Proteolytic Enzymes: Serine and Cysteine Peptidases

765 Pages·2004·14.663 MB·English

by  Abelson J.N., Simon M.I., Barrett A.J. (eds.)

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.
SUNGAM NOSMAHARBA (49), Department of KRAM T. BROWN (27), Biology Department,
Clinical Chemistry, University of Lund, Brookhaven National Laboratory, Upton,
University Hospital, S-221 58 Lund, Swe- New York 37911
den
DAVID J. BUTTLE (37, 38, 45), Department
YGGAM ADAM (19), Centre d'lngdnierie de of Human Metabolism and Clinical Bio-
Prot~ines, Universitd de Liege, Institut de chemistry, University of Sheffield Medi-
Chimie, B6, B-4000 Sart Tilman, LiOge ,1 cal School, Sheffield OIS 2RX, United
Belgium Kingdom
WILLIAM W. BACHOVCHIN (10), Depart- MICHEL NEIT~IRHC (13), Department of Mo-
ment of Biochemistry, Tufts University lecular Neuroendocrinology, Clinical Re-
School of Medicine, Boston, Massachu- search Institute of Montreal, Montreal,
setts 11120 Quebec, Canada H2Q 1R7
ALAN J. TTERRAB (1, 2, 32), Department of
NO~IL CHRISTIAENS (19), Centre d'lngd-
Biochemistry, Strangeways Research
nierie de Prot~ines, Universit~ de Likge,
Laboratory, Cambridge 1BC 4RN,
Institut de Chimie, B6, B-4000 Sart
United Kingdom
Tilman, Lidge ,1 Belgium
DLAREG W. REKCEB (29), Biotechnology Di-
CHIN HA CHUNG (25), Department of Mo-
vision, Lilley Research Laboratories, Lil-
lecular Biology, Seoul National Univer-
ycI Corporate Center, Eli Lilley and Com-
sity, Seoul, Korea
pany, Indianapolis, Indiana 58264
SEMAJ A. COOK (29), Virology Research -iD
YRREK BEMIS (29), Statistical and Mathe-
vision, Lilley Research Laboratories, Lil-
matical Sciences Division, Lilley Re-
ley Corporate Center, Eli Lilley and Com-
search Laboratories, Lilley Corporate
pany, Indianapolis, Indiana 58264
Center, Eli Lilley and Company, Indian-
apolis, Indiana 58264 SEUQCAJ ETTEYOC (19), Centre d'lng~nierie
de Prot~ines, Universit~ de Lidge, lnstitut
ALISON BEVAN (l ,)1 Department of -DiB
de Chimie, B6, B-4000 Sart Tilman, Liege
chemistry, Stanford University School of
,1 Belgium
Medicine, Stanford, California 50349
JENS J. TFOTKRIB (8), Roche Research Cen- Ross E. YEBLAD (21), Department of Chem-
ter, Hoffmann-La Roche Inc., Nutley, istry, Ohio State University, Columbus,
New Jersey 01170 Ohio 01234
KLAUS BREDDAM (8, 18), Department of HANS-ULRICH DEMUTH (48), Department of
Chemistry, Carlsberg Research Labora- Biochemistry, Martin-Luther-University,
tory, DK-2500 Valby, Copenhagen, Den- 99060-D Halle (Saale), Germany
mark HAKIM DJABALLAH (24), Department of
SELRAHC BRENNER (l l), Rosenstiel Center, Biochemistry, University of Leicester,
Brandeis University, Waltham, Massa- Leicester LE1 7RH, United Kingdom
chusetts 02254 ETTELOC DUEZ (19), Centre d'lng~nierie de
DIETER EMMORB (48), Khepri Pharmaceuti- Protdines, Universitd de Liege, Institut de
cals, Inc., South San Francisco, Califor- Chimie, B6, B-4000 Sart Tilman, Liege ,1
ain 08049 Belgium
ix
X CONTRIBUTORS TO VOLUME 244
JOHN J. DUNN (27), Biology Department, BENOIT GRANIER (19), Centre d'Ingdnierie
Brookhaven National Laboratory, Upton, de Prot~ines, Universitd de Lidge, Institut
New York 37911 de Chimie, B6, B-4000 Sart Tilman, Lidge
,1 Belgium
JEAN DUSART (19), Centre d'Ingdnierie de
Protdines, Universitd de Lidge, lnstitut de BEULAH GRAY (3), Department of Microbi-
Chimie, B6, B-4000 Sart Tilman, Lidge ,1 ology, The University of Minnesota, Min-
Belgium neapolis, Minnesota 55455
CLAUDINE FRAIPONT (19), Centre d'lng~- JOHN R. HO1DAL (3), Pulmonary Division,
nierie de Protdines, Universitd de Lidge, University of Utah School of Medicine,
Institut de Chimie, B6, B-4000 Sart Salt Lake City, Utah 84132
Tilman, Lidge ,1 Belgium YUKIO IKEHARA (16), Department of Bio-
chemistry, Fukuoka University School of
JEAN-MARIE FRI~RE (19), Centre d'lng~-
Medicine, Fukuoka 814-01, Japan
nierie de Prot~ines, UniversiN de Lidge,
Institut de Chimie, B6, B-4000 Sart SHIN-ICHI ISHII (42), Faculty of Pharma-
Tilman, Lidge ,1 Belgium ceutical Sciences, Hokkaido University,
Sapporo 060, Japan
TREBOR S. FULLER (l ,)1 Department of Bio-
chemistry, Stanford University School of MARC JAMIN (19), Centre d'lng~nierie de
Medicine, Stanford, California 50349 Prot~ines, UniversiN de Lidge, Institut de
Chimie, B6, B-4000 Sart Tilman, Lidge ,1
ONEROM GALLENI (19), Centre d'Ingdnierie
Belgium
de Protdines, Universitd de Lidge, Institut
WANDA M. JONES (17), The Rockefeller
de Chimie, B6, B-4000 Sart Tilman, Lidge
University, New York, New York 12001
,1 Belgium
DRANREB JORIS (19), Centre d'lngdnierie de
EIRAM-NA~IJ GHUYSEN (19), Centre d'Inge-
Protdines, Universitd de Lidge, Institut de
nierie de Protdines, Universitd de Lidge,
Chimie, B6, B-4000 Sart Tilman, Lidge ,1
Institut de Chimie, B6, B-4000 Sart
Belgium
Tilman, Lidge ,1 Belgium
CHIH-MIN KAM (31), School of Chemistry
WADE GIBSON (28), Virology Laboratories, and Biochemistry, Georgia Institute of
The Johns Hopkins School of Medicine, Technology, Atlanta, Georgia 30332
Baltimore, Maryland 21205
BALK KIM (20), Laboratory of Cell Biology,
ANNAOJ ONADRO1G (29), Virology Research National Cancer Institute, National Insti-
Division, Lilley Research Laboratories, tutes of Health, Bethesda, Maryland
Lilley Corporate Center, Eli Lilley and 29802
Company, Indianapolis, Indiana 46285
SEUNG-HO KIM (23), Laboratory of Cell Bi-
ALFRED L. GREBDLOG (25, 26), Department ology, National Cancer Institute, Na-
of Cell Biology, Harvard Medical School, tional Institutes of Health, Bethesda,
Boston, Massachusetts 02115 Maryland 20892
STUART G. GORDON (39), Department of HEIDRUN KIRSCHKE (34), Institute of Bio-
Pathology, University of Colorado, chemistry, Medical Faculty, Martin-
Health Sciences Center, and the Colo- Luther University, D-06097 Halle, Saale,
rado Cancer Center, Denver, Colorado Germany
26208 MICHAEL D. KRAMER (4), Laboratory for
ENILEUQCAJ SPMAHCDNARG (19), Centre Immunopathology, Institut far lmmunol-
d'Ingdnierie de Prot~ines, Universit~ de ogie, D-59120 Heidelberg, Germany
Lidge, lnstitut de Chimie, B6, B-4000 Sart ALLEN KRANTZ (47), Syntex Discovery Re-
Tilman, Liege l, Belgium search, Palo Alto, California 30349
CONTRIBUTORS TO VOLUME 244 xi
UKOTOK KUI~,CHI (7), Department of Hu- Center, Eli Lilley and Company, Indian-
man Genetics, University of Michigan apolis, Indiana 58264
Medical School, Ann Arbor, Michigan WALTER F. MANGEL (27), Biology Depart-
90184 ment, Brookhaoen National Laboratory,
STEFAN KUZELA*(26), Institute of Molecu- Upton, New York 37911
lar Biology, Slovak Academy of Sciences,
SEMAJ M. MANNING (17), ehT Rockefeller
Bratislava, SIovak Republic
University, New York, New York 12001
JEAN LABUS (29), Biochemical Pharmacol-
URAHEKAT MASAKI (9), Faculty of Agricul-
ogy Research Division, Lilley Research
ture, lbaraki University, Ibaraki ,30-003
Laboratories, Lilley Corporate Center,
Japan
Eli Lilley and Company, Indianapolis, In-
LEAHCIM R. MAURIZI (23, 25), Laboratory
diana 58264
of Cell Biology, National Cancer Insti-
DRANREB EYAKAL (19), Centre d'lng~nierie
tute, National Institutes of Health, Be-
de ProNines, Universitd de Liege, lnstitut
thesda, Maryland 29802
de Chimie, B6, B-4000 Sart Tilman, Liege
,1 Belgium TREBOR DRAN~IM (33), Biotechnology Re-
search Institute, National Research
MIJIN LEE (27), Biology Department,
Council of Canada, Montrdal, ,'cebeuQ
Brookhaven National Laboratory, Upton,
Canada H4P 2R2
New York 37911
YOSHIO MISUMI (16), Department of Bio-
MI~LINA LEYH-BOUILLE (19), Centre
chemistry, Fukuoka University School of
d'lngdnierie de ProNines, Universitd de
Medicine, Fukuoka 814-01, Japan
,egOiL Institut de Chimie, B6, B-4000 Sart
Tilman, Lidge ,1 Belgium DRAHCIR P. LLEHCSREOM (25), Department
of Cell Biology, Harvard Medical School,
HANS-DIETER LIEBIG (40), Department of
Boston, Massachusetts 51120
Biochemistry, Medical Faculty, Univer-
sity to Vienna, A1030 Vienna, Austria KAZUHISA AMAYAKAN (12), Institute of Bi-
ological Sciences and Gene Experiment
LIH-LING LIN (20), Genetics Institute,
Center, University of Tsukuba, lbaraki
Cambridge, Massachusetts 04120
305, Japan
JOHN W. LITTLE (20), Department of Bio-
ANN L. NEWSOME (22), Department of
chemistry and Molecular and Cellular -iB
Neurobiology and Anatomy, Bowman
ology, Universiy of Arizona, Tucson, Ari-
Gray School of Medicine, Wake Forest
zona 12758
University, Winston-Salem, North Caro-
KRAM O. LIVELY (22), Department of Bio- lina 27157
chemistry, Bowman Gray School of Medi-
MARTINE NGUYEN-DIsT~CHE (19), Centre
cine, Wake Forest University, Winston-
d'Ingdnierie de Prot~ines, Universit~ de
Salem, NC 27157
Lidge, lnstitut de Chimie, B6, B-4000 Sart
JENNIFER LUDFORD (28), Virology Labora- Tilman, Liege ,1 Belgium
tories, ehT Johns Hopkins School of Med-
LEAHCIM J. NORTH (36), Department of -iB
icine, Baltimore, Maryland 50212
ological and Molecular Sciences, Univer-
Yu-TING MA (44), Department of Biological sity of Stirling, Stirling FK9 4LA, Ger-
Chemistry, and Molecular Pharmacol- many
ogy, Harvard Medical School, Boston,
DAMAHOM NUSIER (23), Department of
Massachusetts 51120
Neurobiology and Anatomy, Bowman
HPESOJ S. MANETTA (29), Biochemical Gray School of Medicine, Wake Forest
Pharmacology Research Division, Lilley University, Winston-Salem, North -)(raC
Research Laboratories, Lilley Corporate lina 27157
*deceased
xii CONTRIBUTORS TO VOLUME 244
IRONEGIHS OGATA (16), Department of Bio- search, Osaka University, Osaka 565, Ja-
chemistry, Fukuoka University School of pan
Medicine, Fukuoka 814-01, Japan PETER J. YROVAS (24), Department of Bio-
JOZEF NYZSYSKELO (30), OsteoArthritis Sci- chemistry, University of Leicester,
ences, Inc., Cambridge, Massachusetts Leicester LEI 7RH, United Kingdom
93120
ANDREA SCALON1 (17), The Rockefeller
MANUEL C. PEITSCH (5), University of Lau- University, New York, New York 12001
sanne, Institute de Biochemie, CH-1066
HENNING SCHOLZE (35), Department of Bi-
Epalinges, Switzerland
ology~Chemistry, Biochemistry, Univer-
ANDREW G. PLAUT (10), Gastroenterology sity of Osnabrueak, D-49069 Osnabrueck,
Division, Department of Medicine, Tufts Germany
University School of Medicine, and New ECNERWAL B. SCHWARTZ (6), Department
England Medical Center Hospital, Bos- of Internal Medicine, Virginia Common-
ton, Massachusetts 11120 wealth University, Richmond, Virginia
LASZL6 RAGLOP (14), Institute of Enzymol- 89232
ogy, Biological Research Center, Hun-
NABIL G. SEIDAH (13), Biochemical Labo-
garian Academy of Sciences, H-1518 Bu- ratory, J.A. DeSdve Laboratory of Bio-
dapest, Hungary chemical Neuroendocrinology, Clinical
SEMAJ C. SREWOP (30, 31), School of Chem- Research Institute of Montreal, Mon-
istry and Biochemistry, Georgia Institute treal, Quebec, Canada H2W 1R7
of Technology, Atlanta, Georgia 30332 ELLIOTT SHAW (46), Friedrich Miescher-In-
TREBOR R. RANDO (44), Department of Bio- stitut, CH-4002 Basel, Switzerland
logical Chemistry, and Molecular Phar-
SUKRAM M. SIMON (4), Max-Planck-Insti-
macology, Harvard Medical School, Bos-
tute for Immunobiology, D-79108 Frei-
ton, Massachusetts 02115
burg, Germany
N. V. RAO (3), Pulmonary Division, Univer- ARDNEYTAS K. SINGH (23), Laboratory of
sity of Utah School of Medicine, Salt Cell Biology, National Cancer Institute,
Lake City, Utah 84132 National Institutes of Health, Bethesda,
NEIL D. RAWLINGS (2, 32), Strangeways Maryland 29802
Research Laboratory, Cambridge CBI TIM SKERN (40), Department of Biochemis-
4RN, United Kingdom try, Medical Faculty, University of Vi-
S. JAMES NOTGNIMER (18),Institute of Mo- enna, A1030 Vienna, Austria
lecular Biology, University of Oregon, EVETS N. YTALILS (20), Quantum Biotech-
Eugene, Oregon 30479 nologies, Montreal, Quebec, Canada
A. JENNIFER RIVETT (24), Department of H4P 2R2
Biochemistry, University of Leicester, TERAGRAM n. SMITH (20), Department of
Leicester LE1 7RH, United Kingdom Biochemistry, University of Arizona, Tuc-
KENNETH L. ROLAND (20), Department of son, Arizona 12758
Microbiology and Immunology, Emory MICHELE C. SMITH (29), Virology Research
University, Atlanta, Georgia 30322 Division, Lilley Research Laboratories,
WERDNA D. ROWAN (38), Pharmacia Lilley Corporate Center, Eli Lilley and
Biotech Ltd., St. Albans, Herts ALl Company, Indianapolis, Indiana 58264
3A W, United Kingdom ANDREW C. STORER (33), Biotechnology
FUMIO AMAYIKAS (9), Division of Protein Research Institute, National Research
Chemistry, and Research Center for Pro- Council of Canada, Montreal, Quebec,
tein Engineering, Institute for Protein Re- Canada H4P 2R2
CONTRIBUTORS TO VOLUME 244 xiii
TREBGE TANNICH (35), Department of Mo- ELCIRA C. LAERRALLIV (29), Virology Re-
lecular Biology, Bernhard Nocht Institute search Division, Lilley Research Labora-
for Tropical Medicine, D-20359 Ham- tories, Lilley Corporate Center, Eli Lilley
burg, Germany and Company, Indianapolis, Indiana
MARK W. NOSPMOHT (23), Laboratory of 58264
Cell Biology, National Cancer Institute, KRAM KIHCLUKAW (29), Virology Research
National Institutes of Health, Bethesda, Division, Lilley Research Laboratories,
Maryland 29802 Lilley Corporate Center, Eli Lilley and
NANCY A. YRREBNROHT (43), Department Company, Indianapolis, Indiana 58264
of Biochemistry, Merck Research Labo- HPESOJ M. WEBER (41), Department of -iM
ratories, Rahway, New Jersey 56070
crobiology CHUS, University of
YLORAK TIHANY! (41), Department of Mi- Sherbrooke, Sherbrooke, Quebec, Can-
crobiology CHUS, University of ada J1H 5N4
Sherbrooke, Sherbrooke, Quebec, Can-
YNOHTNA R. WELCH (28), Virology Labora-
ada J1H 5N4
tories, ehT Johns Hopkins School of Med-
DIANA L. TOLEDO (27), Biology Depart- icine, Baltimore, Maryland 50212
ment, Brookhaven National Laboratory,
BERND WIEDERANDERS (34), lnstitut ff2r
Upton, New York 37911
Biochemie, Freidrich-Schiller-Universitdt
ADRIAN ODASOR-SERROT (7), Department
Jena, D-07740 Jena, Germany
of Human Genetics, University of Michi-
gan Medical School, Ann Arbor, Michi- JEAN-MARC WILKIN (19), Centre d'lnge-
gan 90184 nierie de Protdines, Universitd de Lidge,
lnstitat de Chimie, B6, B-4000 Sart
WILLIAM R. ZTNAHCST (21), Department of
Tilman, Lidge ,1 Belgium
Chemistry, Ohio State University, Colum-
bus, Ohio 01234 YLREBMIK WORZALLA (27), Biology De-
partment, Brookhaven National Labora-
JUERG TSCHOPP (5), University of Lau-
tory, Upton, New York 37911
sanne, Institute de Biochemie, CH-1066
Epalinges, Switzerland IHSADAT OTOMIHSOY (15), School of Phar-
AKIHIKO TSUJI (7), Department of Human maceutical Sciences, Nagasaki Univer-
Genetics, University of Michigan Medical sity, Nagasaki 852, Japan
School, Ann Arbor, Michigan 90184 WILLY ZORZl (19), Centre d'Ingdnierie de
EKUSIAD TSURU (15), School of Pharmaceu- Protdines, Universitd de Lidge, lnstitut de
tical Sciences, NagasaM University, Na- Chimie, B6, B-4000 Sart Tilman, LiOge ,1
gasaki 852, Japan Belgium
1 CLASSIFICATION OF PEPTIDES I
1 Classification of Peptidases
By ALAN J. TTERRAB
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 1,2991 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.
thgirypoC © 4991 yb cimedacA Press, Inc.
SDOHTEM NI ,YGOLOMYZNE .LOV 442 llA rights of noitcudorper ni yna mrof .devreser
2 SDOHTEM NI YGOLOMYZNE 1
TABLE I
NOITACIFISSALC FO SESAD1TPEP YB EPYT FO NOITCAER DEZYLATAC a
Action Group EC subsection
Exopeptidases
~:)-O-(~-~: Aminopeptidases 11.4.3
~-O-(~:~: Dipeptidyl-peptidases, 41.4.3
~ 2 ~ : tripeptidyl-peptidases
:H:~)-O-(~ Carboxypeptidase s 3.4.16-18
: H - ~ Peptidyl-dipeptidases 51.4.3
Dipeptidases 31.4.3
~ ~ _ ~ Omega peptidases 91.4.3
: ~ ~ : ~ : 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 .)2991(
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, 751 (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 1P 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,