Cazzulo J J, Bravo M, Raimondi A, Engström U, Lindeberg G, Hellman U
Instituto de Investigaciones Bioquímicas Fundación Campomar, Buenos Aires, Argentina.
Cell Mol Biol (Noisy-le-grand). 1996 Jul;42(5):691-6.
Two synthetic peptides, peptide G, with the sequence KEEASSAVVGGPG, consisting of the last 10 amino acid residues of the catalytic domain, plus the first 3 at the C-terminal domain, of cruzipain, and peptide R, with the sequence KEEASSAVVRGPG, were hydrolyzed by the enzyme, as shown by reversed-phase HPLC. Peptide R was the best substrate, with a Vmax/K(m) ratio 6-fold higher as compared with peptide G, in good agreement with previous studies indicating that, in small peptides, cruzipain prefers R or K at P1. The optimal pH values for hydrolysis of peptides G and R were 6.8 and 8.0, respectively. A p-nitroanilide derivative containing the P1-P3 residues, Z-VVR-pNA, was an excellent substrate for cruzipain, with a K(m) value (33 microM at pH 9.0) lower than that for Bz-PFR-pNA (66 microM). These results open the possibility of synthesizing more specific substrates and inhibitors of cruzipain.
两种合成肽，肽G，序列为KEEASSAVVGGPG，由克氏锥虫蛋白酶催化结构域的最后10个氨基酸残基加上C端结构域的前3个氨基酸组成；肽R，序列为KEEASSAVVRGPG，经该酶水解，反相高效液相色谱法显示了这一结果。肽R是最佳底物，其Vmax/K(m)比值比肽G高6倍，这与之前的研究结果高度一致，即小肽中克氏锥虫蛋白酶在P1位点更倾向于R或K。肽G和肽R水解的最佳pH值分别为6.8和8.0。一种含有P1 - P3残基的对硝基苯胺衍生物Z - VVR - pNA是克氏锥虫蛋白酶的优良底物，其K(m)值（pH 9.0时为33 microM）低于Bz - PFR - pNA（66 microM）。这些结果为合成更具特异性的克氏锥虫蛋白酶底物和抑制剂提供了可能性。
