无底物情况下来自Rhodococcus zopfii的腈水解酶中亲核进攻试剂CYS165的活性状态的探究(英文)  

Exploring the active state of the nucleophilic attacking agent CYS165 of nitrilase from Rhodococcus zopfii in the absence of substrate

作　　者：张慧珠[1] 高旭丹 梅晔[1,2,3] Zhang Huizhu;Gao Xudan;Mei Ye(State Key Laboratory of Precision Spectroscopy,School of Physics and Electronic Science,East China Normal University,Shanghai,200241,China;NYU⁃ECNU Center for Computational Chemistry at NYU Shanghai,Shanghai,200062,China;Collaborative Innovation Center of Extreme Optics,Shanxi University,Taiyuan,030006,China)

机构地区：[1]精密光谱学国家重点实验室,华东师范大学物理与电子科学学院,上海200241 [2]华东师范大学⁃上海纽约大学计算化学联合中心,上海200062 [3]山西省极端光学协同创新中心,山西大学,太原030006

出　　处：《南京大学学报(自然科学版)》2025年第1期129-139,共11页Journal of Nanjing University(Natural Science)

基　　金：国家自然科学基金(22073030)

摘　　要：腈水解酶作为一类在工业应用中具有巨大价值的绿色生物催化剂,能够高效地催化腈基化合物转化为羧酸.尽管其应用广泛,但腈水解酶的具体催化机制仍然不明确.先前的研究揭示了腈水解酶活性中心的GLU-LYS-GLU-CYS四联体在催化中起到关键作用,其中CYS残基作为亲核试剂攻击腈基,其硫醇基团的电离是反应中的一个关键步骤,然而,CYS的去质子化过程尚未被清晰揭示.研究聚焦于来自Rhodococcus zopfii(RzNIT)的腈水解酶,并研究了底物尚未进入酶活性位点时CYS165的质子化状态.通过对CYS165去质子化可能路径的详细分析,确认了在无底物状态下RzNIT中的CYS165处于中性状态.这一发现为进一步研究RzNIT的催化机制奠定了基础.Nitrilase,as a class of green biocatalysts of great value in industrial applications,can efficiently catalyze the hydrolysis of nitrile compounds into carboxylic acids.Despite its wide application,the specific catalytic mechanism of nitrilase remains elusive.Previous studies have revealed that the GLU⁃LYS⁃GLU⁃CYS tetrad in the active center of nitrilase plays a pivotal role in the catalysis,where the CYS residue acts as a nucleophile attacking the nitrile,and the ionization of its thiol group is a key step in the reaction.However,the process of deprotonation of CYS has not been clearly illustrated.This study focuses on the nitrilase from Rhodococcus zopfii(RzNIT)and investigates the protonation state of CYS165 when the substrate has not yet entered the enzyme′s active site.Through detailed analysis of possible pathways for CYS165 deprotonation,it is confirmed that CYS165 in RzNIT is in its neutral state in the absence of substrate.This finding lays the foundation for further studies on the catalytic mechanism of RzNIT.

关 键 词：ONIOM(the Own n-Layered Integrated Molecular Orbital and Molecular Mechanics) 腈水解酶 能垒 催化机制 

分 类 号：TQ426.97[化学工程]