Kawabata S, Miura T, Morita T, Kato H, Fujikawa K, Iwanaga S, Takada K, Kimura T, Sakakibara S
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Eur J Biochem. 1988 Feb 15;172(1):17-25. doi: 10.1111/j.1432-1033.1988.tb13849.x.
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 The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively.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).
新合成了74种7-氨基-4-甲基香豆素（Mec）类型为Boc-Xaa-Yaa-Arg-NH-Mec的肽酰胺，并对其进行测试以寻找凝血蛋白酶和胰蛋白酶的特异性底物。这些底物的Xaa和Yaa残基分别被12种和15种不同的氨基酸取代。在这些肽中，发现以下是个别酶的最敏感底物：人α-凝血酶的Boc-Asp(OBzl)-Pro-Arg-NH-Mec（kcat = 160 s-1，Km = 11 microM，kcat/Km = 15,000,000 M-1 s-1）；牛因子Xa的Z-小于Glu-Gly-Arg-NH-Mec（kcat = 19 s-1，Km = 59 microM，kcat/Km = 320,000 M-1 s-1）；牛因子XIIa的Boc-Gln-Gly-Arg-NH-Mec（kcat = 5.8 s-1，Km = 140 microM，kcat/Km = 42,000）；牛活化蛋白C的Boc-Asp(OBzl)-Ala-Arg-NH-Mec（kcat = 9.2 s-1，Km = 120 microM，kcat/Km = 77,000 M-1 s-1）；牛血浆激肽释放酶的Boc-Gly-Phe-Arg-NH-Mec（kcat = 29 s-1，Km = 230 microM，kcat/Km = 130,000 M-1 s-1）。此外，新发现Boc-Glu(OBzl)-Ala-Arg-NH-Mec（kcat = 46 s-1，Km = 370 microM，kcat/Km = 120,000 M-1 s-1）是人因子XIa的良好底物。牛胰蛋白酶能有效水解P2位点含有Ala和Pro的肽-NH-Mec底物。反应性最高的底物是Boc-Gln-Ala-Arg-NH-Mec（kcat = 120 s-1，Km = 6.0 microM，kcat/Km = 20,000,000 M-1 s-1）。
