羧肽酶A催化反应中通用碱机制的证据：亲核试剂的分配研究和H2(18)O动力学同位素效应
Evidence for the general base mechanism in carboxypeptidase A-catalyzed reactions: partitioning studies on nucleophiles and H2(18)O kinetic isotope effects.
作者信息
Breslow R, Chin J, Hilvert D, Trainor G
出版信息
Proc Natl Acad Sci U S A. 1983 Jul;80(14):4585-9. doi: 10.1073/pnas.80.14.4585.
Abstract
Methanol does not detectably compete with water in carboxypeptidase-catalyzed cleavage of any substrate, although it is preferentially reactive in a model for the proposed nucleophilic mechanism for the enzyme that involves an anhydride intermediate. To test for such a common intermediate in the cleavage of related peptide and ester substrates, a method has been developed to examine H2(16)O-H2(18)O kinetic isotope-partitioning effects. The finding that benzoylglycylphenylalanine has an isotope effect of 1.019 +/- 0.002 while benzoylglycyl-beta-L-phenyl-lactate shows a small inverse isotope effect excludes most versions of a nucleophilic mechanism having a common anhydride intermediate. The bulk of the available evidence strongly favors the previously proposed general base mechanism.
摘要
甲醇在羧肽酶催化的任何底物裂解反应中，都不会与水发生可检测到的竞争，尽管在该酶的拟亲核机制模型（涉及酸酐中间体）中，甲醇具有优先反应性。为了检测相关肽和酯底物裂解过程中是否存在这种常见中间体，已开发出一种方法来研究H₂¹⁶O - H₂¹⁸O动力学同位素分配效应。苯甲酰甘氨酰苯丙氨酸的同位素效应为1.019±0.002，而苯甲酰甘氨酰-β-L-苯基乳酸显示出较小的逆同位素效应，这一发现排除了具有常见酸酐中间体的亲核机制的大多数版本。大量现有证据强烈支持先前提出的通用碱机制。
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