principles for Drug Hunters

"Dr. Stein . . . make[s] a difficult topic easy to understand and comprehend."
—Richard Harrison, Wyeth Research
Details on the approaches and applications essential to kinetically characterizing enzyme inhibitors for drug discovery
The ability of enzymes to bring about the transformation of one chemical substance into another makes them the favorite—and most productive—drug target in the pharmaceutical industry. Kinetics of Enzyme Action explores, in depth, the characteristics and applications of these proteins and their vital role in the development of new medicines by placing the subject of enzymology in a historical context that enriches and heightens understanding of this complex science. It introduces readers to a wide range of topics invaluable for helping them discover and kinetically characterize enzyme inhibitors—from simple, one–substrate enzyme reactions to the more complex two–substrate enzymes reactions. Some of the highlights in this book include:A detailed description of enzyme kinetics as applied to drug discoveryA comprehensive collection of theory and pragmatic approaches to guide readers in the science of performing detailed enzyme mechanistic studiesAn outline of the tools and techniques necessary to perform, understand, and interpret detailed kinetic studies for drug discoveryNumerous case studies from scientific literature that bring the topics to life
From discussion of the early developments in the kinetics of enzymatic reactions to fresh insights on contemporary enzymology, Kinetics of Enzyme Action offers comprehensive coverage of a discipline that holds vital significance for all pharmaceutical practitioners.

Spis treści:
Preface xi
1 INTRODUCTION 1
1.1 A Brief History of Enzymology 2
1.2 Goal of Enzymology: The Elucidation of Mechanism 11
1.3 The Emergence of Mechanism from Data 13
2 KINETICS OF SINGLE–SUBSTRATE ENZYMATIC REACTIONS 19
2.1 The Dependence of Initial Velocity on Substrate Concentration and the Requirement for an E: S Complex 19
2.2 Derivation of the Rate Equation for a Single–Substrate Reaction with the Assumption of Rapid Equilibrium 21
2.3 Derivation of Rate Equations Using the Steady–State Assumption 24
2.4 Methods of Enzyme Assay 26
2.5 Enzyme Kinetics Practicum: Assay Development, Experimental Design, Data Collection, and Data Analysis 32
3 KINETICS OF SINGLE–SUBSTRATE ENZYMATIC REACTIONS: SPECIAL TOPICS 41
3.1 Transition State Theory and Free Energy Diagrams 41
3.2 Kinetic Consequences of an Enzyme: Substrate Complex 46
3.3 Reactions with More Than One Intermediary Complex 51
3.4 Deviations from Michaelis–Menten Kinetics 53
3.5 Kinetics of Enzymatic Action on Substrates with Multiple Reactive Centers 65
4 ENZYME INHIBITION: THE PHENOMENON AND MECHANISM–INDEPENDENT ANALYSIS 73
4.1 Enzyme Inhibition: The Phenomenon 74
4.2 Enzyme Inhibition: The First Quantitative Steps 76
4.3 Enzyme–Inhibitor Systems Misbehaving 77
4.4 Case Studies 83
5 KINETIC MECHANISM OF INHIBITION OF ONE–SUBSTRATE ENZYMATIC REACTIONS 89
5.1 Importance in Drug Discovery 89
5.2 Theoretical Considerations 90
5.3 Analysis of Initial Velocity Data for Enzyme Inhibition 96
5.4 Inhibition of One–Substrate, Two–Intermediate Reactions 108
5.5 Inhibition by Depletion of Substrate 111
6 TIGHT–BINDING, SLOW–BINDING, AND IRREVERSIBLE INHIBITION 115
6.1 Importance in Drug Discovery 115
6.2 Tight–Binding Inhibition 116
6.3 Slow–Binding Inhibition 122
6.4 Irreversible Inhibition 137
7 KINETICS OF TWO–SUBSTRATE ENZYMATIC REACTIONS 141
7.1 Importance in Drug Discovery 141
7.2 Basic Mechanisms 142
7.3 Conceptual Understanding of Sequential Mechanisms 143
7.4 Derivation of Rate Equations for Sequential Mechanisms 147
7.5 Ping–Pong Mechanisms 153
7.6 Determining the Kinetic Mechanism for Two–Substrate Reactions 156
7.7 A Conceptual Understanding of the Shapes of Secondary Plots 163
7.8 Mistaken Identity: Rapid Equilibrium Random Versus Steady–State Ordered 168
8 KINETIC MECHANISM OF INHIBITION OF TWO–SUBSTRATE ENZYMATIC REACTIONS 169
8.1 Importance in Drug Discovery 170
8.2 Mechanisms of Inhibition of Two–Substrate Reactions 170
8.3 Inhibition by Substrate Analogs 185
8.4 Analysis of Sequential Reactions in which Inhibitor Binds to Enzyme: Product Complexes 191
8.5 Driving SAR Programs for Two–Substrate Enzymatic Reactions 196
9 ALLOSTERIC MODULATION OF ENZYME ACTIVITY 199
9.1 Mechanisms of Enzyme Modulation 201
9.2 Kinetics of Allosteric Modulation 202
9.3 Meaning of β and γ 208
9.4 Case Studies: Dependence of Allosteric Modulation on Structural Features of the Substrate 212
10 KINETICS–BASED PROBES OF MECHANISM 219
10.1 pH Dependence of Enzymatic Reactions 220
10.2 Temperature Dependence of Enzymatic Reactions 229
10.3 Viscosity Dependence of Enzymatic Reactions 235
10.4 Kinetic Isotope Effects on Enzyme–Catalyzed Reactions 239
APPENDIX A BASIC PRINCIPLES OF CHEMICAL KINETICS 251
A.1 One–Step, Irreversible, Unimolecular Reactions 252
A.2 One–Step, Irreversible, Bimolecular Reactions 253
A.3 One–Step, Reversible Reactions 254
A.4 Two–Step, Irreversible Reactions 257
A.5 Two–Step Reaction, with Reversible First Step 259
APPENDIX B TRANSITION STATE THEORY AND ENZYMOLOGY: ENZYME CATALYTIC POWER AND INHIBITOR DESIGN 263
B.1 Catalytic Power of Enzymes 263
B.2 Transition State Analog Inhibition 268
APPENDIX C SELECTING SUBSTRATE CONCENTRATIONS FOR HIGH–THROUGHPUT SCREENS 275
C.1 Bal ancing the Steady State for One–Substrate Reactions 276
C.2 Balancing the Steady State for Two–Substrate, Rapid Equilibrium–Ordered Enzymatic Reactions 276
C.3 Balancing the Steady State for Two–Substrate, Rapid Equilibrium Random Enzymatic Reactions 279
C.4 Balancing the Steady State for Nonequilibrium Enzymatic Reactions Involving a Second Steady–State Intermediate 282
C.5 Balancing the Steady State for Two–Substrate, Ping–Pong Enzymatic Reactions 283
Index 287

Nota biograficzna:
ROSS L. STEIN received his PhD in physical organic chemistry from Indiana University in 1978, after which he conducted post–doctoral research in mechanistic enzymology at the University of Kansas. His career has included positions at Merck, where he headed the Enzymology Department, and ProScript (acquired by Millennium in 1999) as vice president of biochemistry. At ProScript, Dr. Stein was instrumental in the pre–clinical development and co–inventor of the drug VELCADETM, a proteasome inhibitor used to treat multiple myeloma. In 2001, he accepted a position at Harvard Medical School to establish and head the Laboratory for Drug Discovery in Neurodegeneration. Most recently, from 2009–2011, Dr. Stein was vice president of drug discovery at Sirtris Pharmaceuticals.

Okładka tylna:
"Dr. Stein . . . make[s] a difficult topic easy to understand and comprehend."
—Richard Harrison, Wyeth Research
Details on the approaches and applications essential to kinetically characterizing enzyme inhibitors for drug discovery
The ability of enzymes to bring about the transformation of one chemical substance into another makes them the favorite—and most productive—drug target in the pharmaceutical industry. Kinetics of Enzyme Action explores, in depth, the characteristics and applications of these proteins and their vital role i n the development of new medicines by placing the subject of enzymology in a historical context that enriches and heightens understanding of this complex science. It introduces readers to a wide range of topics invaluable for helping them discover and kinetically characterize enzyme inhibitors—from simple, one–substrate enzyme reactions to the more complex two–substrate enzymes reactions. Some of the highlights in this book include:A detailed description of enzyme kinetics as applied to drug discoveryA comprehensive collection of theory and pragmatic approaches to guide readers in the science of performing detailed enzyme mechanistic studiesAn outline of the tools and techniques necessary to perform, understand, and interpret detailed kinetic studies for drug discoveryNumerous case studies from scientific literature that bring the topics to life
From discussion of the early developments in the kinetics of enzymatic reactions to fresh insights on contemporary enzymology, Kinetics of Enzyme Action offers comprehensive coverage of a discipline that holds vital significance for all pharmaceutical practitioners.