Phillips M A, Kaplan A P, Rutter W J, Bartlett P A
Hormone Research Institute, University of California, San Francisco 94143.
Biochemistry. 1992 Feb 4;31(4):959-63. doi: 10.1021/bi00119a003.
A new strategy of potentially broad application for probing transition-state (TS) analogy in enzymatic systems is described in this paper. The degree to which a series of phosphonate inhibitors act as TS analogues of rat carboxypeptidase A1 has been determined for the wild-type enzyme, for the R127K, R127M, and R127A mutants, and for the R127A mutant in the presence of 0.5 M guanidine hydrochloride. The impact that the mutations have on the inverse second-order rate constants (Km/kcat) for substrate hydrolysis is mirrored by the effect on the inhibition constants (Ki) for the corresponding phosphonate inhibitors. These results demonstrate that the phosphonate moiety mimics some of the electronic as well as the geometric characteristics of the TS. A similar but distinctly separate correlation is observed for tripeptide analogues in comparison to analogues of the dipeptide Cbz-Gly-Phe, reflecting an anomalous mode of binding for the latter system. The selective rate increases and corresponding enhancement in inhibitor binding observed on addition of 0.5 M guanidine hydrochloride to the R127A mutant indicate that the exogenous cation can assume the role played by Arg-127 in stabilizing the TS and in providing substrate selectivity at the P2 position.
本文描述了一种在酶系统中探测过渡态（TS）类似物的具有潜在广泛应用的新策略。已测定了一系列膦酸酯抑制剂作为大鼠羧肽酶A1的TS类似物对野生型酶、R127K、R127M和R127A突变体以及在0.5 M盐酸胍存在下的R127A突变体的作用程度。突变对底物水解的二级反应速率常数（Km/kcat）的影响与对相应膦酸酯抑制剂的抑制常数（Ki）的影响相对应。这些结果表明，膦酸酯部分模拟了TS的一些电子和几何特征。与二肽Cbz-Gly-Phe类似物相比，三肽类似物观察到类似但明显不同的相关性，反映了后者系统的异常结合模式。向R127A突变体中添加0.5 M盐酸胍后观察到的选择性速率增加和抑制剂结合的相应增强表明，外源阳离子可以承担由Arg-127在稳定TS和在P2位置提供底物选择性方面所起的作用。
