Essential Enzyme Kinetics - 1st Edition - ISBN: 9780128218082

Essential Enzyme Kinetics

1st Edition

A Textbook for Molecular Life Scientists

Authors: Daniel Purich
Paperback ISBN: 9780128218082
Imprint: Academic Press
Published Date: 1st June 2020
Page Count: 425
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Description

Essentials of Enzyme Kinetics: A Textbook for Molecular Life Scientists discusses best-practice approaches for using site-directed mutagenesis to explore enzyme catalysis. The book presents a balance of initial-rate methods, fast reaction techniques, and kinetic isotope effect approaches, providing a must-have reference for bioscience workers and investigators who seek a better understanding of enzyme kinetics. Non-biochemists entering careers in Big Pharma will also benefit from the book's content as covered topics have become main drivers in small-molecule drug development.

Key Features

  • Offers practical information on how to work with enzymes and design experiments to identify new inhibitors or activators
  • Provides detailed, step-by-step derivations of rate equations, showing tried-and-true ways to confirm that equations are correct
  • Includes 10-12 problems for each chapter

Readership

Graduate students, postdocs and researchers across the biosciences seeking a better understanding of enzyme kinetics

Table of Contents

1. Introduction 
1.1. Proficiency of Enzyme Catalysis
1.2 Catalytic Strategies Used by Enzymes
1.2-a. Proximity Effects
1.2-b. Desolvation
1.2-c. Electrostatic Destabilization
1.2-d. Managing Intrinsic Binding Energy
1.2-e. Orienting Reactive Groups
1.2-f.  Destabilizing Reactant Ground States
1.2-g. Acid-Base Catalysis
1.2-h. Hydrogen Bonding
1.2-i.  Covalent Catalysis
1.2-j.  Metal Ion Effects
1.2-k. Conformational Flexibility
1.3. Chymotrypsin: A Prototypical Enzyme   

2. Basic Chemical Kinetics
2.1 A Few of the Basics
2.2 Reaction Order & Molecularity
2.3 Integrated Rate Laws   
2.3-a. First-Order Reactions
2.3-b. Series First-Order Reactions
2.3-c. Second-Order Reactions
2.3-d. Pseudo-Second-Order Reactions
2.3-e. Opposing First-Order Reactions
2.4 Barriers to Reaction   
2.4-a. Arrhenius Theory
2.4-b. Smoluchowski Equation
2.4-c. Transition State Theory (TST)
2.5 Reaction Mechanisms 
2.6 Reaction Coordinate Diagrams 
2.7 Energetics of Triose-P Isomerization
2.8 Timescales of Chemical Processes 
2.9 Kinetics versus Dynamics   
2.10 Concluding Remarks 
Problem Set                     

3. Initial-Rate Kinetics of One-Substrate Enzymes
3.1 Michaelis-Menten Kinetics      
3.2 Briggs-Haldane Treatment
3.3 KS and Km in Michaelis and Haldane Treatments
3.4 Meaning of kcat and Vm
3.5 Meaning of kcat/Km
3.6 Two-Intermediate Rate Law
3.7 Persistence of the Steady-State Phase
3.8 Concluding Remarks
Recommended Reading
Problem Set

4. Measuring Initial Velocities of Enzyme-catalyzed Reactions
4.1 The Initial-Rate Experiment 
4.1-a. Lineweaver-Burk Plot
4.2-b. [S]/v versus [S] Plot
4.2 Experimental Design
4.3 Quantifying Product Formation
4.3-a. Absorption Spectroscopy
4.3-b. Fluorescence Spectroscopy
4.3-c. Radiometric Assays
4.3-d. Polarimetry & Circular Dichroism
4.3-e. Manometry
4.3-f.  pH Measurements
4.3-g. Mass Spectroscopy
4.3-h. Light Scattering
4.3-i.  Turbidity
4.3-j. Calorimetry
4.4 Coupled Enzyme Assays
4.5 Statistical Analysis of Enzyme Rate Data 
4.6 Concluding Remarks
Further Reading
Problem Set

5. Multi-substrate Enzyme Kinetic Mechanisms   
5.1 Multi-Substrate Kinetic Mechanisms 
5.2 Theorell-Chance Mechanism
5.3 Ordered Ternary Complex Mechanism
5.4 Rapid-Equilibrium Random Mechanism   
5.5 Ping Pong Bi Bi Mechanism               
5.6 Bisubstrate Initial-Rate Data    
5.7 How Bisubstrate Enzyme Kinetic Mechanisms are Differentiated
5.7-a. Initial-Rate Experiments
5.7-b. Product Inhibition Experiments
5.7-c. Alternative Substrate Experiments
5.7-d. Competitive Inhibition Experiments
5.7-e. Isotope Exchange at Equilibrium
5.7-f. Fast Kinetic Techniques  
5.8 Iso-Mechanisms
5.9 Three-Substrate Kinetic Mechanisms
5.10  Concluding Remarks
Problem Set

6. Fast Kinetic Techniques for Probing Enzyme-Catalyzed Reactions
6.1 A Fuller Picture of Enzyme Catalysis
6.2 Stopped-Flow Technique
6.3 Rapid Mix/Quench Technique
6.4 Global Statistical Analysis
6.5 Relaxation Methods  
6.5-a. Temperature-Jump Technique
6.5-b. Pressure-Jump Technique
6.5-c. Flash Photolysis
6.5-d. Nuclear Magnetic Resonance
6.6 Basics of Chemical Relaxation Theory
6.7 Examples of Fast Reaction Studies 
6.7-a. Aspartate Aminotransferase
6.7-b. Ribonuclease
6.7-c. Antibody-Antigen Interactions
6.8 Concluding Remarks
Further Reading
Problem Set

7. Factors Affecting Enzyme Rates
7.1 pH Effects on Enzyme Kinetics
7.1-a. pH-Rate Behavior
7.1-b. Derivation of pH Functions 
7.2 Temperature Effects on Enzyme Rates
7.3 Pressure Effects on Enzyme Rates
7.4 Activator Effects on Enzyme Rates   
7.4-a. Types of Activators
7.4-b. Activator Rate Equations  
7.4-c. Metal Ion as Enzyme Activators
7.5 Effect of Mutation on Enzyme Kinetics 
7.5-a. Enzymes as Targets for Mutation and Change
7.5-b. Alanine Scanning Mutagenesis
7.5-c. Probing Catalysis by Site-Directed Mutagenesis
7.5-d. Probing Triose-Phosphate Isomerase by Mutagenesis  
7.6 Concluding Remarks

8. Kinetic Isotope Effects
8.1 Kinetic Isotope Effects
8.2 Primary Kinetic Isotope Effects
8.2-a. Basics
8.2-b. Other Factors Influencing Primary KIEs
8.2-c. Effects on Equilibria
8.2-d. Quantum Mechanical Tunneling
8.3 Secondary Kinetic Isotope Effects
8.4 Why Some Enzymatic KIEs Are Masked
8.5 Scope of KIE Measurements
Further Readings  

9. Inhibitor Effects on Enzyme-Catalyzed Reactions
9.1 Reversible versus Irreversible Inhibition
9.2 Reversible Enzyme Inhibitors
9.2-a. Competitive Inhibition
9.2-b. Noncompetitive Inhibition
9.2-c. Uncompetitive Inhibition
9.2-d. Slow, tight-binding Inhibition
9.2-e. Transition-State Inhibitors
9.3 Other Types of Enzyme Inhibition
9.3-a. Product Inhibition
9.3-b. Multi-Substrate Geometric Inhibition
9.3-c. Fragment-based Inhibitor Design
9.3-d. Photoaffinity Inhibition
9.3-e. Mechanism-Based Inhibition
9.4 Concluding Remarks
Problem Set

10. Enzyme Cooperativity
10.1 Cooperativity of K-Systems and V-Systems
10.2 Specific versus Nonspecific Binding
10.3 O2 Interactions with Hemoglobin Drove the Development of Cooperativity Models
10.4 Hill Model for Cooperativity
10.5 Monod-Wyman-Changeux "Concerted Transition" Model for Cooperativity
10.6 Adair-Koshland Sequential Model
10.7 Hysteretic Enzymes
10.8 Concluding Remarks

11. Kinetics of Force-Generating Enzymes

Details

No. of pages:
425
Language:
English
Copyright:
© Academic Press 2020
Published:
1st June 2020
Imprint:
Academic Press
Paperback ISBN:
9780128218082

About the Author

Daniel Purich

Dr. Purich earned his Doctor of Philosophy degree in 1973 for his kinetic characterization of brain hexokinase under the preceptorship of Professor Herbert J. Fromm in the Department of Biochemistry & Biophysics at Iowa State University. As a Staff Research Fellow with Dr. Earl Stadtman at the National Institutes of Health, he conducted research on the cascade of covalent interconverting enzymes that control the glutamine synthetase reaction in Escherichia coli. Dr. Purich subsequently joined the Department of Chemistry at the University of California Santa Barbara where he rose through the ranks as Assistant Professor (1973-78), Associate Professor (1978-82), and Professor (1982-84). At UC Santa Barbara, he was awarded an Alfred P. Sloan Fellow in Chemistry (1978-80), an NIH Research Career Development Award (1978-1983), and the Campus-wide Outstanding Teacher Award (1978). In 1984, he assumed the post of Professor and Chairman of the Department of Biochemistry & Molecular Biology at the University of Florida College of Medicine, and in 1996 he retired as Chairman to resume full-time activities as a professor. Dr. Purich has served on the editorial boards of the Journal of Biological Chemistry (1981-87) and Archives of Biochemistry and Biophysics (1975-85), and as a regular member of the NIH Biochemistry Study Section. Professor Purich succeeds the late Alton Meister as series editor for Advances in Enzymology, and he continues to edit the multi-volume treatise "Enzyme Kinetics & Mechanism" appearing as volumes 63, 64, 87, and 249 in Methods in Enzymology. He has also published Contemporary Enzyme Kinetics and Mechanism (1st ed.,1983; 2nd ed.,1996) based on chapters selected from his Methods volumes.

Affiliations and Expertise

Professor of Biochemistry and Molecular Biology, University of Florida Health Science Center, FL, USA

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