Preface Through the earlier, general volumes on proteolytic enzymes (Vol- umes 19, 45, and 80), Methods in Enzymology made available over 200 authoritative articles on these enzymes and their inhibitors. Since the appearance of the latest of these volumes, however, there have been many profound advances in this field of study. The biomedical importance of proteolytic enzymes, suspected for so long, has been established be- yond reasonable doubt for a number of groups, including the matrix me- talloproteinases, the viral polyprotein-processing enzymes, and the pro- hormone-processing peptidases. The more recent, specialized Volumes 222, 223, and 241 have dealt with some of these areas, but others have remained to be covered. The resurgence of excitement about proteolytic enzymes has inevita- bly resulted in an information explosion, but some of the new understand- ing has also helped us develop novel approaches to the management of the mass of data. As a result, we can now "see the forest for the trees" a little more clearly. Like other proteins, the proteolytic enzymes have benefited from the recent advances in molecular biology, and amino acid sequences are now available for many hundreds of them. These can be used to group the enzymes in families of evolutionarily related members. Also, there has been a major overhaul of the recommended nomenclature for pepti- dases by the International Union of Biochemistry and Molecular Biology. In Volumes 244 and its companion Volume 248 on aspartic and metallo peptidases, the chapters on specific methods, enzymes, and inhibitors are organized within the rational framework of the new systems for classifica- tion and nomenclature. In the peptidases dealt with in this volume, the nucleophilic character of a serine or cysteine residue is at the heart of the catalytic mechanism, whereas the peptidases of the aspartic and metallo types described in Volume 248 depend for their activity on an ionized water molecule. A wide variety of specificities of peptide bond hydrolysis is represented in each set of peptidases, together with an equally wide range of biological functions. ALAN J. BARRETT XV Contributors to Volume 244 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. MAGNUS ABRAHAMSON (49), Department of Clinical Chemistry, University of Lund, University Hospital, S-221 85 Lund, Swe- den MAGGY ADAM (19), Centre d'lngdnierie de Prot~ines, Universitd de Liege, Institut de Chimie, B6, B-4000 Sart Tilman, LiOge 1, Belgium WILLIAM W. BACHOVCHIN (10), Depart- ment of Biochemistry, Tufts University School of Medicine, Boston, Massachu- setts 02111 ALAN J. BARRETT (1, 2, 32), Department of Biochemistry, Strangeways Research Laboratory, Cambridge CB1 4RN, United Kingdom GERALD W. BECKER (29), Biotechnology Di- vision, Lilley Research Laboratories, Lil- Icy Corporate Center, Eli Lilley and Com- pany, Indianapolis, Indiana 46285 KERRY BEMIS (29), Statistical and Mathe- matical Sciences Division, Lilley Re- search Laboratories, Lilley Corporate Center, Eli Lilley and Company, Indian- apolis, Indiana 46285 ALISON BEVAN (l 1), Department of BiD- chemistry, Stanford University School of Medicine, Stanford, California 94305 JENS J. BIRKTOFT (8), Roche Research Cen- ter, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 KLAUS BREDDAM (8, 18), Department of Chemistry, Carlsberg Research Labora- tory, DK-2500 Valby, Copenhagen, Den- mark CHARLES BRENNER (l l), Rosenstiel Center, Brandeis University, Waltham, Massa- chusetts 02254 DIETER BROMME (48), Khepri Pharmaceuti- cals, Inc., South San Francisco, Califor- nia 94080 MARK T. BROWN (27), Biology Department, Brookhaven National Laboratory, Upton, New York 11973 DAVID J. BUTTLE (37, 38, 45), Department of Human Metabolism and Clinical Bio- chemistry, University of Sheffield Medi- cal School, Sheffield SIO 2RX, United Kingdom MICHEL CHRI~TIEN (13), Department of Mo- lecular Neuroendocrinology, Clinical Re- search Institute of Montreal, Montreal, Quebec, Canada H2Q 1R7 LI~ON CHRISTIAENS (19), Centre d'lngd- nierie de Prot~ines, Universit~ de Likge, Institut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium CHIN HA CHUNG (25), Department of Mo- lecular Biology, Seoul National Univer- sity, Seoul, Korea JAMES A. COOK (29), Virology Research Di- vision, Lilley Research Laboratories, Lil- ley Corporate Center, Eli Lilley and Com- pany, Indianapolis, Indiana 46285 JACQUES COYETTE (19), Centre d'lng~nierie de Prot~ines, Universit~ de Lidge, lnstitut de Chimie, B6, B-4000 Sart Tilman, Liege 1, Belgium Ross E. DALBEY (21), Department of Chem- istry, Ohio State University, Columbus, Ohio 43210 HANS-ULRICH DEMUTH (48), Department of Biochemistry, Martin-Luther-University, D-06099 Halle (Saale), Germany HAKIM DJABALLAH (24), Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom COLETTE DUEZ (19), Centre d'lng~nierie de Protdines, Universitd de Liege, Institut de Chimie, B6, B-4000 Sart Tilman, Liege 1, Belgium ix X CONTRIBUTORS TO VOLUME 244 JOHN J. DUNN (27), Biology Department, Brookhaven National Laboratory, Upton, New York 11973 JEAN DUSART (19), Centre d'Ingdnierie de Protdines, Universitd de Lidge, lnstitut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium CLAUDINE FRAIPONT (19), Centre d'lng~- nierie de Protdines, Universitd de Lidge, Institut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium JEAN-MARIE FRI~RE (19), Centre d'lng~- nierie de Prot~ines, UniversiN de Lidge, Institut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium ROBERT S. FULLER (l 1), Department of Bio- chemistry, Stanford University School of Medicine, Stanford, California 94305 MORENO GALLENI (19), Centre d'Ingdnierie de Protdines, Universitd de Lidge, Institut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium JI~AN-MARIE GHUYSEN (19), Centre d'Inge- nierie de Protdines, Universitd de Lidge, Institut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium WADE GIBSON (28), Virology Laboratories, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205 JOANNA G1ORDANO (29), Virology Research Division, Lilley Research Laboratories, Lilley Corporate Center, Eli Lilley and Company, Indianapolis, Indiana 46285 ALFRED L. GOLDBERG (25, 26), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 STUART G. GORDON (39), Department of Pathology, University of Colorado, Health Sciences Center, and the Colo- rado Cancer Center, Denver, Colorado 80262 JACQUELINE GRANDCHAMPS (19), Centre d'Ingdnierie de Prot~ines, Universit~ de Lidge, lnstitut de Chimie, B6, B-4000 Sart Tilman, Liege l, Belgium BENOIT GRANIER (19), Centre d'Ingdnierie de Prot~ines, Universitd de Lidge, Institut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium BEULAH GRAY (3), Department of Microbi- ology, The University of Minnesota, Min- neapolis, Minnesota 55455 JOHN R. HO1DAL (3), Pulmonary Division, University of Utah School of Medicine, Salt Lake City, Utah 84132 YUKIO IKEHARA (16), Department of Bio- chemistry, Fukuoka University School of Medicine, Fukuoka 814-01, Japan SHIN-ICHI ISHII (42), Faculty of Pharma- ceutical Sciences, Hokkaido University, Sapporo 060, Japan MARC JAMIN (19), Centre d'lng~nierie de Prot~ines, UniversiN de Lidge, Institut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium WANDA M. JONES (17), The Rockefeller University, New York, New York 10021 BERNARD JORIS (19), Centre d'lngdnierie de Protdines, Universitd de Lidge, Institut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium CHIH-MIN KAM (31), School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332 BALK KIM (20), Laboratory of Cell Biology, National Cancer Institute, National Insti- tutes of Health, Bethesda, Maryland 20892 SEUNG-HO KIM (23), Laboratory of Cell Bi- ology, National Cancer Institute, Na- tional Institutes of Health, Bethesda, Maryland 20892 HEIDRUN KIRSCHKE (34), Institute of Bio- chemistry, Medical Faculty, Martin- Luther University, D-06097 Halle, Saale, Germany MICHAEL D. KRAMER (4), Laboratory for Immunopathology, Institut far lmmunol- ogie, D-59120 Heidelberg, Germany ALLEN KRANTZ (47), Syntex Discovery Re- search, Palo Alto, California 94303 CONTRIBUTORS TO VOLUME 244 xi KOTOKU KUI~,CHI (7), Department of Hu- man Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109 STEFAN KUZELA*(26), Institute of Molecu- lar Biology, Slovak Academy of Sciences, Bratislava, SIovak Republic JEAN LABUS (29), Biochemical Pharmacol- ogy Research Division, Lilley Research Laboratories, Lilley Corporate Center, Eli Lilley and Company, Indianapolis, In- diana 46285 BERNARD LAKAYE (19), Centre d'lng~nierie de ProNines, Universitd de Liege, lnstitut de Chimie, B6, B-4000 Sart Tilman, Liege 1, Belgium MIJIN LEE (27), Biology Department, Brookhaven National Laboratory, Upton, New York 11973 MI~LINA LEYH-BOUILLE (19), Centre d'lngdnierie de ProNines, Universitd de LiOge, Institut de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium HANS-DIETER LIEBIG (40), Department of Biochemistry, Medical Faculty, Univer- sity to Vienna, A1030 Vienna, Austria LIH-LING LIN (20), Genetics Institute, Cambridge, Massachusetts 02140 JOHN W. LITTLE (20), Department of Bio- chemistry and Molecular and Cellular Bi- ology, Universiy of Arizona, Tucson, Ari- zona 85721 MARK O. LIVELY (22), Department of Bio- chemistry, Bowman Gray School of Medi- cine, Wake Forest University, Winston- Salem, NC 27157 JENNIFER LUDFORD (28), Virology Labora- tories, The Johns Hopkins School of Med- icine, Baltimore, Maryland 21205 Yu-TING MA (44), Department of Biological Chemistry, and Molecular Pharmacol- ogy, Harvard Medical School, Boston, Massachusetts 02115 JOSEPH S. MANETTA (29), Biochemical Pharmacology Research Division, Lilley Research Laboratories, Lilley Corporate Center, Eli Lilley and Company, Indian- apolis, Indiana 46285 WALTER F. MANGEL (27), Biology Depart- ment, Brookhaoen National Laboratory, Upton, New York 11973 JAMES M. MANNING (17), The Rockefeller University, New York, New York 10021 TAKEHARU MASAKI (9), Faculty of Agricul- ture, lbaraki University, Ibaraki 300-03, Japan MICHAEL R. MAURIZI (23, 25), Laboratory of Cell Biology, National Cancer Insti- tute, National Institutes of Health, Be- thesda, Maryland 20892 ROBERT MI~NARD (33), Biotechnology Re- search Institute, National Research Council of Canada, Montrdal, Quebec', Canada H4P 2R2 YOSHIO MISUMI (16), Department of Bio- chemistry, Fukuoka University School of Medicine, Fukuoka 814-01, Japan RICHARD P. MOERSCHELL (25), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 KAZUHISA NAKAYAMA (12), Institute of Bi- ological Sciences and Gene Experiment Center, University of Tsukuba, lbaraki 305, Japan ANN L. NEWSOME (22), Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Caro- lina 27157 MARTINE NGUYEN-DIsT~CHE (19), Centre d'Ingdnierie de Prot~ines, Universit~ de Lidge, lnstitut de Chimie, B6, B-4000 Sart Tilman, Liege 1, Belgium MICHAEL J. NORTH (36), Department of Bi- ological and Molecular Sciences, Univer- sity of Stirling, Stirling FK9 4LA, Ger- many MOHAMAD NUSIER (23), Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Car()- lina 27157 *deceased xii CONTRIBUTORS TO VOLUME 244 SHIGENORI OGATA (16), Department of Bio- chemistry, Fukuoka University School of Medicine, Fukuoka 814-01, Japan JOZEF OLEKSYSZYN (30), OsteoArthritis Sci- ences, Inc., Cambridge, Massachusetts 02139 MANUEL C. PEITSCH (5), University of Lau- sanne, Institute de Biochemie, CH-1066 Epalinges, Switzerland ANDREW G. PLAUT (10), Gastroenterology Division, Department of Medicine, Tufts University School of Medicine, and New England Medical Center Hospital, Bos- ton, Massachusetts 02111 LASZL6 POLGAR (14), Institute of Enzymol- ogy, Biological Research Center, Hun- garian Academy of Sciences, H-1518 Bu- dapest, Hungary JAMES C. POWERS (30, 31), School of Chem- istry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332 ROBERT R. RANDO (44), Department of Bio- logical Chemistry, and Molecular Phar- macology, Harvard Medical School, Bos- ton, Massachusetts 02115 N. V. RAO (3), Pulmonary Division, Univer- sity of Utah School of Medicine, Salt Lake City, Utah 84132 NEIL D. RAWLINGS (2, 32), Strangeways Research Laboratory, Cambridge CBI 4RN, United Kingdom S. JAMES REMINGTON (18),Institute of Mo- lecular Biology, University of Oregon, Eugene, Oregon 97403 A. JENNIFER RIVETT (24), Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom KENNETH L. ROLAND (20), Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322 ANDREW D. ROWAN (38), Pharmacia Biotech Ltd., St. Albans, Herts ALl 3A W, United Kingdom FUMIO SAKIYAMA (9), Division of Protein Chemistry, and Research Center for Pro- tein Engineering, Institute for Protein Re- search, Osaka University, Osaka 565, Ja- pan PETER J. SAVORY (24), Department of Bio- chemistry, University of Leicester, Leicester LEI 7RH, United Kingdom ANDREA SCALON1 (17), The Rockefeller University, New York, New York 10021 HENNING SCHOLZE (35), Department of Bi- ology~Chemistry, Biochemistry, Univer- sity of Osnabrueak, D-49069 Osnabrueck, Germany LAWRENCE B. SCHWARTZ (6), Department of Internal Medicine, Virginia Common- wealth University, Richmond, Virginia 23298 NABIL G. SEIDAH (13), Biochemical Labo- ratory, J.A. DeSdve Laboratory of Bio- chemical Neuroendocrinology, Clinical Research Institute of Montreal, Mon- treal, Quebec, Canada H2W 1R7 ELLIOTT SHAW (46), Friedrich Miescher-In- stitut, CH-4002 Basel, Switzerland MARKUS M. SIMON (4), Max-Planck-Insti- tute for Immunobiology, D-79108 Frei- burg, Germany SATYENDRA K. SINGH (23), Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 TIM SKERN (40), Department of Biochemis- try, Medical Faculty, University of Vi- enna, A1030 Vienna, Austria STEVE N. SLILATY (20), Quantum Biotech- nologies, Montreal, Quebec, Canada H4P 2R2 MARGARET n. SMITH (20), Department of Biochemistry, University of Arizona, Tuc- son, Arizona 85721 MICHELE C. SMITH (29), Virology Research Division, Lilley Research Laboratories, Lilley Corporate Center, Eli Lilley and Company, Indianapolis, Indiana 46285 ANDREW C. STORER (33), Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada H4P 2R2 CONTRIBUTORS TO VOLUME 244 xiii EGBERT TANNICH (35), Department of Mo- lecular Biology, Bernhard Nocht Institute for Tropical Medicine, D-20359 Ham- burg, Germany MARK W. THOMPSON (23), Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 NANCY A. THORNBERRY (43), Department of Biochemistry, Merck Research Labo- ratories, Rahway, New Jersey 07065 KAROLY TIHANY! (41), Department of Mi- crobiology CHUS, University of Sherbrooke, Sherbrooke, Quebec, Can- ada J1H 5N4 DIANA L. TOLEDO (27), Biology Depart- ment, Brookhaven National Laboratory, Upton, New York 11973 ADRIAN TORRES-ROSADO (7), Department of Human Genetics, University of Michi- gan Medical School, Ann Arbor, Michi- gan 48109 WILLIAM R. TSCHANTZ (21), Department of Chemistry, Ohio State University, Colum- bus, Ohio 43210 JUERG TSCHOPP (5), University of Lau- sanne, Institute de Biochemie, CH-1066 Epalinges, Switzerland AKIHIKO TSUJI (7), Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109 DAISUKE TSURU (15), School of Pharmaceu- tical Sciences, NagasaM University, Na- gasaki 852, Japan ELCIRA C. VILLARREAL (29), Virology Re- search Division, Lilley Research Labora- tories, Lilley Corporate Center, Eli Lilley and Company, Indianapolis, Indiana 46285 MARK WAKULCHIK (29), Virology Research Division, Lilley Research Laboratories, Lilley Corporate Center, Eli Lilley and Company, Indianapolis, Indiana 46285 JOSEPH M. WEBER (41), Department of Mi- crobiology CHUS, University of Sherbrooke, Sherbrooke, Quebec, Can- ada J1H 5N4 ANTHONY R. WELCH (28), Virology Labora- tories, The Johns Hopkins School of Med- icine, Baltimore, Maryland 21205 BERND WIEDERANDERS (34), lnstitut ff2r Biochemie, Freidrich-Schiller-Universitdt Jena, D-07740 Jena, Germany JEAN-MARC WILKIN (19), Centre d'lnge- nierie de Protdines, Universitd de Lidge, lnstitat de Chimie, B6, B-4000 Sart Tilman, Lidge 1, Belgium KIMBERLY WORZALLA (27), Biology De- partment, Brookhaven National Labora- tory, Upton, New York 11973 TADASHI YOSHIMOTO (15), School of Phar- maceutical Sciences, Nagasaki Univer- sity, Nagasaki 852, Japan WILLY ZORZl (19), Centre d'Ingdnierie de Protdines, Universitd de Lidge, lnstitut de Chimie, B6, B-4000 Sart Tilman, LiOge 1, Belgium [1] CLASSIFICATION OF PEPTIDES I [1] Classification of Peptidases By ALAN J. BARRETT Introduction The establishing of a system for classification and nomenclature can be seen as a clear indication of the "coming of age" of a branch of science. The introduction of the Linnaean system for naming and classifying organ- isms in the eighteenth century and the invention of a system of nomencla- ture for enzymes in the 1950s were such landmarks, and their value has been obvious. However, the peptide hydrolases (peptidases) have never fitted comfortably into the system of classification and nomenclature intro- duced for enzymes in general, in which the characteristics of the reaction catalyzed are all important, and this has stimulated efforts to develop other forms of classification for them. Three major criteria are currently in use for the classification of the peptidases: (1) the reaction catalyzed, (2) the chemical nature of the cata- lytic site, and (3) the evolutionary relationship, as revealed by structure. The purpose of this chapter is to describe how these criteria are used, and to consider their strengths and weaknesses. Classification by Reaction Catalyzed The classification and naming of enzymes by reference to the reactions they catalyze has become the underlying principle of the enzyme nomen- clature of the International Union of Biochemistry and Molecular Biology (see Ref. 1, pp. 5-8). In this method, knowledge of the systematic chemical name of the substrate and the type of reaction that it undergoes allows one to derive a systematic name for the enzyme and to decide how it should be classified. In Enzyme Nomenclature 1992,1 class 3 contains the hydrolases, and subclass 3.4 contains all of the enzymes that we are concerned with here, the peptide hydrolases or peptidases. Subclass 3.4 is further divided primarily on the basis of the reactions catalyzed (Table I). The sub-subclasses of peptidases fall into two sets, comprising the exopeptidases and the endopeptidases. The exopeptidases act only near the ends of polypeptide chains. Those acting at a free N terminus liberate a single amino acid residue, a dipeptide or a tripeptide (aminopeptidases, 1 Nomenclature Committee of the International Union of Biochemistry and Molecular Biol- ogy, "Enzyme Nomenclature 1992." Academic Press, Orlando, Florida, 1992. Copyright © 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 244 All rights of reproduction in any form reserved. 2 METHODS IN ENZYMOLOGY [1] TABLE I CLASSIFICATION OF PEPT1DASES BY TYPE OF REACTION CATALYZED a Action Group EC subsection Exopeptidases ~:)-O-(~-~: Aminopeptidases 3.4.11 ~-O-(~:~: Dipeptidyl-peptidases, 3.4.14 ~2~: tripeptidyl-peptidases :H:~)-O-(~ Carboxypeptidase s 3.4.16-18 :H-~ Peptidyl-dipeptidases 3.4.15 Dipeptidases 3.4.13 ~~_~ Omega peptidases 3.4.19 :~~:~: Endopeptidases 3.4.21-24 and 99 a Open circles represent amino acid residues and filled circles are the residues comprising the blocks of one, two, or three terminal amino acids that are cleaved off by these enzymes. The triangles indicate the blocked termini that provide substrates for some of the omega peptidases. Further subdivisions of the carboxy- peptidases and the endopeptidases have been made on the basis of catalytic type, as shown in Table III. dipeptidyl-peptidases, and tripeptidyl-peptidases, respectively). The exo- peptidases acting at a free C terminus liberate a single residue or a dipeptide (carboxypeptidases and peptidyl-dipeptidases, respectively). Other exo- peptidases are specific for dipeptides (dipeptidases), or remove terminal residues that are substituted, cyclized, or linked by isopeptide bonds (peptide linkages other than those of a-carboxyl to ~-amino groups) (omega peptidases). Endopeptidases act preferentially in the inner regions of peptide chains, away from the termini, and the presence of free s-amino or a-carboxyl groups has a negative effect on the activity of the enzyme. The oligopeptidases are a subset of the endopeptidases that are confined to action on oligopeptide or polypeptide substrates smaller than proteins, although the size ranges of the substrates differ between the individual en- zymes. 2 2 A. J. Barrett and N. D. Rawlings, Biol. Chem. Hoppe-Seyler 373, 353 (1992). [1] CLASSIFICATION OF PEPTIDES 3 Methods The classification of peptidases according to their mode of action on substrates has proved more useful for the exopeptidases than for the endopeptidases, as may be judged from the fact that sub-subclasses only of exopeptidases are distinguished in this way in Enzyme Nomenclature 1992 (see also Ref. 3). Synthetic substrates have become increasingly important in both assay and characterization of peptidases in recent years. Factors responsible for this shift away from dependence on natural substrates for many purposes include the discovery of convenient leaving groups, such as 7-amino-4- methylcoumarin, especially for serine and cysteine peptidases, and the recognition of the ability of dipeptide and tripeptide sequences to confer high sensitivity as well as selectivity on the substrates (illustrated by Ref. 4). The use of larger peptide substrates has been facilitated by the increased availability of high-performance, reversed-phase chromatography and the development of quenched fluorescence substrates (reviewed by KnightS). As a result of improvements in the methods of peptide synthesis it is commonly possible to assemble a range of artificial substrates, allowing the specificity of a newly discovered peptidase to be explored in some detail.* The omega peptidases are best thought of as exopeptidases, because they hydrolyze terminal residues, but some of them act on atypical resi- dues that do not bear free a-carboxyl or a-amino groups. Examples would be peptidyl-glycinamidase (EC 3.4.19.2), the pyroglutamyl-peptidases (EC 3.4.19.3; EC 3.4.19.6), and N-formylmethionyl-peptidase (EC 3.4.19.7). The extended substrate-binding sites of many endopeptidases tend to 3 j. K. McDonald and A. J. Barrett, "Mammalian Proteases: a Glossary and Bibliography. Volume 2: Exopeptidases," pp. 1-6. Academic Press, London, 1986. 4 A. J. Barrett and H. Kirschke, this series, Vol. 80, p. 535. C. G. Knight, this series, Vol. 248, Chapter 2. * In describing the specificity of peptidases, use is made of a model in which the catalytic site is considered to be flanked on one or both sides by specificity subsites, each able to accommodate the side chain of a single amino acid residue. These sites are numbered from the catalytic site, S l Sn toward the N terminus of the structure, and S 1 ' Sn' toward the C terminus. The residues they accommodate are numbered P1 Pn, and PI' Pn', respectively, as follows: Substrate:-P3-P2-P1 + PI '-P2'-PY- Enzyme: -$3-$2-S1 * SI'-S2'-S3'- This scheme is based on that initially described by I. Schechter and A. Berger, Biochem. Biophys. Res. Commun. 27, 157 (1967) and slightly modified in "Enzyme Nomenclature 1992," p. 372. Academic Press, Orlando, 1992. The peptide bond cleaved (the scissile bond) is indicated by the crossbar symbol (-~), and the catalytic site of the enzyme is denoted by an asterisk. 4 METHODS IN ENZYMOLOGY [1] require larger synthetic substrates for their characterization than do those of the exopeptidases. The binding site of an endopeptidase normally will not accommodate a free N or C terminus, whereas one or both is essential for a typical exopeptidase. Errors in the characterization ofpeptidases with substrates have arisen primarily from the use of impure enzyme preparations, so that the first set of products was rapidly converted into others. This can give the impres- sion of an enzyme quite different from any that is actually present. For example, there was a case in which Z-Ala-Ala-Phe-NHMec [Z, benzyloxy- carbonyl; NHMec, 7-O-methyl)coumarylamide] was cleaved to release the free fluorescent aminomethylcoumarin. This is what chymotrypsin does, and the natural conclusion was that a chymotrypsin-like endopepti- dase was present. However, more detailed analysis showed that an initial cleavage at the -Ala÷Phe- bond (probably by neprilysin, EC 3.4.24.11) was followed by hydrolysis of the Phe-NHMec product by an aminopepti- dase. It is a help toward avoiding such pitfalls if efforts are made to identify the first products of cleavage of the substrate, as can be done by a timed series of high-performance liquid chromatograms (HPLC) or thin- layer chromatograms of the products as the reaction proceeds. A second type of problem has come with the technology that now facilitates the synthesis of oligopeptide substrates. Initially, it was natural to assume that the endopeptidases that cleave these substrates would also act on the same sequences when they occurred in proteins. This has proved not always to be the case, however, because the members of the oligopeptidase subset of endopeptidases will act only on the small substrates. Information about the reaction catalyzed by an individual peptidase can form a basis for naming as well as classifying it, when the specificity clearly depends on the identity of one or, at most, two amino acid residues. An exopeptidase is commonly named by reference to the type of reaction it catalyzes (aminopeptidase, etc.; see Table I). When the enzyme shows a marked preference for a particular amino acid residue in the P1 or Pl' position, the name of this may form a qualifier (e.g., "prolyl aminopepti- dase") (Table II). The names currently recommended I for many aminopep- tidases, dipeptidases, carboxypeptidases, and omega peptidases are de- rived in this way. For other exopeptidases the specificity is too complex to form a qualifier for the name, however, and then alphabetical or numeri- cal serial names such as "dipeptidyl-peptidase I," "dipeptidyl-peptidase II," "peptidyl-dipeptidase A," and "peptidyl-dipeptidase B" are used. Endopeptidases that show a clear preference for a particular amino acid residue in the P1 or PI' position also can be named with reference to this ("glycyl endopeptidase," "peptidyl-Lys endopeptidase"). It is important to note that such a term is a name for the reaction catalyzed, [...]... classification and naming of many peptidases on the basis of reaction catalyzed have been recognized for 6 [1] METHODS IN ENZYMOLOGY T A B L E III SUBDIVISION OF CARBOXYPEPTIDASES AND ENDOPEPTIDASES ACCORDING TO CATALYTIC TYPE a Group of Peptidases Carboxypeptidases Serine- type carboxypeptidases Metallocarboxypeptidase s Cysteine- type carboxypeptidases Endopeptidases Serine endopeptidases Cysteine endopeptidases... Introduction Proteolytic enzymes dependent on a serine residue for catalytic activity are widespread and very numerous Serine peptidases are found in viruses (Table I), bacteria, and eukaryotes, and they include exopeptidases, endopeptidases, oligopeptidases, and omega peptidases By the criteria we use to distinguish families of peptidases (see [1] in this volume), over 20 families of serine peptidases. .. reagents for the serine peptidases because they are mostly selective for endopeptidases of the chymotrypsin family Several of the inhibitors are easy to obtain and use, however, and seldom give false-positive reactions, so they deserve consideration Cysteine- Type Peptidases Many of the cysteine peptidases that are encountered are cysteine endopeptidases of the papain or calpain families, and these are... mechanism were used by peptidases, and this observation has been developed into a practical system for classification 7,8 In the enzyme list, l the carboxypeptidases and the endopeptidases are divided into sub-subclasses on the basis of catalytic mechanism (Table III) The serine- type peptidases have an active center serine involved in the catalytic process, the cysteine- type peptidases have a cysteine residue... [14] in this volume), and an important subset of the subtilisin family (see [2] and [13] in this volume) Thiol-dependent metallopeptidases include thimet oligopeptidase and other members of this family, 19as well as insulysin2° and aminopeptidase p.21 Several of the thiol-dependent serine peptidases and metallopeptidases contain a cysteine residue close to the catalytic site (see [2] and [18] in this volume;... endopeptidases include those in Refs 11 and 12 A simple protocol for the identification of the catalytic type of peptidase by use of inhibitors is given in Table IV Serine- Type Peptidases The reagent of choice for the recognition of a serine peptidase is 3,4-DCI This is safe and convenient to use, and reacts rapidly and irreversibly with a wide range of serine peptidases (see [31] in this volume) Serine. .. sometimes been treated as if it were a type-specific inhibitor for cysteine peptidases, but in fact it is a potent inhibitor of many serine peptidases also Reviews of the inhibitors of cysteine peptidases have been provided by Rich24 and Shaw 25 Aspartic-Type Peptidases The aspartic-type peptidases (all known examples of which are endopeptidases) are reversibly inhibited by pepstatin (Ki values in the... at pH 7, 250.15 DFP and PMSF may inhibit cysteine peptidases, but the effect is reversed by thiol compounds such as dithiothreitol Synthetic inhibitors of serine peptidases have been comprehensively reviewed by Powers and colleagues (Ref 14; see [30] and [31] in this volume) Such protein inhibitors of serine peptidases as soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin cannot... Chapters in this volume and in Volume 288 will reflect the results of classification ofpeptidases by family These will summarize the distinctive characteristics of over 20 families of serine peptidases (see [2]), nearly as many families of cysteine peptidases (see [32]), about 30 families of m e t a l l o p e p t i d a s e s , 3 4 and also a number of families of aspartic peptidases and those of as yet... catalytic serine and lysine residues in the known members of various families of serine peptidases The codes for clans and families are as in Table II [2] FAMILIES OF SERINE PEPTIDASES 23 peptidase ($30) have the same order of catalytic residues as chymotrypsin The Neisseria IgA-specific serine endopeptidase ($6), hepatitis C virus NS3 endopeptidase ($29), cattle diarrhea virus p80 endopeptidase ($31), and . Group of Peptidases EC sub-subclass Carboxypeptidases Serine- type carboxypeptidases Metallocarboxypeptidase s Cysteine- type carboxypeptidases Endopeptidases Serine endopeptidases Cysteine. recognition of a serine peptidase is 3,4-DCI. This is safe and convenient to use, and reacts rapidly and irreversibly with a wide range of serine peptidases (see [31] in this volume). Serine peptidases. deserve consideration. Cysteine- Type Peptidases. Many of the cysteine peptidases that are encountered are cysteine endopeptidases of the papain or calpain families, and these are mostly susceptible