Enzymes A Practical Introduction to Structure, Mechanism, and Data Analysis

by Copeland, Robert A.

• ISBN13: 9780471359296
• ISBN10: 0471359297
• Edition: 2nd
• Format: Hardcover
• Copyright: 2000-04-10
• Publisher: Wiley-VCH

Summary

Fully updated and expanded-a solid foundation for understanding experimental enzymology.This practical, up-to-date survey is designed for a broad spectrum of biological and chemical scientists who are beginning to delve into modern enzymology. Enzymes, Second Edition explains the structural complexities of proteins and enzymes and the mechanisms by which enzymes perform their catalytic functions. The book provides illustrative examples from the contemporary literature to guide the reader through concepts and data analysis procedures. Clear, well-written descriptions simplify the complex mathematical treatment of enzyme kinetic data, and numerous citations at the end of each chapter enable the reader to access the primary literature and more in-depth treatments of specific topics.This Second Edition of Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis features refined and expanded coverage of many concepts, while retaining the introductory nature of the book. Important new features include: A new chapter on protein-ligand binding equilibria Expanded coverage of chemical mechanisms in enzyme catalysis and experimental measurements of enzyme activity Updated and refined discussions of enzyme inhibitors and multiple substrate reactions Coverage of current practical applications to the study of enzymology Supplemented with appendices providing contact information for suppliers of reagents and equipment for enzyme studies, as well as a survey of useful Internet sites and computer software for enzymatic data analysis, Enzymes, Second Edition is the ultimate practical guide for scientists and students in biochemical, pharmaceutical, biotechnical, medicinal, and agricultural/food-related research.

Author Biography

ROBERT A. COPELAND, PhD, is Director of Chemical Enzymology at the DuPont Pharmaceuticals Company and Adjunct Professor of Biochemistry and Biophysics at the University of Pennsylvania School of Medicine.

Table of Contents

Preface	xi
Acknowledgments	xiii
Preface to the First Edition	xv
A Brief History of Enzymology	1	(10)
Enzymes in Antiquity	2	(1)
Early Enzymology	3	(1)
The Development of Mechanistic Enzymology	4	(1)
Studies of Enzyme Structure	5	(2)
Enzymology Today	7	(1)
Summary	8	(3)
References and Further Reading	10	(1)
Chemical Bonds and Reactions in Biochemistry	11	(31)
Atomic and Molecular Orbitals	11	(12)
Thermodynamics of Chemical Reactions	23	(6)
Acid--Base Chemistry	29	(3)
Noncovalent Interactions in Reversible Binding	32	(3)
Rates of Chemical Reactions	35	(6)
Summary	41	(1)
References and Further Reading	41	(1)
Structural Components of Enzymes	42	(34)
The Amino Acids	42	(11)
The Peptide Bond	53	(2)
Amino Acid Sequence or Primary Structure	55	(2)
Secondary Structure	57	(5)
Tertiary Structure	62	(3)
Subunits and Quaternary Structure	65	(3)
Cofactors in Enzymes	68	(3)
Summary	71	(5)
References and Further Reading	74	(2)
Protein--Ligand Binding Equilibria	76	(33)
The Equilibrium Dissociation Constant, Kd	76	(2)
The Kinetic Approach to Equilibrium	78	(2)
Binding Measurements at Equilibrium	80	(8)
Graphic Analysis of Equilibrium Ligand Binding Data	88	(6)
Equilibrium Binding with Ligand Depletion (Tight Binding Interactions)	94	(1)
Competition Among Ligands for a Common Binding Site	95	(1)
Experimental Methods for Measuring Ligand Binding	96	(11)
Summary	107	(2)
References and Further Reading	108	(1)
Kinetics of Single-Substrate Enzyme Reactions	109	(37)
The Time Course of Enzymatic Reactions	109	(2)
Effects of Substrate Concentration on Velocity	111	(2)
The Rapid Equilibrium Model of Enzyme Kinetics	113	(2)
The Steady State Model of Enzyme Kinetics	115	(5)
The Significance of Kcat and Km	120	(4)
Experimental Measurement of Kcat and Km	124	(9)
Other Linear Transformations of Enzyme Kinetic Data	133	(3)
Measurements at Low Substrate Concentrations	136	(1)
Deviations from Hyperbolic Kinetics	137	(4)
Transient State Kinetic Measurements	141	(4)
Summary	145	(1)
References and Further Reading	145	(1)
Chemical Mechanisms in Enzyme Catalysis	146	(42)
Substrate--Active Site Complementarity	147	(4)
Rate Enhancement Through Transition State Stabilization	151	(3)
Chemical Mechanisms for Transition State Stabilization	154	(24)
The Serine Proteases: An Illustrative Example	178	(6)
Enzymatic Reaction Nomenclature	184	(2)
Summary	186	(2)
References and Further Reading	186	(2)
Experimental Measures of Enzyme Activity	188	(78)
Initial Velocity Measurements	188	(16)
Detection Methods	204	(19)
Separation Methods in Enzyme Assays	223	(15)
Factors Affecting the Velocity of Enzymatic Reactions	238	(19)
Reporting Enzyme Activity Data	257	(1)
Enzyme Stability	258	(5)
Summary	263	(3)
References and Further Reading	263	(3)
Reversible Inhibitors	266	(39)
Equilibrium Treatment of Reversible Inhibition	268	(2)
Modes of Reversible Inhibition	270	(3)
Graphic Determination of Inhibitor Type	273	(9)
Dose--Response Curves of Enzyme Inhibition	282	(5)
Mutually Exclusive Binding of Two Inhibitors	287	(4)
Structure--Activity Relationships and Inhibitor Design	291	(12)
Summary	303	(2)
References and Further Reading	303	(2)
Tight Binding Inhibitors	305	(13)
Identifying Tight Binding Inhibition	305	(2)
Distinguishing Inhibitor Type for Tight Binding Inhibitors	307	(3)
Determining Ki for Tight Binding Inhibitors	310	(3)
Use of Tight Binding Inhibitors to Determine Active Enzyme Concentration	313	(2)
Summary	315	(3)
References and Further Reading	316	(2)
Time-Dependent Inhibition	318	(32)
Progress Curves for Slow Binding Inhibitors	321	(4)
Distinguishing Between Slow Binding Schemes	325	(5)
Distinguishing Between Modes of Inhibitor Interaction with Enzyme	330	(2)
Determining Reversibility	332	(2)
Examples of Slow Binding Enzyme Inhibitors	334	(14)
Summary	348	(2)
References and Further Reading	349	(1)
Enzyme Reactions with Multiple Substrates	350	(17)
Reaction Nomenclature	350	(2)
Bi Bi Reaction Mechanisms	352	(5)
Distinguishing Between Random and Compulsory Ordered Mechanisms by Inhibition Pattern	357	(3)
Isotope Exchange Studies for Distinguishing Reaction Mechanisms	360	(2)
Using the King--Altman Method to Determine Velocity Equations	362	(2)
Summary	364	(3)
References and Further Reading	366	(1)
Cooperativity in Enzyme Catalysis	367	(18)
Historic Examples of Cooperativity and Allostery in Proteins	368	(5)
Models of Allosteric Behavior	373	(6)
Effects of Cooperativity on Velocity Curves	379	(3)
Sigmoidal Kinetics for Nonallosteric Enzymes	382	(1)
Summary	383	(2)
References and Further Reading	384	(1)
Appendix I. Suppliers of Reagents and Equipment for Enzyme Studies	385	(2)
Appendix II. Useful Computer Software and Web Sites for Enzyme Studies	387	(4)
Index	391