Kinetic analysis of macromolecules : a practical approach / edited by Kenneth A. Johnson.

Format:

Book

Contributor:

Johnson, K. (Kenneth), 1931-

Series:

Practical approach series ; no. 267.

Practical approach series ; no. 267

Language:

English

Subjects (All):

Enzyme kinetics.

Physical Description:

xx, 256 pages : illustrations ; 26 cm.

Place of Publication:

Oxford ; New York : Oxford University Press, 2003.

Contents:

1 Introduction to kinetic analysis of enzyme systems / Kenneth A. Johnson 1

1 Steady-state kinetic analysis: what you learn 2

2 Enzyme inhibition patterns 4

2.1 Active site titration 4

3 Data fitting by nonlinear regression 5

3.1 Transient kinetic methods 6

4 Substrate binding kinetics 7

5 Measuring the dissociation rate by competition 9

6 Kinetics of a pre-steady-state burst 10

7 Kinetics of a single turnover 12

8 Kinetics of a lag: what is a lag time? 13

9 Site-directed mutagenesis and proper analysis of mutants 15

10 Global fitting of kinetic and equilibrium data by computer simulation 15

2 Detection and characterization of enzyme intermediates: utility of rapid chemical quench methodology and single enzyme turnover experiments / Karen S. Anderson 19

2 Criteria for establishing enzyme intermediates 20

3 Rapid chemical quench methodology 21

4 Single turnover experiments for the detection of enzyme intermediates 23

5 Selected examples of enzyme studies using a rapid chemical quench strategy to detect and characterize enzyme intermediates 24

5.2 PEP-utilizing enzymes that catalyze C-O bond cleavage of phosphoenol pyruvate 24

5.3 A covalent phosphoryl cysteine enzyme intermediate in a tyrosine phosphatase 33

5.4 Tryptophan synthase: probing substrate channelling in a bifunctional enzyme and searching for indole as an intermediate 36

6 New directions and approaches for detecting and characterizing enzyme intermediates 41

6.1 Mass spectrometry: a novel approach to the detection of enyzme intermediates 41

6.2 Recent advances in mass spectroscopy to study biomolecules 42

3 Kinetic analysis of ribozyme cleavage / Philip C. Bevilacqua, Trevor S. Brown, Durga M. Chadalavada, Amy Diegelman-Parente, Rieko Yajima 49

2 Initial considerations for the system and experiments 50

2.1 Choosing and preparing the system 50

2.2 Choosing the experiment 54

2.3 Deriving kinetic equations 55

2.4 Simulating data 59

3 Kinetic analysis of ribozymes using radiolabelled RNA 62

3.1 Introduction: advantages and disadvantages of radiolabelled methods 62

3.2 Preparing ribozymes and RNA substrates 62

3.3 A few guidelines for purifying and handling RNA 67

3.4 Radiolabelling ribozymes 68

3.5 Slow (hand-mixing) kinetics 69

3.6 Fast (quench-flow mixing) kinetics 72

4 Kinetic analysis of ribozymes: overview of optical techniques 72

4 Kinetics analysis of DNA-protein interactions by time-resolved synchrotron X-ray footprinting / Gauri M. Dhavan, Mark R. Chance, Michael Brenowitz 75

1 Preparation of materials and choice of experimental conditions 76

2 Determining the X-ray exposure required for optimal DNA cutting 76

3 Conducting a quench flow hydroxyl radical experiment 79

4 Data analysis 83

5 Transient-state kinetics and computational analysis of transcription initiation / Smita S. Patel, Rajiv P. Bandwar, Mikhail K. Levin 87

1.1 RNA polymerase 88

1.2 Minimal pathway 88

2 Experimental approach 89

2.1 Fluorimetric methods 89

2.2 DNA 91

2.3 Protein 93

3 Equilibrium fluorescence measurement 95

3.1 Fluorescence of T7 RNAP-DNA complex 95

3.2 Equilibrium dissociation constant 96

4 Kinetics of RNAP and promoter DNA binding 99

4.1 Stopped flow kinetics of p-dsDNA binding 99

4.2 Dissociation rate constant 101

4.3 Stopped flow kinetics of dsDNA promoter binding 103

4.4 DNA binding in the presence of initiating GTP 105

5 Kinetics of initiating GTP binding 107

6 Radiometric assay for RNA synthesis 108

7 Data simulation and fitting 112

7.1 Model building and simulation 113

7.2 Data fitting 119

6 A fluorescent sensor to assay inorganic phosphate / Martin R. Webb 131

2 P[subscript i] sensor 131

2.2 Preparation and characterization 133

3 An enzymic assay for P[subscript i] 137

4 P[subscript i] mop 139

5 Choice of assay method 140

5.1 Summary of different types of phosphate assay 140

5.2 Fluorescence versus absorbance 140

6 Practical considerations 142

6.2 Minimizing contaminant P[subscript i] 142

6.3 Other considerations for using the P[subscript i] sensor 143

6.4 Limitations of the P[subscript i] sensor 146

7 Measurement of P[subscript i] formation kinetics using the P[subscript i] sensor 147

7 What to do if there is no signal: using competition experiments to determine binding parameters / Nicolas Thoma, Roger S. Goody 153

2 Determination of equilibrium constants of unlabelled ligands 153

3 Use of transient experiments to determine rate constants of unlabelled molecules 159

3.1 Competitive displacement 159

3.2 Competitive association kinetics 161

8 The biotin regulatory system

kinetic, thermodynamic, and structural interrogation of a biological switch / Dorothy Beckett 171

2 The participants in the biotin regulatory system 173

3 Protein-small ligand binding studies 175

3.1 Kinetic methods for measurement of binding of the biotin and bio-5'-AMP to BirA 176

3.2 Kinetic stability of BirA - small ligand complexes 176

4 An enzymatic assay to measure dissociation of the BirA - bio-5'-AMP complex 178

5 Kinetic measurements of bimolecular association of BirA with small ligands: evidence of ligand-induced conformational changes 181

6 Low resolution structural analysis of the system 183

7 Functional studies of flexible loops in BirA 185

7.1 The glycine-rich loop and small ligand binding 185

7.2 A flexible loop in biotin and bio-5'-AMP binding 186

7.3 Flexible loops in DNA binding and dimerization 187

8 Bio-5'-AMP binding promotes dimerization of BirA 189

9 Involvement of flexible loops in site-specific DNA binding and dimerization 191

10 X-ray crystallographic determination of the dimer structure 191

11 The structural basis of the biological switch 193

12 Kinetics of DNA binding and the functional switch in BirA 194

13 Measurement of association of holoBirA with bioO 197

14 Data analysis 200

9 Time resolved spectral analysis / Bruce A. Palfey 203

2 Experimental design 204

2.1 Sample concentration and quality 204

2.2 Reaction conditions 204

2.3 Types of kinetic experiments for spectral analysis 208

2.4 Anaerobic experiments 209

3 Methods for collecting spectral data 218

3.1 Single-wavelength traces 218

3.2 Rapid scanning 219

4 Calculating spectra 220

4.1 Global analysis 221

4.2 Singular value decomposition 223

10 Kinetic analysis of enzyme mutants for identification of catalytic residues / Yansheng Wei, Lusong Luo, Wenhai Zhang, Debra Dunaway-Mariano 229

1.1 Statement of purpose 229

1.2 Defining the catalytic site of the enzyme 229

1.3 Defining the reaction steps catalysed by the enzyme 230

1.4 Targeting potential catalytic residues 232

1.5 Screening mutants for transient kinetic analysis 233

2 Kinetic analysis 234

2.1 Defining the kinetic mechanism: an overview 234

2.2 Defining rate constants for substrate and product binding and release steps 235

2.3 Determining time courses for single turnover reactions 241

2.4 Extracting microscopic rate constants from single turnover kinetic data 244

2.5 Comparison of turnover rates derived from microscopic rate constants to experimental steady-state turnover rates 245

3 Interpretation of results 248

3.1 Structure determinations of mutants 248

3.2 Additional mutants to be analyzed 249.

Notes:

Includes bibliographical references and index.

ISBN:

0198524935

0198524943

OCLC:

52486305