Laboratory Guide to Enzymology

Informationen zum Autor 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. Klappentext An accessible guide to understanding the foundations of enzymology at its application in drug discoveryEnzymes 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 activationLaboratory 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. Zusammenfassung LABORATORY GUIDE TO ENZYMOLOGYAn accessible guide to understanding the foundations of enzymology at its application in drug discoveryEnzymes 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 activationLaboratory Guide to Enzymology is ideal for all pharmaceutical and biomedical researchers working in enzymology and assay development, as well as advanced students in...

Auteur

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.

Texte du rabat

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.

Contenu

Preface xiii

Acknowledgments xv

1 Introduction to Proteins and Enzymes 1

1.1 Protein Structure 1

1.1.1 Primary Structure 1

1.1.2 Secondary Structure 1

1.1.3 Tertiary Structure 5

1.1.4 Quaternary Structure 6

1.1.5 Protein Structure Prediction 6

1.2 Enzymes 9

References 15

2 Binding Equilibria and Kinetics 17

2.1 Introduction to Chemical Kinetics 17

2.2 Introduction to Binding Kinetics 22

2.3 Ligand Binding to Single Binding Site 23

2.4 Kinetic Approach to Equilibrium 26

2.5 Methods for Measuring Protein-ligand Binding 27

2.6 Ligand Depletion (Tight Binding) 29

2.8 Ligand Competition 34

References 35

3 Protein QC and Handling 37

3.1 Introduction 37

3.2 Confirming Protein Identity 37

3.2.2 Peptide Mapping 38

3.2.3 Edman Sequencing 39

3.3 Protein Purity 39

3.3.1 Sds-page 40

3.3.2 Dynamic Light Scattering (DLS) 40

3.3.3 Analytical Gel Filtration 41

3.3.4 Analytical Ultracentrifugation 42

3.4 Concentration 43

3.4.1 UV-Vis Spectrum 44

3.4.2 Bradford Assay 44

3.5 Functionality 46

3.5.1 Ligand Binding 46

3.5.1.1 Itc 46

3.5.1.2 Spr 46

3.5.2 Functional Studies 46

3.6 Stability 48

3.6.1 Differential Scanning Calorimetry (DSC) 48

3.6.2 Differential Scanning Fluorimetry (DSF) 49

3.6.3 Circular Dichroism 51

3.6.4 Selwyn's Test 51

References 52

4 Buffers and their Use in the Study of Enzyme Mechanism 55

4.1 Introduction 55

4.1.1 Ionization and pK a 55

4.1.2 pH 57

4.1.3 Henderson-Hasselbalch Equation 57

4.1.4 Buffers 58

4.2 Buffering Capacity 60

4.3 Ionic Strength 60

4.4 Change in pH with Temperature 62

4.5 Choice of Buffer 63

4.6 Characteristics of Ionizing Groups in Proteins 65

4.7 Effect of pH on Enzyme Activity 66

4.7.…