Kinetics of Enzyme Action

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

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.

"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)

"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)