Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens
Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens
复制标题
DOI：
10.1074/jbc.m113.545293
复制
发表时间：
2014-04-18
影响因子：
4.8
通讯作者：
Withers, Stephen G.
中科院分区：
文献类型：
--
作者：
Jongkees, Seino A. K.;Yoo, Hayoung;Withers, Stephen G.
背景：来自GH88的不饱和葡萄糖醛酸水解酶(UGL)是参与糖胺多糖降解的细菌酶，是毒力因子。结果：给出了与目前公认的UGL机制不一致的力学数据。结论：提出了一个修订的机制来解释到目前为止发表的所有机制数据。意义：了解UGL的作用机制可能有助于设计抑菌剂。在CAZy分类系统中，对CAZy分类系统中糖苷水解酶家族88的产气荚膜梭菌不饱和葡萄糖醛酸水解酶催化水合引发的水解酶的细节进行了实验。直接对酶反应的H-1核磁共振监测没有检测到溶液中积累的反应中间产物，表明初始水化产物在酶上发生了重排。使用1，1-二氟底物基于机理捕获酶上中间体的尝试失败了，因为探针太过失活，无法被酶翻转。碳1和碳4上的重氢取代所产生的动力学同位素效应为水合反应中分开的第一个不可逆步骤和总的速率决定步骤提供了证据，并提出了两种可能的机制来解释这些结果。根据催化残基在酶活性部位的位置，缺乏2-脱氧-2-氟底物的有效周转，以及多次尝试确认涉及共价糖基酶中间体的更简单的机制，最可信的机制是涉及碳1和碳2上含有环氧化物的中间体的机制。
Background: Unsaturated glucuronyl hydrolases (UGL) from GH88 are bacterial enzymes involved in the degradation of glycosaminoglycans and are virulence factors. Results: Mechanistic data inconsistent with the currently accepted mechanism of UGL are presented. Conclusion: A revised mechanism is proposed to explain all mechanistic data published to date. Significance: Understanding of the mechanism of UGL may allow design of bacteriostatic agents.Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct H-1 NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2.