Ghosh S S, Wu Y Q, Mobashery S
Department of Chemistry, Wayne State University, Detroit, Michigan 48202.
J Biol Chem. 1991 May 15;266(14):8759-64.
N-(Cyanoacetyl)-L-phenylalanine (compound 1) and N-(3-chloropropionyl)-L-phenylalanine (compound 2) were studied as the first peptidic mechanism-based inactivators (suicide substrates) for the zinc protease carboxypeptidase A (CPA). A crucial deprotonation on the methylene alpha to the amide carbonyl of 1 and 2 has been suggested to lead to the transient formation of a ketenimine and an alpha, beta-unsaturated amide, respectively. Subsequently, it is proposed that these key intermediates trap an active site nucleophile, resulting in covalent modification of the protein. In competition with the inactivation process, the enzyme hydrolyzes the amide bonds in these molecules. Partition ratios of 1180 +/- 40 and 1680 +/- 60 were determined for 1 and 2, respectively. N-Acrolyl-L-phenylalanine (compound 4), the putative intermediate from 2, was independently studied to test the validity of the mechanistic scheme and was observed to be an active site-directed inactivator of CPA. A solvent deuterium isotope effect of 1.39 +/- 0.02 was noted for inactivation by 2 and one of 1.31 +/- 0.01 for its hydrolysis, in keeping with a proposed promoted water hydrolytic pathway for peptide hydrolysis by CPA (Christanson, D. W., and Lipscomb, W. N. (1989) Acc. Chem. Res. 22, 62-69). Details of the kinetic analysis and design concepts are discussed.
N-（氰乙酰基）-L-苯丙氨酸（化合物1）和N-（3-氯丙酰基）-L-苯丙氨酸（化合物2）作为锌蛋白酶羧肽酶A（CPA）的首批基于肽机制的失活剂（自杀底物）进行了研究。有人提出，化合物1和2酰胺羰基α位亚甲基上的关键去质子化分别导致酮亚胺和α，β-不饱和酰胺的瞬时形成。随后，有人提出这些关键中间体捕获活性位点亲核试剂，导致蛋白质的共价修饰。在与失活过程的竞争中，该酶水解这些分子中的酰胺键。化合物1和2的分配比分别测定为1180±40和1680±60。对化合物2的假定中间体N-丙烯酰基-L-苯丙氨酸（化合物4）进行了独立研究，以检验该机制方案的有效性，并观察到它是CPA的活性位点导向失活剂。化合物2失活的溶剂氘同位素效应为1.39±0.02，其水解的溶剂氘同位素效应之一为1.31±0.01，这与CPA促进肽水解的水水解途径一致（克里斯滕森，D. W.，和利普斯科姆，W. N.（1989年）《化学研究述评》22，62 - 69）。讨论了动力学分析的细节和设计概念。
