Kinetics of Enzyme Action

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.

• Preface xi1 Introduction 11.1 A Brief History of Enzymology 21.2 Goal of Enzymology: The Elucidation of Mechanism 111.3 The Emergence of Mechanism from Data 132 Kinetics of Single-substrate Enzymatic Reactions 192.1 The Dependence of Initial Velocity on Substrate Concentration and the Requirement for an E : S Complex 192.2 Derivation of the Rate Equation for a Single-Substrate Reaction with the Assumption of Rapid Equilibrium 212.3 Derivation of Rate Equations Using the Steady-State Assumption 242.4 Methods of Enzyme Assay 262.5 Enzyme Kinetics Practicum: Assay Development, Experimental Design, Data Collection, and Data Analysis 323 Kinetics of Single-substrate Enzymatic Reactions: Special Topics 413.1 Transition State Theory and Free Energy Diagrams 413.2 Kinetic Consequences of an Enzyme : Substrate Complex 463.3 Reactions with More Than One Intermediary Complex 513.4 Deviations from Michaelis–Menten Kinetics 533.5 Kinetics of Enzymatic Action on Substrates with Multiple Reactive Centers 654 Enzyme Inhibition: the Phenomenon and Mechanism-independent Analysis 734.1 Enzyme Inhibition: The Phenomenon 744.2 Enzyme Inhibition: The First Quantitative Steps 764.3 Enzyme-Inhibitor Systems Misbehaving 774.4 Case Studies 835 Kinetic Mechanism of Inhibition of One-substrate Enzymatic Reactions 895.1 Importance in Drug Discovery 895.2 Theoretical Considerations 905.3 Analysis of Initial Velocity Data for Enzyme Inhibition 965.4 Inhibition of One-Substrate, Two-Intermediate Reactions 1085.5 Inhibition by Depletion of Substrate 1116 Tight-binding, Slow-binding, and Irreversible Inhibition 1156.1 Importance in Drug Discovery 1156.2 Tight-Binding Inhibition 1166.3 Slow-Binding Inhibition 1226.4 Irreversible Inhibition 1377 Kinetics of Two-substrate Enzymatic Reactions 1417.1 Importance in Drug Discovery 1417.2 Basic Mechanisms 1427.3 Conceptual Understanding of Sequential Mechanisms 1437.4 Derivation of Rate Equations for Sequential Mechanisms 1477.5 Ping-Pong Mechanisms 1537.6 Determining the Kinetic Mechanism for Two-Substrate Reactions 1567.7 A Conceptual Understanding of the Shapes of Secondary Plots 1637.8 Mistaken Identity: Rapid Equilibrium Random Versus Steady-State Ordered 1688 Kinetic Mechanism of Inhibition of Two-substrate Enzymatic Reactions 1698.1 Importance in Drug Discovery 1708.2 Mechanisms of Inhibition of Two-Substrate Reactions 1708.3 Inhibition by Substrate Analogs 1858.4 Analysis of Sequential Reactions in which Inhibitor Binds to Enzyme : Product Complexes 1918.5 Driving SAR Programs for Two-Substrate Enzymatic Reactions 1969 Allosteric Modulation of Enzyme Activity 1999.1 Mechanisms of Enzyme Modulation 2019.2 Kinetics of Allosteric Modulation 2029.3 Meaning of β and γ 2089.4 Case Studies: Dependence of Allosteric Modulation on Structural Features of the Substrate 21210 Kinetics-based Probes of Mechanism 21910.1 pH Dependence of Enzymatic Reactions 22010.2 Temperature Dependence of Enzymatic Reactions 22910.3 Viscosity Dependence of Enzymatic Reactions 23510.4 Kinetic Isotope Effects on Enzyme-Catalyzed Reactions 239Appendix A Basic Principles of Chemical Kinetics 251A.1 One-Step, Irreversible, Unimolecular Reactions 252A.2 One-Step, Irreversible, Bimolecular Reactions 253A.3 One-Step, Reversible Reactions 254A.4 Two-Step, Irreversible Reactions 257A.5 Two-Step Reaction, with Reversible First Step 259Appendix B Transition State Theory and Enzymology: Enzyme Catalytic Power and Inhibitor Design 263B.1 Catalytic Power of Enzymes 263B.2 Transition State Analog Inhibition 268Appendix C Selecting Substrate Concentrations for High-throughput Screens 275C.1 Balancing the Steady State for One-Substrate Reactions 276C.2 Balancing the Steady State for Two-Substrate, Rapid Equilibrium-Ordered Enzymatic Reactions 276C.3 Balancing the Steady State for Two-Substrate, Rapid Equilibrium Random Enzymatic Reactions 279C.4 Balancing the Steady State for Nonequilibrium Enzymatic Reactions Involving a Second Steady-State Intermediate 282C.5 Balancing the Steady State for Two-Substrate, Ping-Pong Enzymatic Reactions 283Index 287

"Kinetics of Enzyme Action: Essential Principles for Drug Hunters provides a needed resource for pharmaceutical scientists whose job it is to discover and kinetically characterize enzyme inhibitors. This book starts with the most basic principles pertaining to simple, one-substrate enzyme reactions and their inhibitors, and progresses to a thorough treatment of two-substrate enzymes and their inhibitors." (News Blaze, 18 October 2011)