Albeck A, Kliper S
Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel.
Biochem J. 1997 Mar 15;322 ( Pt 3)(Pt 3):879-84. doi: 10.1042/bj3220879.
Peptidyl epoxides are time- and concentration-dependent selective cysteine protease inhibitors. The lack of recovery of enzymic activity and the retention of 1 molar equivalent of radioactive inhibitor associated with the enzyme on dialysis, shown in this study, indicate that they form a covalent irreversible equimolar complex with the enzyme. It is also shown that the peptidyl epoxide inhibitors alkylate the active-site thiol. This alkylation only occurs when the enzyme is in its native conformation, as the denatured enzyme does not undergo alkylation by the inhibitor to any appreciable extent. Finally, the inactivation process is compared with a model reaction between a peptidyl epoxide and a protected cysteine in neutral and basic aqueous media. The inactivation of cathepsin B by Cbz-Phe-(O-benzyl)-Thr-epoxide is accelerated by 5.5 orders of magnitude relative to the rate of the model reaction at pH 10.0 and 25 degrees C, and estimated to be at least 10(8) times faster than the model reaction at pH 7.0. These results, in conjunction with the selectivity exhibited by peptidyl epoxides at all levels, point to a mechanism-based inhibition, and may have mechanistic implications regarding the catalysis carried out by cysteine proteases.
肽基环氧化物是时间和浓度依赖性的选择性半胱氨酸蛋白酶抑制剂。本研究表明，酶活性未能恢复，且透析时与酶结合的放射性抑制剂保留了1摩尔当量，这表明它们与酶形成了共价不可逆等摩尔复合物。研究还表明，肽基环氧化物抑制剂使活性位点的巯基烷基化。这种烷基化仅在酶处于天然构象时发生，因为变性酶不会被抑制剂显著烷基化。最后，将失活过程与肽基环氧化物和受保护的半胱氨酸在中性和碱性水性介质中的模型反应进行了比较。在pH 10.0和25℃下，Cbz-Phe-(O-苄基)-Thr-环氧化物使组织蛋白酶B失活的速度相对于模型反应的速度加快了5.5个数量级，据估计在pH 7.0时比模型反应至少快10^8倍。这些结果，连同肽基环氧化物在各个水平上表现出的选择性，表明是基于机制的抑制作用，并且可能对半胱氨酸蛋白酶的催化作用具有机制方面的意义。
