Proteolytic Enzymes A Practical Approach

by Beynon, Robert; Bond, Judith S.

• ISBN13: 9780199636624
• ISBN10: 0199636621
• Edition: 2nd
• Format: Paperback
• Copyright: 2001-03-22
• Publisher: Oxford University Press

Summary

Like the popular first edition, this new edition of Proteolytic Enzymes emphasizes practical aspects of the handling, characterization, inhibition, and use of proteolytic enzymes giving general advice and specific examples. The text and protocols have been thoroughly updated to take account ofthe advances made in the last 10 years in both the increased understanding of the role of peptidases in many critical cellular processes e.g. apoptosis and new technological developments e.g. in recombinant protein expression, protein sequencing, and structural studies. The topics covered are:nomenclature and classification; purification; assay methods; determination of mechanism; inhibition and prevention of unwanted proteolytic activity; characterizing natural inhibitors; proteolytic enzymes in peptide mapping and primary structure elucidation by mass spectrometry and Edman sequencing;limited proteolysis as a structural probe; synthetic function. This book will be as invaluable as the first edition in providing ideas and protocols for scientists either studying proteases or using proteases as a research tool.

Table of Contents

Preface	v
List of protocols	xv
Abbreviations	xvii
Proteolytic enzymes: nomenclature and classification	1	(22)
Alan J. Barrett
Introduction	1	(1)
Terminology and nomenclature	1	(3)
Peptidase and related terms	1	(2)
Specificity-subsite terminology	3	(1)
Catalytic type	3	(1)
Homology	3	(1)
The EC classification of peptidases	4	(8)
What is the EC system?	4	(2)
What information does the EC list contain?	6	(1)
When and how can a newly-discovered peptidase be added to the EC list?	7	(5)
The MEROPS system for the classification of peptidases	12	(7)
Families	13	(4)
Clans	17	(1)
Individual peptidases	17	(1)
Uses of the MEROPS system	18	(1)
Steps one might take on discovering a new peptidase	19	(4)
Acknowledgement	20	(1)
References	20	(3)
Purification of proteolytic enzymes	23	(22)
Sherwin Wilk
Introduction	23	(1)
Prelude to purification	24	(2)
Assay	24	(2)
Initial considerations	26	(4)
Source	26	(1)
Buffer composition	26	(1)
Membrane-bound or soluble?	26	(1)
Membrane-bound enzymes	27	(2)
Endogenous inhibitors and activators	29	(1)
General scheme for purification of proteolytic enzymes	30	(3)
Initial steps	30	(2)
Intermediate and final steps	32	(1)
Specialized techniques for proteolytic enzymes	33	(4)
Peptidyl aldehyde affinity chromatography	33	(1)
Affinity columns for cysteine proteinases	34	(1)
Affinity columns for trypsin-like enzymes	34	(1)
Affinity columns for metalloproteinases	35	(1)
Other affinity columns	35	(2)
Optimization of the purification protocol	37	(1)
Determination of homogeneity	38	(1)
Purification of the proteasome; EC 3.4.99.46	38	(4)
Conclusions: many roads lead to Rome	42	(3)
References	42	(3)
Protease assay methods	45	(32)
Gautam Sarath
Michael G. Zeece
Alan R. Penheiter
Introduction	45	(1)
Assays with natural substrates	45	(10)
Endopeptidase assays	45	(10)
Exopeptidase assays	55	(1)
Assays with synthetic substrates	55	(14)
Endopeptidase and aminopeptidase substrates	55	(1)
Spectrophotometric assays	56	(3)
Fluorimetric assays	59	(3)
Miscellaneous fluorimetric methods	62	(2)
Carboxypeptidase substrates	64	(1)
Radiometric assays	64	(1)
HPLC assays for peptidases	65	(2)
Capillary electrophoretic analyses of proteases	67	(2)
Solid-phase protease assays	69	(8)
Gel electrophoretic methods	70	(3)
Plate assays	73	(1)
Miscellaneous solid-phase assays	73	(1)
Assays for histochemical studies	74	(1)
Acknowledgements	75	(1)
References	75	(2)
Determination of protease mechanism	77	(28)
Ben M. Dunn
Introduction---the importance of mechanistic classification	77	(6)
The serine peptidases	78	(2)
The cysteine peptidases	80	(1)
The aspartic peptidases	81	(1)
The metallopeptidases	81	(2)
Methods for determining the mechanistic class	83	(8)
Classification based on `standard' inhibitors	83	(3)
Chemical modification/identification	86	(1)
Site-directed mutagenesis	87	(1)
Mechanistic distinctions---intermediates	88	(3)
Kinetic studies to probe the mechanism in more detail	91	(6)
Notes on `ideal' assays	91	(1)
Kinetic determination of Km and kcat	92	(3)
pH dependence of the kinetic parameters	95	(1)
Solvent deuterium isotope effects	96	(1)
Transition state analogues and substrate alteration	97	(1)
Determination of primary specificity of a protease	97	(8)
Degradation of standard proteins and peptides	98	(1)
Cleavage of homologous synthetic peptides	99	(3)
References	102	(3)
Inhibition of proteolytic enzymes	105	(26)
Guy S. Salvesen
Hideaki Nagase
Introduction: which inhibitors?	105	(1)
Principles for using irreversible and reversible inhibitors	106	(1)
General structure of synthetic inhibitors	106	(1)
How to live with crossreactivity	106	(1)
Practical use of inhibition constants	107	(2)
Irreversible inhibitors	107	(1)
Reversible inhibitors	108	(1)
Non-specific inhibitors	109	(3)
α-Macroglobulins	109	(1)
Peptide aldehydes	110	(1)
Peptide chloromethyl ketones	110	(1)
Metal chelators	111	(1)
Class-specific inhibitors	112	(10)
Serine proteases	112	(4)
Cysteine proteases	116	(3)
Proteasome	119	(1)
Metalloproteases	120	(2)
Aspartic proteases	122	(1)
Inhibitors as active-site titrants	122	(3)
Cysteine proteases	122	(2)
Serine proteases	124	(1)
Protease inhibitors in cell culture	125	(1)
Suppression of proteolysis	126	(1)
Therapeutic value of protease inhibitors	127	(4)
References	128	(3)
Finding, purification and characterization of natural protease inhibitors	131	(18)
Hideaki Nagase
Guy S. Salvesen
Introduction	131	(1)
The meaning of inhibition	131	(1)
Finding protease inhibitors	132	(5)
Screening of inhibitors from natural sources	132	(1)
Finding inhibitors by reverse zymography	133	(2)
Finding inhibitors from DNA sequences	135	(2)
Combinatorial protease inhibitors	137	(1)
Purification of natural protease inhibitors	137	(1)
Use of the target enzyme as an affinity ligand	137	(1)
Conventional purification	137	(1)
Reverse zymography	138	(1)
Characterization of inhibitors: inhibition kinetics	138	(8)
The importance of kinetics	138	(1)
IC50 and percentage inhibition	138	(1)
Practical inhibitor kinetics	139	(1)
Reversible inhibitors	140	(3)
Irreversible inhibitors	143	(3)
Practical applications of protein inhibitors	146	(3)
Acknowledgements	146	(1)
References	146	(3)
Mass spectrometry of proteolysis-derived peptides for protein identification	149	(38)
Bernhard Kuster
Andrej Shevchenko
Matthias Mann
Introduction	149	(1)
Mass spectrometry	150	(5)
Methods of ionization	150	(2)
Mass analysis	152	(1)
Tandem mass spectrometry	153	(2)
Interrogation of sequence databases using mass spectrometry data	155	(6)
Choice of protease	155	(2)
Peptide mass mapping	157	(2)
Database identification via tandem mass spectrometry	159	(2)
Analytical strategy for the identification or cloning of proteins using mass spectrometry	161	(20)
Low-level protein preparation for characterization by mass spectrometry	161	(2)
Protein visualization	163	(2)
Proteolytic cleavage of gel separated proteins	165	(2)
Protein identification by MALDI peptide mass mapping	167	(5)
Peptide sequencing by nanoelectrospray tandem mass spectrometry	172	(5)
Towards cloning of proteins using mass spectrometry data	177	(4)
Post-translationally modified proteins	181	(2)
Concluding remarks	183	(4)
Acknowledgements	183	(1)
References	183	(4)
Using proteinases for Edman sequence analysis and peptide marking	187	(24)
John Shannon
Introduction	187	(1)
The need for digesting proteins to peptides	187	(1)
Substrate preparation	188	(5)
Purification techniques	188	(1)
Sample requirements	189	(2)
Reduction and alkylation of proteins	191	(2)
Digestion	193	(7)
Choice of proteinase	193	(3)
Conditions for proteolytic digestions	196	(3)
Digestion of proteins isolated on polyacrylamide gels	199	(1)
Monitoring a reaction	200	(1)
Analysis of the proteolytic digestion	200	(11)
Mass spectrometry	200	(1)
Electrophoresis	200	(1)
Ion exchange	200	(1)
Reverse-phase chromatography	201	(8)
Data interpretation	209	(1)
Acknowledgments	209	(1)
References	209	(2)
Prevention of unwanted proteolysis	211	(22)
Michael J. North
Robert J. Beynon
Introduction	211	(2)
Proteolytic susceptibility of native proteins	213	(1)
Intrinsic factors determining the susceptibility of proteins to proteolysis	213	(1)
The influence of other molecules on susceptibility to proteolysis	213	(1)
Properties of endogenous proteinases	214	(1)
Identification of proteolysis as a problem	214	(3)
Changes in protein properties	214	(2)
Mimicking an effect with added proteinases	216	(1)
Checking samples for proteinase activity	216	(1)
Inhibition of proteinases	217	(10)
Outline of approaches for reducing proteinase activity	217	(1)
Suppression of endogenous proteinase activity	217	(1)
Preventing proteolysis by denaturation	217	(3)
Use of proteinase inhibitors	220	(7)
Removal of proteinases	227	(6)
Choice of starting material	227	(3)
Cell disruption and fractionation	230	(1)
Selective removal of proteinases during purification	231	(1)
Acknowledgements	231	(1)
References	231	(2)
Proteolysis of native proteins as a structural probe	233	(32)
Simon Hubbard
Robert J. Beynon
Introduction	233	(2)
Factors influencing susceptibility	235	(5)
Molecular recognition and limited proteolysis	235	(1)
Prediction of nicksites	236	(3)
A tool to aid in prediction of sites of limited proteolysis	239	(1)
Experimental considerations	240	(9)
Choice of proteinase	242	(3)
Ratio of proteinase to substrate	245	(1)
Solution conditions	245	(1)
Determination of site of proteolysis	246	(1)
Strategies for limited proteolysis experiments	247	(2)
Analysis of limited proteolysis data and simulations	249	(16)
Obtaining quantitative data	250	(4)
Analysing the data by non-linear curve fitting	254	(3)
Example reaction schemes	257	(1)
Simulations and modelling	258	(6)
References	264	(1)
Proteases in peptide synthesis	265	(28)
Volker Kasche
Introduction	265	(2)
Enzyme properties influencing the product yield and steric purity in protease catalysed peptide synthesis	267	(4)
Kinetically controlled synthesis	267	(3)
Equilibrium-controlled peptide synthesis	270	(1)
Selecting the optimal protease	271	(7)
Purity of the protease	271	(1)
P1 and P1' specificity	272	(6)
Factors controlling the yield and steric purity in the synthesis of a peptide bond with a given enzyme	278	(8)
Protection of the P1' and activation of the P1-carboxyl group	279	(1)
pH	279	(2)
Temperature	281	(1)
Ionic strength	282	(1)
Solvent composition	283	(1)
Peptide synthesis in suspensions with solid product or substrate	284	(2)
Planning a protease-catalysed synthesis of a peptide bond	286	(2)
What enzyme?	286	(1)
Equilibrium-controlled or kinetically controlled synthesis?	287	(1)
Free or immobilized enzyme?	287	(1)
Experimental methods for protease-catalysed peptide synthesis	288	(1)
Enzyme purity and purification	288	(1)
Enzyme immobilization	288	(1)
Substrates and buffers	288	(1)
Monitoring the synthesis; purification of products	289	(1)
Optimizing the yield	289	(1)
Proteases in peptide synthesis: limitations and perspectives	289	(4)
References	291	(2)
A1 The Schechter and Berger nomenclature for proteinase subsites	293	(2)
A2 Some commercially available proteases	295	(22)
A3 Commercially available proteinase inhibitors	317	(14)
A4 List of suppliers	331	(6)
Index	337