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 Volume 244 on peptidases of serine and cysteine type and in this volume on aspartic, metallo, and other peptidases, the chapters on spe- cific methods, enzymes, and inhibitors are organized within the rational framework of the new systems for classification and nomenclature. The peptidases of the aspartic and metallo types dealth with in this volume depend for their activity on the nucleophilic activity of an ionized water molecule, unlike the enzymes, described in Volume 244, in which the nucleophilic character of a serine or cysteine residue is at the heart of the catalytic mechanism. 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 248 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. ANGELA ANASTASI (43), Department of Pa- thology, The Medical School, St. Luke's Hospital, Msida, Malta DAVID S. AULD (14), Center for Biochem- ical and Biophysical Sciences and Medi- cine, and Department of Pathology, Har- vard Medical School and Brigham and Women's Hospital, Boston, Massachu- setts 02115 HI~LRN BARELLI (36), Institat de Pharma- cologie Moldculaire et Cellulaire, CNRS, Universitd de Nice-Sophia Antipolis, F-06560 Valbonne, France ALAN J. BARRETT (7, 13, 32, 43), Depart- ment of Biochemistry, Strangeways Re- search Laboratory, Cambridge CB1 4RN, United Kingdom ANDREW B. BECKER (44), Department of Molecular Pharmacology, Stanford Uni- versity School of Medicine, Stanford, California 94305 JUDD BERMAN (3), Department of Medici- nal Chemistry, Glaxo Inc. Research Insti- tute, Research Triangle Park, North Car- olina 27709 D. MARK BICKETT (3), Department of Bio- chemistry, Glaxo Inc. Research Institute, Research Triangle Park, North Carolina 27709 JOSEPH G. BIETH (5), INSERM Unitd 392, Laboratoire d'Enzymologie, Universitd Louis Pasteur de Strasbourg, F-67400 Illkirch, France J6N B. BJARNASON (21, 22), The Science In- stitute, University of lceland, IS-107 Rey- kjavik, Iceland JUDITH S. BOND (20), Department of Bio- chemistry and Molecular Biology, Col- lege of Medicine, Pennsylvania State University, Hershey, Pennsylvania 17033 JACQUES BOUVIER (37), Animal Health De- partment, Ciba-Geigy Ltd., CH-1566 St. Aubin, Switzerland MOLLY A. BROWN (32), Department of BiD- chemistry, Strangeways Research Labo- ratory, Cambridge CB1 4RN, United Kingdom MICHAEL BRUNNER (46), Institut fiir Phy- siologische Chemie, Universitiit Miin- chen, D-80336 Miinchen 2, Germany DAVID J. BUTTLE (4), Department of BiD- chemistry, Strangeways Research Labo- ratory, Cambridge CBI 4RN, United Kingdom PAUL CANNON (25), Institute of Biochemis- try and Cell Biology, Syntex Discovery Research, Paid Alto, California 94303 NIAMH X. CAWLEY (9), Laboratory of Developmental Neurobiology, National Institute of Child Health and Human De- velopment, N1H and Department of Biochemistry, Uniformed Services, Uni- versity of the Health Sciences, Bethesda, Maryland 20892 FRI~DI~RIC CHECLER (36), lnstitut de Phar- macologie Moleculaire et Cellulaire, CNRS, UniversiN de Nice-Sophia Anti- pulis, F-06560 Valbonne, France VALI~RIE CHESNEAU (45), Laboratoire de Biochimie des Signaux R~gulateurs, Cel- lulair et Mol~culaires, Unitd de Recher- ches Associ~e au Centre National de la Scientifique, Universitd Pierre et Marie Curie, 75006 Paris, France PAUL COHEN (45), Laboratoire de Biochi- mie des Signaux Rdgulateurs, Cellulair et Mol#culaires, Unitd de Recherches Asso- cide au Centre National de la Scientifi- que, Universitd Pierre et Marie Curie, 75006 Paris, France ix X CONTRIBUTORS TO VOLUME 248 CHRISTOPHER A. CONLIN (34), Department of Biological Sciences, Mankato State University, Mankato, Minnesota 56002 PIERI~E CORVOL (18), lnstitut National de la Sant~ et de la Recherche Medicale, Col- ldge de France, 75005 Paris, France THOMAS CRABBE (28), Celltech Therapeu- tics Ltd., Slough SL1 4EN, United King- dom PHILIPPE CRINE (17), D~partement de Bio- chimie, Facult~ de Mddecine, Universitd de Montrdal, Montrdal, Canada H3C 3J7 PAMELA M. DANDO (32), Department of Biochemistry, Strangeways Research Laboratory, Cambridge CB1 4RN, United Kingdom PASCALE DAUCH (36), Institut de Pharma- cologie Moleculaire et Cellulaire, CNRS, Universitd de Nice-Sophia Antipolis, 1:-06560 Valbonne, France PETER A. DEDDISH (41), Departments of Pharmacology and Anesthesiology, Uni- versity of Illinois College of Medicine, Chicago, Illinois 60612 MARIANNA DIOSZEGI (25), Institute of Bio- chemistry and Cell Biology, Syntex Dis- covery Research, Palo Alto, California 94303 VINCENT DIVE (36), D~partment d'lng~- nierie et d'Etude des Prot~ines, C.E.N. de Saclay, Laboratoire de Structure des Pro- tdines en Solution, 91191 GiflYvette, France ROBERT ETGES (37), Department of Bio- chemistry, University of Puerto Rico, San Juan, Puerto Rico 00936 STEPHAN FISCHER (51), Boehringer Mann- heim GmbH, D-82372 Penzberg, Ger- many THIERRY FOULON (45), Laboratoire de Bio- chimie des Signaux R~gulateurs, Cellulair et Mol~culaires, Unit~ de Recherches As- soci~e au Centre National de la Scientifi- que, Universit~ Pierre et Marie Curie, 75006 Paris, France MARIE-CLAUDE FOURNII~-ZALUSKI (17), D~partement de Pharmacochimie Mol~- culaire et Structurale, Institut National de la Sante et de la Recherche Medicale, Universit~ Rend Descartes, 75270 Paris Cedex 06, France JAY W. Fox (21, 22), Department of Micro- biology, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22908 URSULA GEUSS (51), Boehringer Mannheim GmbH, D-68305 Mannheim, Germany PAUL GLYNN (23), Medical Research Coun- cil Toxicology Unit, University of Leices- ter, Leicester LE1 9HN, United Kingdom MICHAEL GREEN (3), Department of Medic- inal Chemistry, Glaxo Inc. Research In- stitute, Research Triangle Park, North Carolina 27709 MARIE-LuISE HAGMANN (51), Boehringer Mannheim GmbH, D-82372 Penzberg, Germany CHRISTOPHER J. HANDLEY (4), Department of Biochemistry and Molecular Biology, Monash University, Clayton Victoria 3168, Australia Louis B. HERSH (16), Department of Bio- chemistry, College of Medicine, Univer- sity of Kentucky, Lexington, Kentucky 40536 LINDA HOWARD (23), Department of Cell Biology and Lombardi Cancer Center, Georgetown University School of Medi- cine, Washington 20007 GRAZIA ISAYA (33), Department of Genet- ics, Yale University School of Medicine, New Haven, Connecticut 06510 KARL E. KADLER (49, 50), Department of Biological Sciences, Research Division of Biochemistry, University of Manchester, Manchester M13 9PT, United Kingdom TAKASHI KAGEYAMA (8), Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, In- uyama, Aichi 484, Japan FRANTISEK KALOUSEK (33), Department of Genetics, Yale University School of Medi- cine, New Haven, Connecticut 06510 CONTRIBUTORS TO VOLUME 248 xi CHIH-MIN KAM (1), School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332 EERAT KESSLER (48), Maurice and Gabriela Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Hashomer 52621, Israel C. GRAHAM KNIGHT (2, 6, 32), Department of Cell Adhesion and Signalling, Strange- ways Research Laboratory, Cambridge CB1 4RN, United Kingdom GEORG-B URKHARD KRESSE (51), Boehringer Mannheim GmbH, D-82372 Penzberg, Germany CHINGWEN LI (16), Department of Bio- chemistry, College of Medicine, Univer- sity of Kentucky, Lexington, Kentucky 40536 SAMANTHA J. LIGHTFOOT (49), Department of Biological Sciences, Research Division of Biochemistry, University of Manches- ter, Manchester M13 9PT, United King- dom XINLI LIN (11), Protein Studies Program, Oklahoma Medical Research Founda- tion, Oklahoma City, Oklahoma 73104 REGGIE Y.C. LO (47), Department of Micro- biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Y. PENG LOH (9), Section on Cellular Neu- robiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, and Department of Biochemistry, Uniformed Services, University of the Health Sci- ences, Bethesda, Maryland 20892 HIROSHI MAEDA (24), Department of Micro- biology, Kumamoto University Medical School, Kumamoto 860, Japan GERARD M. MCGEEHAN (3), Department of Biochemistry, Glaxo Inc. Research Labo- ratories, Research Triangle Park, North Carolina 27709 NORMAN McKIE (32), Department of Bio- chemistry, Strangeways Research Labo- ratory, Cambridge CB1 4RN, United Kingdom ALAN MELLORS (47), Department of Chem- istry and Biochemistry, Guelph-Waterloo Centre for Graduate Work in Chemistry, University of Guelph, Guelph, Ontario, Canada NIG 2W1 CHARLES G. MILLER (34), Department of Microbiology, University of Illinois at Ur- bana-Champaign, Urbana, Illinois 61801 VI~RONIQUE MONNET (35), 1NRA Centre De Recherches De Jouy-en-Josas, Station De Recherches Laitidres, Domaine de Vilvert, 78352 Jouy-en-Josas, Cedex, France CESARE MONTECUCCO (39), Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Universita di Pa- dora, 75-35100 Padova, Italy KAZUYUKI MORIHARA (15)~ Institute of Ap- plied Life Sciences, Graduate School, University of East Asia, Yamaguchi 751, Japan GILLIAN MURPHY (28, 30), Department of Cell and Molecular Biology, Strangeways Research Laboratory, Cambridge CB1 4RN, United Kingdom HIDEAKI NAGASE (27), Department of Bio- chemistry and Molecular Biology, Uni- versity of Kansas Medical Center, Kan- sas City, Kansas 66160 WALTER NEUPERT (46), Institut fiir Phy- siologische Chemie, Universit~it Miin- chen, D-80336 Miinchen 2, Germany FLORENCE NOBLE (17), D~partement de Pharmacochimie Molgcalaire et Strac- turale, Institut National de la Sante et de la Recherche Medicale, Centre, National de la Recherche Scientifique, Universitg Rend Descartes, 75270 Paris Cedex 06, France ADRIAN R. PIEROTTI (45), Department of Biological Sciences, Glasgow Caledonian University, Glasgow GL4 OBA, Scotland ANDREW G. PLAUT (38), Department of Medicine, Gastroenterology Division, Tufts University School of Medicine and New England Medical Center, Boston, Massachusetts 02111 xii CONTRIBUTORS TO VOLUME 248 JAMES C. POWERS (1), School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332 ANNIK PRAT (45), Laboratoire de Biochimie des Signaux Rdgulateurs, Cellulair et Mo- Idculaires, Unitd de Recherches Associde au Centre National de la Scientifique, Universitd Pierre et Marie Curie, 75006 Paris, France NEIL D. RAWLINCS (7, 13, 32), Department of Biochemistry, Strangeways Research Laboratory, Cambridge CB1 4RN, United Kingdom BERNARD P. ROQUES (17), Ddpartement de Pharmacochimie Moldculaire et Struc- turale, Institut National de la Santd et de la Recherche Medicale, Centre National de la Recherche Scientifique, Unioersitd Rend Descartes, 75270 Paris Cedex 06, France RICHARD A. ROTH (44), Department of Mo- lecular Pharmacology, Stanford Univer- sity School of Medicine, Stanford, Cali- fornia 94305 KRISHNAN SANKARAN (12), Department of Microbiology and Immunology, Uni- formed Services, University of the Health Sciences, Bethesda, Maryland 20814 GIAMPIETRO SCHIAVO (39), Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Universitd di Pa- dora, 75-35100 Padova, Italy PASCAL SCHNEIDER (37), Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland ATSUSHI SERIZAWA (32), Sapporo Research Laboratory, Snow Brand Milk Products Co., Ltd., Sapporo 065, Japan RANDAL A. SKIDGEL (40, 41), Department of Pharmacology and Anesthesiology, University of lllinois College of Medicine, Chicago, Illinois 60612 FLORENT SOUBRIER (18), Institut National de la Santd et de la Recherche Medicale, Colldge de France, 75005 Paris, France VALENTIN M. STEPANOV (42), Protein Chemistry Laboratory, Institute of Micro- bial Genetics, Moscow 113545, Russia WALTER STOCKER (19), Zoologisches lnsti- tut der Universitiit Heidelberg, Physiolo- gie, Im Neuenheimer FeN, D-69120 Hei- delberg, Germany KENJI TAKAHASHI (10), Department of Bio- physics and Biochemistry, Faculty of Sci- ence, The University of Tokyo, Tokyo 113, Japan FULONG TAN (41), Departments of Pharma- cology and Anesthesiology, University of Illinois College of Medicine, Chicago, Illi- nois 60612 JORDAN TANG (11), Oklahoma Medical Re- search Foundation, Protein Studies Pro- gram, Oklahoma City, Oklahoma 73104 HARALD TSCHESCHE (26), Biochemistry De- partment, University Bielefeld, D-33615, Bielefeld Germany HAROLD E. VAN WART (25), Institute of Biochemistry and Cell Biology, Syntex Discovery Research, PaiD Alto, Califor- nia 94303 BRUNO VINCENT (36), Institut de Pharma- cologie Moldculaire et Cellulaire, CNRS, Universitd de Nice-Sophia Antipolis, F-06560 Valbonne, France JEAN PIERRE VINCENT (36), Institut de Pharmacologie Moldculaire et Cellulaire, CNRS, Universitd de Nice-Sophia Anti- polls, F-06560 Valbonne, France ROD B. WATSON (49), Department of Bio- logical Sciences, Research Division of Biochemistry, University of Manchester, Manchester M13 9PT, United Kingdom FRANCES WILLENBROCK (30), Department of Biochemistry, Queen Mary and West- field College, University of London, Lon- don E1 4NS, United Kingdom TRAcY A. WILLIAMS (18), Institut National de la Santd et de la Recherche Medicale, Colldge de France, 75005 Paris, France JEFFREY S. WISEMAN (3), Department of Biochemistry, Glaxo Inc. Research Insti- tute, Research Triangle Park, North Car- olina 27709 J. FREDERICK WOESSNER, JR. (29, 31), De- partment of Biochemistry and Molecular Biology, University of Miami, School of Medicine, Miami, Florida 33101 CONTRIBUTORS TO VOLUME 248 xiii RUSSELL L. WOLZ (20), Department of Bio- chemistry and Molecular Biology, Col- lege of Medicine, Pennsylvania State University, Hershey, Pennsylvania 17033 ANDREW WRIGHT (38), Department of Mo- lecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111 HENRY C. Wu (12), Department of Microbi- ology and Immunology, Uniformed Ser- vices, University of the Health Sciences, Bethesda, Maryland 20814 ROBERT ZWILLING (19), Zoologisches Insti- tut der Universitiit Heidelberg, Physiolo- gie lm Neuenheimer Feld, D-69120 Hei- delberg, Germany [ 1 ] PEPTIDE THIOESTER SUBSTRATES 3 [1] Peptide Thioester Substrates for Serine Peptidases and Metalloendopeptidases By JAMES C. POWERS and CHIH-MIN KAM Introduction Synthetic peptide substrates are widely used in biochemical and physiological studies of proteolytic enzymes. Synthetic peptide substrates can be used to detect enzyme during isolation, to assay enzyme activity, to determine enzyme concentrations, to investigate enzyme specificity, and to determine inhibitor potency. The three most commonly used synthetic substrates are peptide 4-nitroanilides, peptide thioesters, and peptide derivatives of 7-amino-4-methylcoumarin. Amino acid and peptide thioesters are sensitive substrates for serine peptidases and metalloendo- peptidases because the substrates have high kcat/Km values for enzymatic hydrolysis rates and low background hydrolysis rates, and the thiol- leaving group can be easily detected at low concentrations. Cleavage of the thioester bond can be monitored continuously by reaction with a thiol reagent such as 4,4'-dithiodipyridine or 5,5'-dithiobis (2-nitrobenzoic acid) contained in the assay mixture to produce a chromogenic com- pound. 1"2 Alternately, the thiol detection reagent can be used after the reaction has been concluded. Synthetic peptide thioester substrates have been used for substrate mapping of elastases, chymotrypsin-like enzymes, coagulation enzymes, and complement proteins, and they are also useful for detecting various new serine peptidase activities in cell extracts such as lymphocyte and natural killer cell granules. Several synthetic peptide thioesters have been used to monitor enzyme activities of various metal- loendopeptidases such as collagenases, stromelysin, gelatinase, and ther- molysin. 1 D. R. Grassetti and J. F. Murray, Jr., Arch. Biochem. Biophys. 119, 41 (1967). D. A. Farmer and J. H. Hageman, J. Biol. Chem. 250, 7366 (1975). Copyright © 1995 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 248 All rights of reproduction in any form reserved. 4 METHODS [ 11 Synthetic Methods Materials. CDI, 3 DCC, HOBt, and benzyl mercaptan can be obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). All Boc amino acids can be obtained from Chemical Dynamics Corp. (South Plainfield, NJ), Bachem Bioscience, Inc. (Philadelphia, PA), and numerous other sources. Boc-Ala-Ala-AA-SBzl. Boc-AA-SBzl derivatives are prepared by cou- pling Boc-AA-OH and benzyl mercaptan using the DCC/HOBt method. Boc-AA-SBzl can then be deblocked with HC1 in dioxane or ethyl acetate to give HC1. AA-SBzl which is further coupled with Boc-Ala-Ala-OH to give the final product Boc-Ala-Ala-AA-SBzl. 4,5 When the amino acid AA is glutamic acid or aspartic acid, the side-chain carboxyl group is protected with a tert-butyl group, which along with the Boc group can be removed by trifluoroacetic acid or HC1 in ethyl acetate. 5 Boc-Ala-Ala-Nva-SBzl. To prepare Boc-Ala-Ala-Nva-SBzl, 4 Boc-Nva- OH (2.17 g, 10 mmol) is dissolved in dry THF (10 ml), CDI (1.62 g, 10 mmol) is added, and the reaction is stirred at 0 ° for 45 min. Benzyl mercaptan (1.16 ml, 10 mmol) is added, and the reaction mixture is allowed to warm to 25 ° overnight. The solvent is removed under reduced pressure, ethyl acetate (20 ml) is added, and the organic solution is washed with 10% (w/v) citric acid, 4% (w/v) NaHCO3, and saturated aqueous NaC1. The ethyl acetate solution is dried over MgSO4, then filtered, and the solvent is removed. Boc-Nva-SBzl is solidified with hexane or petroleum ether (80% yield), produces one spot on thin-layer chromatography (TLC) [CHC13 : methanol (9 : 1, v/v)], and is used for subsequent reaction without further purification. Boc-Nva-SBzl (0.97 g, 3 mmol) is treated with 10 equivalents of 2.2 N HC1 in dioxane and allowed to stir at 25 ° for 45 min. The solvent is then removed by evaporation, and ether is added to solidify the product. The resulting Nva-SBzl. HCI is dried in vacuo and used for subsequent steps 3 AA, Amino acid residue; Abz, 2-aminobenzoyl; AMC, 7-amino-4-methylcoumarin; Boc, tert- butyloxycarbonyl; Bu-i, isobutyl; Bzl, benzyl; CDI, 1,1'-carbonyldiimidazole; DCC, N,N'- dicyclohexylcarbodiimide; DMF, dimethylformamide; EDC, 1-ethyl-3-[3-(dimethylamino)- propyl]carbodiimide hydrochloride; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesul- fonic acid; HOBt, N-hydroxybenzotriazole; MES, 2-(N-morpholino)ethanesulfonic acid; Mu. N-morpholinocarbonyl; Nba, 4-nitrobenzylamine; NNap-OCH3, 1-methoxy-3-naph- thylamine; SBzI(C1), SCH2C6H4-4-CI; Suc, succinyl; THF, tetrahydrofuran; Tricine, N-[tris(hydroxymethyl)methyl]glycine; Z, benzyloxycarbonyl. 4 j. Wo Harper, R. R. Cook, J. Roberts, B. J. Mclaughlin, and J. C. Powers, Biochemistry 23, 2995 (1984). s S. Odake, C M. Kam, L. Narasimhan, M. Poe, J. T. Blake, O. Krahenbuhl, J. Tschopp, and J. C. Powers, Biochemistry 30, 2217 (1991). [ 1 ] PEPTIDE THIOESTER SUBSTRATES 5 without further purification. Nva-SBzl-HCI (0.78 g, 3 mmol), Boc-Ala- Ala-OH (0.78 g, 3.0 mmol), HOBt (0.61 g, 4.5 mmol), and triethylamine (0.42 ml, 3.0 mmol) are dissolved in 10 ml of DMF, and the solution is cooled to - 10 ° in an ice-water-salt bath. DCC (0.68 g, 3.3 mmol) is added, and the reaction mixture is stirred at - 10 ° for 2 hr and at 25 ° overnight. Dicyclohexylurea is removed by filtration, DMF is removed by evaporation, and the residue is dissolved in ethyl acetate and washed as described above. The final product is recrystallized from CHC13-petroleum ether with cool- ing, mp 140°-141°; TLC Rf of 0.61 [CHCI3 : methanol (9 : 1, v/v)]. Analysis calculated for C23H35N3OsS: C, 59.38; H, 7.58; N, 9.02. Found: C, 59.58; H, 7.62; N, 9.14. Boc-Ala-Ala-Asp-SBzl. To prepare Boc-Ala-Ala-Asp-SBzl, 5 to a THF solution (60 ml) of Boc-Asp(O-tert-Bu)-OH (11.6 g, 40 mmol) is added HOBt hydrate (3.1 g, 20 mmol), benzyl mercaptan (5.2 ml, 44 mmol), and DCC (9.9 g, 48 mmol) in 15 ml of THF at -5 °. After stirring at 0 ° for 12 hr and at room temperature for 24 hr, the reaction mixture is filtered and the solvent removed by evaporation. Ethyl acetate is added to the residue, and the solution is washed successively with 1 N HCI, 10% (w/v) Na2CO3, and a saturated NaC1 solution. The organic layer is dried over MgSO4, and the solvent is removed by evaporation. The crude product is purified by silica gel chromatography using ethyl acetate :n-hexane (1:10, v/v) as an eluant to give Boc-Asp(O-tert-Bu)-SBzl (13.5 g, 85% yield) as a pale yellow oil. An ethyl acetate solution (70 ml) saturated with HC1 is added to Boc- Asp(O-tert-Bu)-SBzl (4.0 g, 8.8 mmol) at 0 °, and the solution is stirred at 25 ° for 2.5 hr. The solvent is removed, and ethyl acetate is added to the residue to give a white precipitate of H-Asp-SBzl. HC1 which is filtered and dried in vacuo (2.1 g, 87% yield). To a THF solution (20 ml) of Boc-Ala-Ala-OH (0.52 g, 2.0 mmol) is added successively N-methylmorpholine (0.22 ml, 2.0 mmol) and isobutyl chloroformate (0.26 ml, 2.0 mmol) at -15 °. After stirring for 2 min and addition of a cold THF solution (3 ml) of triethylamine (0.56 ml, 4 mmol), the mixture is added to a DMF solution (1 ml) of H-Asp-SBzl • HC1 (0.55 g, 2 mmol) at -15 °. After stirring for 1 hr, the reaction mixture is quenched by the addition of 1 N HC1 (4 ml) and is then concentrated in vacuo. An ethyl acetate solution of the residue is washed with water and dried over MgSO4. The solvent is removed by evaporation, and the crude product is purified by silica gel chromatography using CHC13:methanol (50:1, v/v) as an eluant and solidified with n-hexane to give the final product (0.67 g, 70% yield) as a white powder; mp 72°-79°; TLC Re of 0.52 (CHC13 : metha- nol : CH3COOH, 80 : 10 : 5, v/v/v). Analysis calculated for C22H31OTN3S " 0.25H20-0.25C6H14: C, 55.61; H, 6.95; N, 8.28. Found: C, 55.71; H, 7.00; N, 7.94. 6 METHODS [ 11 Lysine- or Arginine-Containing Thioesters. Z-Lys-SBzl is prepared by coupling Z-Lys-OH with benzyl mercaptan using the DCC method. 6 Simple N-blocked arginine thioesters are synthesized by coupling an N-blocked arginine derivative with a thiol using either the DCC-HOBt or DCC meth- ods. 7 Di- and tripeptide thiol esters are then prepared by deblocking the Boc-Arg-SR with HC1 in dioxane followed by coupling with the appropriate peptide acid using the pentachlorophenyl active ester method. The arginine side chain is protonated with HC1 during the synthesis of arginine thioesters; therefore, it is not necessary to protect the guanidino group with other re- agents. Z-Arg-SBzl. HCl. To prepare Z-Arg-SBzl. HC1, 8 Z-Arg-OH-HC1 (3.45 g, 10 mmol) is dissolved in DMF, HOBt (1.35 g, 1.0 mmol) is added, and the solution is cooled to 0 °. Benzyl mercaptan (1.24 g, 10 mmol) and DCC (2.16 g, 10.5 mmol) are then added. The reaction mixture is stirred overnight at 0 °, followed by removal of the dicyclohexylurea by filtration and the solvent by evaporation. The crude product is purified by flash column chromatography on silica gel (32-64/xm) using 15% methanol in CHCI3 as an eluant. The product is obtained as a white foam after trituration with petroleum ether and drying in vacuo (20% yield); TLC Rf of 0.64 [CHC13 : methanol : CH3COOH (10 : 3 : 1, v/v/v)]. Analysis calculated for C21HzTN403Cl" 0.5H20: C, 54.83; H, 6.14; N, 12.18. Found: C, 54.76, H, 6.10; N, 12.40. Thioester Substrates for Metalloendopeptidases. Thioester substrates for metalloendopeptidases typically contain a thioester bond in the interior of a peptide sequence and require additional synthetic steps. The synthesis usually involves coupling of an N-terminal peptide fragment with the thiol- containing fragment using standard peptide condensation methods to form the thioester bond. For example, CH3CO-Pro-Leu-Gly-SLeu-Leu - Gly-OCzH5 is prepared from CH3CO-Pro-Leu-GIy-OH and HSCH[CHzCH(CH3)z]-CO-Leu-GIy-OC2H5 using the DCC-HOBt method. The required HSCH[CH2CH(CH3)z]-CO-Leu-Gly-OCzH5 is syn- thesized by coupling L-a-mercaptoisocaproic acid and Leu-Gly-OCzH5 us- ing EDC-HOBt. 9 Boc-Abz-Gly-Pro-Leu-SCHzCO-Pro-Nba. Boc-Abz-Gly-Pro-OH and Leu-SCH2CO-Pro-Nba • HC1 are synthesized by standard peptide coupling methods. Boc-Abz-Gly-Pro-OH (0.78 g, 2 mmol) and Leu-SCH2CO-Pro- Nba. HC1 (0.94 g, 2 mmol) are then coupled using the mixed anhydride 6 G. D. Green and E. Shaw, Anal Biochem. 93, 223 (1979). 7 B. J. McRae, K. Kurachi, R. L. Heimark, K. Fujikawa, E. W. Davie, and J. C. Powers, Biochemistry 20, 7196 (1981). R. R. Cook, B. J. McRae, and J. C. Powers, Arch. Biochem. Biophys. 234, 82 (1984). 9 H. Weingarten, R. Martin, and J. Feder, Biochemistry 24, 6730 (1985). [...]... p H 7.5, 10% DMSO, and at 25 ° for factor D, C2a~ and Bb, and 0.1 M HEPES, 10 m M CaC12, buffer, pH 7.5, 9% DMSO, and 30 ° for C l r and Cls The units for kcat, Kin, and kcat/Km are see 1, tzM, and M -1 sec -1 NH, No hydrolysis; SH, slow hydrolysis b C.-M Kam, B J McRae, J W Harper, M M Niemann, J E Volanakis, and J C Powers, J Biol Chem 262, 3444 (1987) ': B J McRae, T.-Y Lin, and J C Powers, J Biol... coefficients are usually greater than 0.99 Assay for Metalloendopeptidases.The enzymatic hydrolysis rates of thioesters by metallopeptidases are measured similarly to those for serine peptidases, and a typical assay for Clostridium histolyticum collagenase followsJ ° The hydrolysis rate is measured in 50 m M Tricine, p H 7.5, containing 10 m M CaCI2,0.5% (v/v) DMSO, and 2.4% (v/v) methanol at 25 ° To a cuvette... 4,4'-dithiodipyridine (Aldrithiol-4) and 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent), and Tricine can be obtained from Aldrich and other sources HEPES is purchased from Research Organics, Inc (Cleveland, OH) Several amino acid and peptide thioester substrates are commercially available, and some are listed in Table I Chymotrypsin, trypsin, porcine pancreatic elastase (PPE), and Clostridium histolyticum... 7.5, 9-10% D M S O , and at 25 ° T h e units for kcat, Kin, and kcat/Km are sec i,/xM, and 106 M -1 sec 1 Boc-Ala-Ala-Phe-SBzl is not hydrolyzed by H L E and PPE b Data were obtained from J W Harper, R R Cook, J Roberts, B J Mclaughlin, and J C Powers, Biochemistry 23, 2995 (1984) c D a t a were obtained from C.-M Kam, J E Kerrigan, K M Dolman, R Goldschmeding, A V o n d e m Borne, and J C Powers, FEBS... T Tanaka, K Cho, and R R Cook, Biochem J 220, 569 (1984) 25 C.-M K a m , G P Vlasuk, D E Smith, K E Arcuri, and J C Powers, Thromb Haemostasis 64, 133 (1990) [ 1] PEPTIDETHIOESTERSUBSTRATES 13 complement peptidases 19'2° are shown in Tables IV and V The Lys- or Arg-containing thioesters are hydrolyzed very rapidly by trypsin with kcat/ Km values of 10 6 M 1 sec-1, and Z-Arg-SBzl and Z-Lys-SBzl are... O , and at 25 ° for all substrates except for Z-Lys-SBzl for which 0.1 M Tris, 0.1 M NaCI, buffer, p H 8.0, was used The units for kc,t, Kin, and kc~JKm are sec ~,/zM, and 106 M 1 see 1 NH, No hydrolysis h G D G r e e n and E Shaw, Anal Biochem 93, 223 (1979) ' R R Cook, B J McRae, and J C Powers, Arch Biochem Biophys 234, 82 (1984)• ,l B J McRae, K Kurachi, R L Heimark, K Fujikawa, E W Davie, and. .. elastase (HLE) and human cathepsin G can be obtained from Athens Research and Technology, Inc (Athens, GA) Proteinase 3 (human neutrophil) was provided by Dr Koert Dolman of the University of Amsterdam Rat mast cell peptidase I and II (RMCP I and RMCP II) were obtained from Drs Richard Woodbury and Hans Neurath of the University of Washington Bovine thrombin, factor IXa, factor Xa, factor XIa, and factor... s e r i n e p e p t i d a s e s t e s t e d 5'27 Thioester Substrates for Metallopeptidases T h e c o m p o u n d L e u - S C z H 5 is t h e first r e p o r t e d t h i o e s t e r s u b s t r a t e f o r a metallopeptidase, leucine aminopeptidase, u Other peptide thioesters that are hydrolyzed by human and bacterial metallopeptidases are shown in T a b l e V I 9'1°'28-3° C H 3 C O - P r o - L e u... Gly-Pro-Leu-SGly-Pro, Pro-Leu-Gly-SLeu-Leu-Gly, or Pro-Leu-Ala-SNva-Trp sequences are hydrolyzed by metalloendopeptidases such as collagenase and stromelysin CH3CO-Pro-Leu-Ala-SNva-Trp NH2 has been shown to be hydrolyzed not only by fibroblast collagenase but also by other metalloendopeptidases such as stromelysin, gelatinase, and thermolysin.3° Several peptide thioesters such as Boc-Abz-Gly-ProLeu-SGly-Pro-Nba... Both the substrate and the product are intensely fluorescent, but with different excitation and A J Barrett and H Kirschke, this series, Vol 80, p 535 2 M Z i m m e r m a n , E Yurewicz, and G Patel, A n a l Biochem 70, 258 (1976) 20 METHODS [2] emission wavelengths Thus, glutaryl-Phe-NH-Mec has a Mx of 325 nm and kern of 395 nm, whereas 7-amino-4-methylcoumarin has a ken of 345 nm and ~kemof 445 rim . and Molecular Biology. In Volume 244 on peptidases of serine and cysteine type and in this volume on aspartic, metallo, and other peptidases, the chapters on spe- cific methods, enzymes, and. Serine Peptidases and Metalloendopeptidases By JAMES C. POWERS and CHIH-MIN KAM Introduction Synthetic peptide substrates are widely used in biochemical and physiological studies of proteolytic. DMSO, and at 25 ° for factor D, C2a~ and Bb, and 0.1 M HEPES, 10 mM CaC12, buffer, pH 7.5, 9% DMSO, and 30 ° for Clr and Cls. The units for kcat, Kin, and kcat/Km are see 1, tzM, and M