title: Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin.
abstract: Seventy-four peptide amides of 7-amino-4-methylcoumarin (Mec) of the type Boc-Xaa-Yaa-Arg-NH-Mec were newly synthesized and tested to find specific substrates for blood-clotting proteases and trypsin. The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively. Among these peptides, the followings were found to be most sensitive substrates for individual enzymes: Boc-Asp(OBzl)-Pro-Arg-NH-Mec (kcat = 160 s-1, Km = 11 microM, kcat/Km = 15,000,000 M-1 s-1) for human alpha-thrombin, Z-less than Glu-Gly-Arg-NH-Mec (kcat = 19 s-1, Km = 59 microM, kcat/Km = 320,000 M-1 s-1) for bovine factor Xa, Boc-Gln-Gly-Arg-NH-Mec (kcat = 5.8 s-1, Km = 140 microM, kcat/Km = 42,000) for bovine factor XIIa, Boc-Asp(OBzl)-Ala-Arg-NH-Mec (kcat = 9.2 s-1, Km = 120 microM, kcat/Km = 77,000 M-1 s-1) for bovine activated protein C, and Boc-Gly-Phe-Arg-NH-Mec (kcat = 29 s-1, Km = 230 microM, kcat/Km = 130,000 M-1 s-1) for bovine plasma kallikrein. Moreover, Boc-Glu(OBzl)-Ala-Arg-NH-Mec (kcat = 46 s-1, Km = 370 microM, kcat/Km = 120,000 M-1 s-1) was newly found as a good substrate for human factor XIa. Bovine trypsin effectively hydrolyzed peptide-NH-Mec substrates containing Ala and Pro at the P2 site. The most reactive substrate was Boc-Gln-Ala-Arg-NH-Mec (kcat = 120 s-1, Km = 6.0 microM, kcat/Km = 20,000,000 M-1 s-1).
authors.affiliation: Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kawabata
authors.forename: T
authors.initials: T
authors.lastname: Miura
authors.forename: T
authors.initials: T
authors.lastname: Morita
authors.forename: H
authors.initials: H
authors.lastname: Kato
authors.forename: K
authors.initials: K
authors.lastname: Fujikawa
authors.forename: S
authors.initials: S
authors.lastname: Iwanaga
authors.forename: K
authors.initials: K
authors.lastname: Takada
authors.forename: T
authors.initials: T
authors.lastname: Kimura
authors.forename: S
authors.initials: S
authors.lastname: Sakakibara
mesh_terms: D000596:Amino Acids; D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects*; D001779:Blood Coagulation Factors / Q000378:metabolism; D002417:Cattle; D003374:Coumarins / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D009842:Oligopeptides / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011486:Protein C / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001779:Blood Coagulation Factors; D003374:Coumarins; D009842:Oligopeptides; D011486:Protein C; C112975:tert-butoxycarbonyl-isoleucyl-glutamyl-glycyl-arginyl-amidomethylcoumarin; D010447:Peptide Hydrolases; D014357:Trypsin; D013917:Thrombin