The Current Status of the -Chymotrypsin Mechanism
The Current Status of the -Chymotrypsin Mechanism
复制标题
胰凝乳蛋白酶机制的现状
DOI：
10.1021/ja01072a020
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发表时间：
1964
影响因子：
15
通讯作者：
F. Kézdy
中科院分区：
文献类型：
--
作者：
M. L. Bender;F. Kézdy
总结了胰凝乳蛋白酶催化特定酯、酸和酰胺底物水解过程中酰基酶中间体假设的证据。比较根据相对速率数据确定的各个速率常数、pH 对动力学的影响、醇解数据和同位素氧交换数据。酯水解的超平衡常数和其他比较表明这些数据的一致性。讨论了各个酰化和脱酰化步骤的特点：（1）根据结构对反应性影响的研究，这些步骤可以被表征为亲核反应；（2）在酰化过程中动力学上看到两个质子基团，pX为7和9，而在脱酰化过程中动力学上看到一两个质子基团，其中一个为pX。 7 和另一个变量 pX。范围从8.3到；（3）酰化的质子基团初步鉴定为（两个）咪唑基团和N端异亮氨酸的氨基；（4）脱酰基化的质子基团初步鉴定为（两个）咪唑基团和脱酰基亲核基团；（5）咪唑基团在两者中充当质子转移的催化剂基于模型研究和氧化氘同位素效应的酰化和脱酰化；(6)根据现有的动力学和分光光度论证以及先前的分离研究，丝氨酸羟基在第一步被酰化；(7)脱酰化反应在水中是一级反应； (8)在脱酰化过程中未观察到可检测到的中间体。间硝基苯甲酰基-α-胰凝乳蛋白酶的（分子内）脱酰化在动力学上类似于5-硝基水杨酸对硝基苯酯的（分子内）非酶一般碱催化水解。基于上述数据以及对称性和微观可逆性的要求，提出了胰凝乳蛋白酶催化的机理，具体体现为式(1)。 10和/或11。这种涉及酰化和脱酰化中四面体加成中间体的形成和分解的机制将咪唑的催化描述为涉及从亲核试剂（分别是丝氨酸或水）中除去质子以及向羰基氧原子提供质子。这种涉及两个咪唑基团的循环过程是与所有已知实验数据一致的一种机制。
The evidence for the postulation of acyl-enzyme intermediates in-chymotrypsin-catalyzed hydrolysis of specificester, acid, and amide substrates is summarized. Individual rate constants determined from rela-tive rate data, the effect of pH on the kinetics, alcoholysis data, and isotopic oxygen exchange, data are com-pared. The over-allequilibrium constant of ester hydrolysis and other comparisons indicate the consistency of these data. The characteristics of theindividual acylation and deacylation steps are discussed:(1) these steps may be characterized as nucleophilic reactions on the basis of studies on the effect of structure on re-activity;(2) two prototropic groups are seen kinetically in acylation, with pX.'s of 7 and 9, while one or two prototropic groups are seen kinetically in deacylation, one with a pX. of 7 and the other a variable pX. ranging from 8.3 to;(3) the prototropic groups of acylation are tentatively identified as (two) imidazole (s) and an-amino group of N-terminal isoleucine, respectively;(4) the prototropic groups of deacylation are tenta-tively identified as (two) imidazole group (s) and the nucleophile of deacylation, respectively;(5) the imidazole group (s) serve as catalyst for transferring protons in both acylation and deacylation on the basis of model studies and deuterium oxide isotope effects;(6) the serine hydroxyl group is acylated in the first step on the basis of present kinetic and spectrophotometric arguments and previous isolation studies;(7) the deacylation reac-tion is first order in water; and (8) no detectable intermediate may be observed in deacylation. The (intra-molecular) deacylation of m-nitrobenzoyl-a-chymotrypsin is kinetically similar to the (intramolecular) non-enzymatic general base-catalyzed hydrolysis of^-nitrophenyl 5-nitrosalicylate. On the basis of the above data and the requirements of symmetry and microscopic reversibility, a mechanism for-chymotrypsin catalysis is proposed embodied in eq. 10 and/or 11. This mechanism involving the formation and decomposition of tetrahedral addition intermediates in both acylation and deacylation describes the catalysis by imidazole (s) as involving both removal of a proton from the nucleophile (serine or water, respectively) and donation of a pro-ton to the carbonyl oxygen atom. This cyclic process involving both imidazole group (s) is the one mechanism consistent with all known experimental data.