Specificity of Proteolysis

Borivoj Keil Specificity of Proteolysis With 17 Figures and 20 Tables Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Hong Kong Barcelona Budapest

Prof. Dr. Borivoj Keil Institut Pasteur 28, Rue du Docteur Roux F-75015 Paris ISBN 978-3-642-48382-0 DOl 10.1007/978-3-642-48380-6 ISBN 978-3-642-48380-6 (ebook) Library of Congress Cataloging-in-Publication Data Keil, Borivoj. Specificity of proteolysis / Borivoj Keil. p. cm. Includes bibliographical references and indexes. 1. Proteolytic enzymes. I. Title. QP609.P78K45 1992 574. 19'256--dc20 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microftlm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9,1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. e Springer-Verlag Berlin Heidelberg 1992 Softcover reprint of the hardcover 1 st edition 1992 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any mformation about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Media conversion: Elsner & Behrens GmbH, Oftersheim 31/3145-543210- Printed on acid-free paper

Preface Three main sources of information on the specificity of proteinases can be distinguished: studies to characterize proteolytic cleavages in the living systems, projects aimed at the determination of protein sequences; and the use of synthetic substrates. In the first case, the investigators characterize a new proteinase and determine bonds cleaved in its natural substrate. A suitable assay is eventually elaborated using a synthetic substrate. In the second case, pure proteinases are used to cleave an unfolded or modified protein to fragments, suitable for subsequent sequence analysis. An important volume of dispersed facts was accumulated in bibliography from these two categories of study. Their collection and evaluation in a computerized system was the aim at the creation of the data bank LYSIS*, which served simultaneously as source of data for the monography "Specificity of Proteolysis". The two publications are independent but complementary. "Specificity of Proteolysis" gives a short review of actual trends in the field and rapid information on catalytic properties of 295 endopeptidases in the classical form of text, tables and figures. Despite the considerable volume of data, compressed in the available space, an effort was made to maintain the content within limits of a readable text for those who prefer to browse through a printed book rather than to consult the screen. On the other hand, the data bank LYSIS, managed by its userfriendly program DIGEST, leads to more detailed information. Its final goal is to make available published data on cleavages produced in all protein and peptide substrates by all actually known endopeptidases, under the condition that the cleaved bonds have been identified. This in turn allows the evaluation by appropriate software, of how the probability of a given cleavage may be influenced by the amino acid sequence of the corresponding substrate, the nature of resistant bonds etc. Besides the data comprised in "Specificity of Proteolysis", LYSIS contains addition- The data bank LYSIS is available as a diskette set: Keil B, Tong TN (1992) LYSIS. Springer, Berlin, Heidelberg, New York. (ISBN 3-540-14123-5 Springer-Verlag Berlin Heidelberg New York) (ISBN 0-387-14123-5 Springer-Verlag New York Berlin Heidelberg)

VI Preface al data on the sequences and on chemical modifications of protein substrates. The book and the first release of LYSIS contain data on 295 endopeptidases, 1082 protein substrates, and 6300 degradation cases compiled from 2200 publications. It does not cover exhaustively the whole pool of data accumulated in the last 40 years; nevertheless, its actual structure is open to gradual completion by both data and methodology, provided that this first attempt will be found helpful. April 1992 Borivoj Keil

Contents 1 Introduction..................................... 1 1 Nomenclature and Conventions... 3 2.1 EC Numbers... 3 2.2 Enzyme Names... 3 2.3 Enzyme and Substrate Codes... 4 2.4 Subsite Nomenclature... 6 2.5 Bibliography... 6 3 Data Treatment... 7 3.1 Data Bank LYSIS... 7 3.2 Statistical Approach to Specificity... 8 3.2.1 Calculations.............................. 9 3.2.2 Sources of Errors and Limits... 10 3.2.3 Influence of Subsites on Chymotryptic Cleavages... 11 3.2.4 Roles of Subsites P 4-P 3' in Cleavages by Pepsin... 14 3.2.5 Fixation Preference of Chymotrypsin and Pepsin... 15 3.2.6 Predictions of Cleavage Probability... 16 3.2.7 Comparison with Kinetic Study... 17 4 Standard Polypeptide Substrates... 19 4.1 Choice of Standard Polypeptides... 19 4.2 Available Data on Cleavages of Insulin Chains and Glucagon... 22 4.3 Repartition of Cleavage-Susceptible Bonds... 35 4.4 Binding Sites - Proposal for Fixation Site Types... 35 4.5 Influence of Subsites... 40

VIn Contents 5 Essential Substrate Residues for Action of Endopeptidases... 43 5.1 Basic Residue... 43 5.1.1 Arg in PI... 43 5.1.2 Arg or Lys in PI (Arg>Lys)... 66 5.1.3 Lys in PI (Lys>Arg)... 77 5.1.4 Lys in PI'... 84 5.1.5 Arg in P 2 and Gly in PI... 86 5.1.6 A Pair of Basic Residues... 86 5.1.7 Basic or Aromatic Residue in PI... 92 5.2 Acidic Residue... 93 5.2.1 Glu in PI (Glu>Asp)... 94 5.2.2 Asp in PI or PI'... 99 5.3 Neutral Residue... 101 5.3.1 Leu or Val in PI... 102 5.3.2 Aromatic or Hydrophobic Residue in PI... 107 5.3.3 Aromatic or Hydrophobic Residue in PI (Acidic ph) 126 5.3.4 Aromatic or Hydrophobic Residue in PI and PI'... 130 5.3.5 Hydrophobic Residue in P 2 145 5.3.6 Hydrophobic Residue in PI'... 155 5.3.7 Hydrophobic Residue in P 3'... 171 5.3.8 Small Neutral Residue in PI... 174 5.3.9 Miscellaneous Neutral Residues in PI... 182 5.3.10 Neutral Residues in Both PI and PI'... 186 5.4 Proline Residue... 206 5.4.1 Pro in PI... 206 5.4.2 Pro in P 2'. 214 5.5 Alpha-Epsilon Peptide Bond... 215 5.6 Peptidases with Occasional Endopeptidase Activity... 215 5.7 Vague or Insufficient Information on Specificity... 218 5.8 No Information on Bond Specificity... 227 6 Comments....................................... 229 6.1 Frequently Used Proteinases and Restriction Proteinases... 229 6.2 Group of Microbial Proteinases... 231

IX References... 233 Appendices... 283 A Tabular Index of LYSIS Enzyme Codes... 283 B Tabular Index of LYSIS Protein Codes... 295 Subject Index... 325