Overview

LABORATORY GUIDE TO ENZYMOLOGY An accessible guide to understanding the foundations of enzymology at its application in drug discovery Enzymes are highly specialized proteins necessary for performing specific biochemical reactions essential for life in all organisms. In disease, the functioning of these enzymes can become altered and, therefore, enzymes represent a large class of key targets for drug discovery. In order to successfully target dysfunctional enzymes pharmaceutically, the unique mechanism of each enzyme must be understood through thorough and in-depth kinetic analysis. The topic of enzymology can appear challenging due its interdisciplinary nature combining concepts from biology, chemistry, and mathematics. Laboratory Guide to Enzymology brings together the theory of enzymology and associated lab-based work to offer a practical, accessible guide encompassing all three scientific disciplines. Beginning with a brief introduction to proteins and enzymes, the book slowly immerses the reader into the foundations of enzymology and how it can be used in drug discovery using modern methods of experimentation. The result is a detailed but highly readable volume detailing the basis of drug discovery research. Laboratory Guide to Enzymology readers will also find: Descriptions of key concepts in enzymology Examples of drugs targeting different enzymes via different mechanisms Detailed discussion about many areas of enzymology such as binding and steady-state kinetics, assay development, and enzyme inhibition and activation Laboratory Guide to Enzymology is ideal for all pharmaceutical and biomedical researchers working in enzymology and assay development, as well as advanced students in the biochemical or biomedical sciences looking to develop a working knowledge of this field of research.

Full Product Details

Author:   Geoffrey A. Holdgate ,  Antonia Turberville ,  Alice Lanne
Publisher:   John Wiley & Sons Inc
Imprint:   John Wiley & Sons Inc
Weight:   0.676kg
ISBN:  

9781394179794

ISBN 10:   1394179790
Pages:   304
Publication Date:   01 March 2024
Audience:   College/higher education ,  Postgraduate, Research & Scholarly
Format:   Paperback
Publisher's Status:   Active
Availability:   Available To Order  
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Table of Contents

Chapter 1 – Introduction to protein and enzymes 1.1. Structure of protein 1.1.1. Primary structure 1.1.2. Secondary structure  1.1.2.1. The alpha helix  1.1.2.2. Beta sheet  1.1.2.3. Loops  1.1.3. Tertiary structure  1.1.3.1. Domains, folds and motifs  1.1.4. Quaternary Structure  1.1.5. Protein Structure Prediction  1.2. Enzymes  1.2.1. Properties of Enzymes  1.2.1.1. Catalysis  1.2.1.2. Specificity  1.2.1.3. Regulation  1.2.1.4. Stability  1.3. References  Chapter 2 – Binding and kinetics 2.1. Introduction to chemical kinetics  2.1.1. Zero order reactions  2.1.2. First order reactions  2.1.3. Second order reactions  2.1.4. Pseudo-first order reactions  2.1.5. Temperature dependence of rate constants  2.2. Introduction to binding kinetics  2.3. Ligand binding to single binding site  2.3.1. Specific vs non-specific binding  2.4. Kinetic approach to equilibrium  2.5. Methods for measuring protein ligand binding  2.6. Ligand depletion (tight-binding)  2.7. Ligand binding to multiple binding sites  2.7.1. Identical independent binding sites  2.7.2. Non-identical independent binding sites  2.8. Ligand competition 2.9.        References  Chapter 3 – Protein QC and handling 3.1. Introduction 3.2. Confirming protein identity  3.2.1. Intact mass measurement by mass spectrometry (MS)  3.2.2. Peptide mapping  3.2.3. Edman sequencing  3.3. Protein purity  3.3.1. SDS-PAGE  3.3.2. Dynamic light scattering (DLS)  3.3.3. Analytical Gel Filtration  3.3.4. Analytical Ultracentrifugation  3.4. Concentration  3.4.1. UV-Vis Spectrum  3.4.2. Bradford assay  3.5. Functionality  3.5.1. Ligand binding 3.5.1.1. ITC  3.5.1.2. SPR  3.5.2. Functional studies  3.6. Stability  3.6.1. Differential scanning calorimetry (DSC)  3.6.2. Differential scanning fluorimetry (DSF)  3.6.3. Circular dichroism  3.6.4. Selwyn’s test  3.7. References  Chapter 4- Buffers 4.1. Introduction  4.1.1. Ionisation and pKa  4.1.2. pH  4.1.3. Henderson-Hasselbalch Equation  4.1.4. Buffers  4.2. Buffering capacity  4.3. Ionic strength  4.4. Change in pH with temperature  4.5. Choice of buffer  4.6. Characteristics of ionising groups in proteins  4.7. Effect of pH on enzyme activity  4.7.1. Assumptions required for the analysis of pH dependence  4.7.2. General rate equation for pH dependence  4.7.3. Fitting pH dependence  4.7.3.1. Bell shaped curves – double ionising system  4.7.3.2. Bell shaped curves – singularly ionising systems  4.8. Effect of solvent and ionic strength  4.9. References  Chapter 5 – Steady-state assays and their design 5.1. Introduction  5.2. The pre-steady state  5.3. Steady-state assays  5.4. Assay Development  5.4.1. Requirements for method development  5.4.2. Different type of enzymes assays  5.4.2.1. Direct continuous assay  5.4.2.2. Indirect assays  5.4.2.3. Discontinuous indirect assays  5.4.2.4. Continuous indirect assays  5.4.2.5. Coupled assays  5.5. Blank rates  5.6. The assay development process  5.6.1. Initial assay scoping  5.6.2. Substrate dependence  5.6.2.1. Non-Michaelian kinetics  5.6.2.2. Multiple substrates  5.6.3. Plate type  5.7. Assay optimisation  5.7.1. Factorial Experimental Design (FED)  5.7.2. Coupling enzyme considerations  5.8. Kinetic characterisation  5.8.1. Substrate concentration  5.9. Assay adaptability  5.9.1. Tool compounds  5.9.2. DMSO tolerance  5.9.3. Assay stability  5.9.4. Triage assays  5.10. Assay Evaluation (validation)  5.10.1. Calculations  5.10.2. Assessing plate uniformity  5.10.3. Acceptable assay performance criteria  5.11. References Chapter 6 – Enzyme inhbition 6.1. Introduction  6.2. Substrate and product inhibition  6.3. Reversibility  6.3.1. Testing for irreversible inhibition  6.3.2. Rapidly reversible inhibition  6.4. The IC50 value 6.4.1. Determining the IC50 value  6.4.2. Use of pIC50  6.4.3. Comparison of potency  6.4.4. Concentration response curve analysis  6.4.4.1. Bell-shaped behaviour  6.4.4.2. Weakly active compounds  6.4.4.3. Steep curves  6.4.4.4. Partial curves  6.4.4.5. Noisy data  6.5. Identity of substrate  6.6. Effect of Enzyme concentration – Tight-binding inhibition  6.7. Slow-binding inhibition  6.7.1. Progress curves for slow-binding inhibition   6.8. Slow, tight-binding inhibition  6.8.1. Conditions where detection of slow-binding may be precluded  6.9. Irreversible inhibition  6.10. Presence of two inhibitors  6.11. Non-specific Inhibition  6.11.1. Common technology hitters  6.11.1.1. UV-light Interference  6.11.1.2. Detection system interference  6.11.2. Chemical reactivity  6.11.3. Aggregators  6.11.4. Redox reactivity  6.11.4.1. Oxidation of aromatic amino acid residues  6.11.4.2. Protein unfolding  6.11.5. Denaturation  6.11.6. Metal ion contamination 6.12 References Chapter 7 – Enzyme activation 7.1. Introduction  7.2. Mechanisms for enzyme activation  7.2.1. Essential activation  7.2.1.1. Essential cationic activation  7.2.2. Non-essential activation  7.2.2.1. Non-essential cationic activation  7.2.3. A comment on Nomenclature: K-type and V-type classification  7.2.4. De-inhibition  7.3. Challenges for identifying non-essential enzyme activators  7.3.1. Enzymes have evolved to be active  7.3.2. Lack of tool compounds  7.3.3. Maintaining steady state  7.3.4. Mechanistic considerations  7.3.5. Assay design and variability  7.4. Addressing the challenges of activator discovery  7.5. References  Chapter 8 – Mechanism of action 8.1. Introduction  8.2. Mechanisms of inhibition  8.2.1. Competitive Inhibition 8.2.2. Mixed noncompetitive and pure noncompetitive inhibition 5 8.2.3. Uncompetitive inhibition  8.3. Choosing between different types of Inhibition  8.4. Interpretation of Mechanism of Inhibition  8.5. Effect of multiple substrates and assignment of mechanism  8.6. Binding site may not equal mechanism  8.6.1. Substrate competitive inhibitors at allosteric sites  8.6.2. Noncompetitive binding giving competitive inhibition  8.6.3. Competitive binding giving uncompetitive inhibition  8.7. Specificity  8.7.1. Effect of increasing [S]  8.7.2. Effect of [I]  8.8. Activation Mechanisms and comparison with Inhibition  8.9. References  Chapter 9 – Data analysis 9.1. Introduction  9.2. Statistical analysis of enzyme kinetic data  9.3. Least squares fitting  9.4. Non-linear regression  9.5. Weighting of experimental data  9.6. Evaluation of potential different models  9.6.1. Distribution of residuals  9.6.1.1. The runs test  9.6.2. Magnitude of standard errors  9.6.3. Quantitative evaluation  9.6.3.1. F-test  9.6.3.2. Aikake information criterion  9.6.3.3. Absolute goodness of fit  9.7. Minimum significant ratio  9.8. Two independent variables  9.8.1. Global fitting  9.8.2. Reducing and repeating and reducing only  9.8.2.1. Reducing only  9.8.2.2. Reducing and repeating  9.9         References Chapter 10 – Molecular interactions 10.1. Introduction  10.2. Binding affinity is a function of difference energy  10.3. Interactions between charged or polar groups  10.3.1. Electrostatic interactions  10.3.2. Hydrogen bonds  10.4. Non-polar interactions  10.4.1. Van der Waals interactions  10.4.2. Hydrophobic interactions  10.5. Changes in hydrophobicity on chemical substitution  10.6. Entropy  10.7. References  Chapter 11- Applications in drug discovery 11.1. Introduction  11.2. Pre screening  11.2.1. Enzyme considerations  11.2.2. Substrate considerations  11.2.3. Enzyme mechanism considerations  11.3. Post screening  11.3.1. Measuring potency  11.3.2. Reversibility  11.3.3. Inhibitor mechanistic characterisation  11.3.3.1. Combining enzyme kinetics and biophysics  11.3.4. Tight binding 11.3.5. Undesired mechanisms  11.4. In vitro vs in cellulo enzyme kinetics  11.5. References  Appendix 1 - Basic maths and statistic A1.1. Algebra  A1.2. Fractions  A1.3. Indices  A1.4. Logarithms  A1.5. Quadratic equations  A1.6. Straight lines  A1.7. Functions  A1.8. Inequalities  A1.9. Differentiation  A1.10. Integration  A1.11. Series A1.12. Statistics  A1.13. Propagation of errors  A1.14. Using logged values  A1.15. Precision, accuracy, significant figures & rounding  A1.16. Dimensional analysis  Appendix 2 – Key formulas A2.1 Assay reagent calculations  A2.2 Assay statistics  A2.3 Curve fitting and calculation of parameters  A2.4 Thermodynamics  Appendix 3 – Constants, prefixes, conversions A3.1 Useful Physical Constants  A3.2 Prefixes used in the SI system  A3.3 Conversion factors  A3.4 Greek Alphabet  Appendix 4 – Common symbols and abbrviatins and their units A4.1. Common symbols  A4.2. Common abbreviations  Appendix 5 – Glossary Appendix 6 – Key derivations A6.1. Langmuir Isotherm, assuming rapid equilibrium A6.2. Michaelis-Menten, assuming rapid equilibrium A6.3. Michaelis-Menten, assuming steady state A6.4. Two substrate reactions A6.5. Dose response equation to calculate Ki’ A6.6. Derivation of Substrate inhibition, assuming rapid equilibrium A6.7. Competing ligands, assuming rapid equilibrium A6.8. Tight-binding A6.9. Single exponential, with first order rate equation A6.10. Protein double ionisation A6.11. Protein single ionisation A6.12. Derivation of the integrated rate equation for slow-binding inhibition described by mechanism B A6.13. Essential activation A6.14. Non-essential activation Appendix 7 – Useful software packages for analysing kinetic data A7.2. Web based tools  A7.3. Other useful online resources 

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Author Information

Geoffrey A. Holdgate is Senior Principal Scientist in Discovery Sciences for BioPharmaceuticals Research and Development at AstraZeneca. Antonia Turberville, PhD, is a Senior Scientist in Discovery Sciences for Biopharmaceuticals R&D at AstraZeneca. Alice Lanne, PhD, is a Senior Scientist in Discovery Sciences for BioPharmaceuticals R&D at AstraZeneca.

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