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1st Edition - December 1, 2021
Author: Daniel L. Purich
Essential Enzyme Kinetics: A Textbook for Molecular Life Scientists describes the theoretical basis and best-practice approaches for using initial-rate, fast reaction, and kinetic… Read more
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Immediately download your ebook while waiting for your print delivery. No promo code is needed.
Essential Enzyme Kinetics: A Textbook for Molecular Life Scientists describes the theoretical basis and best-practice approaches for using initial-rate, fast reaction, and kinetic isotope effect experiments to define enzyme catalysis. Because a detailed knowledge of enzyme transition-states is the main driver for the rational design of slow, tight-binding inhibitors destined to become tomorrow's small-molecule drugs, Essential Enzyme Kinetics is the must-have reference for chemists, biochemists, and pharmacologists intent on pursuing careers in Big Pharma. Given the interdisciplinary nature of contemporary drug development, this book provides a lucid short-course that will also benefit nonspecialists seeking to understand the scope and reach of modern enzyme kinetics.
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
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