Mobashery S, Ghosh S S, Tamura S Y, Kaiser E T
Department of Chemistry, Wayne State University, Detroit, MI 48202.
Proc Natl Acad Sci U S A. 1990 Jan;87(2):578-82. doi: 10.1073/pnas.87.2.578.
(R)-2-Benzyl-5-cyano-4-oxopentanoic acid (compound 4) was studied as a mechanism-based inactivator (suicide substrate) for the zinc protease carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1). This compound was designed rationally based on the knowledge of the active site topology and the reported stereospecific proton exchange on ketonic substrate analogue (R)-3-(p-methoxybenzoyl)-2-benzylpropanoic acid [Sugimoto, T. & Kaiser, E. T. (1978) J. Am. Chem. Soc. 100, 7750-7751] by CPA. It is suggested that enzymic deprotonation on the C-5 methylene moiety may result in the transient formation of a ketenimine as the key intermediate that partitions between turnover and enzyme inactivation. The enzyme inactivation exhibited pseudo-first-order kinetics, was irreversible, and could be fully prevented in the presence of the reversible inhibitor benzyl-succinate. The inactivation rate constant, kintact, was evaluated to be 0.083 +/- 0.003 min-1 and kcat was measured at 1.78 +/- 0.06 min-1. In turn, a partition ratio of 28 +/- 3 was calculated. The reversible inhibitor constant (Ki) was measured at 1.8 +/- 0.5 microM, indicative of a high affinity for compound 4 shown by CPA; however, Km for the turnover process was determined at 4.93 +/- 0.43 mM. Kinetic analysis and labeling by the radioactive form of the inactivator suggested that the stoichiometry for protein modification by compound 4 approaches a 1:1 ratio.
(R)-2-苄基-5-氰基-4-氧代戊酸（化合物4）被作为锌蛋白酶羧肽酶A（CPA；肽基-L-氨基酸水解酶，EC 3.4.17.1）的基于机制的失活剂（自杀底物）进行研究。该化合物是基于对活性位点拓扑结构的了解以及所报道的CPA对酮底物类似物(R)-3-(对甲氧基苯甲酰基)-2-苄基丙酸[Sugimoto, T. & Kaiser, E. T. (1978) J. Am. Chem. Soc. 100, 7750 - 7751]的立体特异性质子交换而合理设计的。有人提出，C-5亚甲基部分的酶促去质子化可能导致烯酮亚胺作为关键中间体的瞬时形成，该中间体在周转和酶失活之间进行分配。酶失活表现出假一级动力学，是不可逆的，并且在可逆抑制剂苄基琥珀酸存在下可以完全防止。失活速率常数kintact经评估为0.083±0.003 min⁻¹，kcat测定为1.78±0.06 min⁻¹。相应地，计算出分配比为28±3。可逆抑制剂常数（Ki）测定为1.8±0.5 μM，表明CPA对化合物4具有高亲和力；然而，周转过程的Km测定为4.93±0.43 mM。动力学分析和失活剂放射性形式的标记表明，化合物4对蛋白质修饰的化学计量比接近1:1。
