Summary
Fourteen years ago when Alan Fersht's Enzyme Structure and Mechanism was published, the field of protein engineering was in its infancy. Since then, spectacular advances in determining biological structure, manipulating genes, engineering proteins, sequencing whole genomes, and computing have led not just to an expansion of protein science, but to its utter transformation. We have entered a new era of protein design, sparked by the convergence of protein folding and enzymology.
Fersht's Structure and Mechanism in Protein Science is a defining exploration of this new era, an expert depiction of the core principles of protein structure, activity, and mechanism as understood and applied today. A thorough recasting of Fersht's previous text, the book takes a more general look at mechanisms in protein science, emphasizing the unity of concepts in folding and catalysis and the importance of the relationships between basic chemistry, kinetics, thermodynamics, and structure.
By concentrating on fundamental principles and the physical and chemical processes behind them, Structure and Mechanism in Protein Science makes the basic formulas, kinetics, and thermodynamics of protein engineering easier to understand and apply. Up-to-date, authoritative, and full with relevant examples, it provides a solid introduction to a sprawling, still-growing field.
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Table of Contents
Structure and Mechanism in Protein Science
A Guide to Enzyme Catalysis and Protein Folding
Alan Fersht (Cambridge U.)
1. The Three-dimensional Structure of Proteins
The primary structure of proteins
Methods for determination of three-dimensional structure
The three-dimensional structure of proteins
Protein diversity
Higher levels of organization: Multienzyme complexes
The structure of enzyme-substrate complexes
Flexibility and conformational mobility of proteins
2. Chemical Catalysis
Transition state theory
Principles of catalysis
Covalent catalysis
Structure-activity relationships
The principle of microscopic reversibility or detailed balance
The principle of kinetic equivalence
Kinetic isotope effects
Summary of classical factors of enzyme catalysis
3. The Basic Equations of Enzyme Kinetics
Steady state kinetics
The significance of the Michaelis-Menten parameters
Graphical representation of data
Inhibition
Nonproductive binding
kcat/KM = k2/Ks
Competing Substrates
Reversibility: The Haldene equation
Breakdown of the Michaelis-Menten equation
Multisubstrate systems
Useful kinetic shortcuts
Thermodynamic cycles
4. Measurement and Magnitude of Enzymatic Rate Constants
Part 1 Methods for measurement: An introduction to pre-steady state kinetics
Rapid mixing and sampling techniques
Flash photolysis
Relaxation methods
Analysis of pre-steady state and relaxation kinetics
The absolute concentration of enzymes
Part 2 The magnitude of rate constants for enzymatic processes
Upper limits on rate constants
Enzymatic rate constants and rate-determining processes
5. The pH Dependence of Enzyme Catalysis
Ionization of simple acids and bases: The basic equations
The effect of ionizations of groups in enzymes on kinetics
Modifications and breakdown of the simple theory
The influence of surface charge on pKa's of groups in enzymes
Graphical representation of data
Illustrative examples and experimental evidence
Direct titration of groups in enzymes
The effect of temperature, polarity of solvent, and ionic strength on pKa's
Highly perturbed pKa's on enzymes
6. Practical Kinetics
Kinetic methods
Plotting kinetic data
Determination of protein-ligand dissociation constants
Plotting binding data
Computer fitting of data
Statistics, errors of observation of accuracy
Appendix: Measurement of protein concentration
7. Detection of Intermediates in Reactions by Kinetics
Pre-steady state vs. steady state kinetics
Chymotrypsin: Determination of intermediates by stopped-flow spectrophotometry, steady state kinetics, and product partitioning
Further examples of detection of intermediates by partition and kinetic experiments
Aminoacyl-tRNA synthetases: Detection of intermediates by quenched flow, steady state kinetics, and isotope exchange
Detection of conformational changes
The future
8. Stereochemistry of Enzymatic Reactions
Optical activity and chirality
Examples of stereospecific enzyme reactions
Detection of intermediates from retention or inversion of configuration at chiral centers
The chiral methyl group
Chiral phosphate
Stereoelectronic control of enzymatic reactions
9. Active-site-directed and Enzyme-activated Irreversible Inhibitors: Affinity Labels and Suicide Inhibitors
Chemical modifications of proteins
Active-site-directed irreversible inhibitors
Enzyme-activated irreversible inhibitors
10. Conformational Change, Allosteric Regulation, Motors, and Work
Positive cooperativity
Mechanisms of allosteric interactions and cooperativity
Negative cooperativity and half-of-the-sites reactivity
Quantitative analysis of cooperativity
Molecular mechanism of cooperative binding to hemoglobin
Regulation of metabolic pathways
Phosphofructokinase and control by allosteric feedback
Glycogen phosporylase and control by phosphorylation
G proteins - molecular switches
Motor proteins
ATP synthesis by rotary catalysis: ATP synthase and F1-ATPase
11. Forces Between Molecules and Enzyme-Substrate Binding Energies
Interactions between nonbonded atoms
The binding energies of proteins and ligands
Experimental measurements of incremental energies
Entropy and binding
Enthalpy-entropy compensation
Summary
12. Enzyme-substrate Complementarity and the Use of Binding Energies in Catalysis
Utilization of enzyme-substrate binding energy in catalysis
Experimental evidence for the utilization of binding energy in catalysis and enzyme-transition state complementarity
Evolution of the maximum rate: Strong binding of the transition state and weak binding of the substrate
Molecular mechanisms for the utilization of binding energy
Effects of rate optimization on accumulation of intermediates and internal equilibria in enzymes
13. Specificity and Editing Mechanisms
Limits on specificity
Editing or proofreading mechanisms
The cost of accuracy
14. Recombinant DNA Technology
The structure and properties of DNA
Cloning enzyme genes for overproduction
Site-specific mutagenesis for rational design
Random mutagenesis and repertoire selection
15. Case Studies of Enzyme Structure and Mechanism
The dehydrogenases
The proteases
Ribonucleases
Lysozyme
Some generalizations
16. Protein Engineering
Part 1: The Dissection of the structure, activity, and mechanism of an enzyme--the tyrosyl-tRNA synthetase
Mechanistic goals
The tyrosyl-tRNA synthetase
Requirements for systematic site-directed mutagenesis studies
Choice of mutation
Strategy-free energy profiles and difference energy diagrams
Results from difference energy diagrams for the activation of tyrosine
Relationship between apparent binding energies from difference energies and incremental binding energies
Probing evolution - "reverse genetics"
Linear free energy relationships in binding energies
Probing the gross structure and symmetry of the enzyme by metagenesis
Measuring the free energy of hydrolysis of Tyr-AMP
Part 2: Redesigning an enzyme - subtilisin
Subtilisin
Dissection of the catalytic triad and the oxyanion binding site
Redesigning specificity
Engineering of stability and other properties
17. Protein Stability
Protein denaturation
Structure of the denatured state
Measurement of changes in stability
Energetics of formation of structure
Stability-activity trade-off?
Prediction of three-dimensional structure from primary structure
18. Kinetics of Protein Folding
Kinetics of folding
Two-state kinetics
Multi-state kinetics
Transition state in protein folding
Introduction to f-value analysis
1H/2H-exchange methods
Folding of peptides
19. Folding Pathways and Energy Landscapes
Levinthal's paradox
Folding of CI2
The Nucleation-Condensation mechanism
Folding of barnase
Folding pathway of Barstar at ms resolution
Unified folding scheme?
Insights from theory
Optimization of folding rates
Molecular Chaperones
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