A mechanistic study of Trichoderma reesei Cel7B catalyzed glycosidic bond cleavage.
A mechanistic study of Trichoderma reesei Cel7B catalyzed glycosidic bond cleavage.
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DOI：
10.1021/jp403999s
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发表时间：
2013-07
期刊：
影响因子：
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通讯作者：
Yu Zhang-;Shihai Yan;L. Yao
中科院分区：
文献类型：
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作者：
Yu Zhang-;Shihai Yan;L. Yao
用ONIOM研究了里氏木霉Cel7B催化对硝基苯基乳糖苷水解的机理。在糖基化和脱糖基化步骤中，反应以一致的方式进行，这意味着亲核攻击和糖苷键断裂同时发生。糖基化步骤是限速的，势垒为18.9千卡/摩尔，与根据本文测量的kCAT得出的实验值相当。通过保守突变研究了四个残基R108、Y146、Y170和D172的功能，它们形成了涉及底物的氢键网络。突变株R108K、Y146F、Y170F和D172N使酶活力降低约150-8000倍。分子动力学模拟表明，这些突变破坏了氢键网络，导致底物偏离活性结合，阻碍了质子从E201到O4(+1)的转移或从E196到C1(-1)的亲核攻击。
An ONIOM study is performed to illustrate the mechanism of Trichoderma reesei Cel7B catalyzed p-nitrophenyl lactoside hydrolysis. In both the glycosylation and deglycosylation steps, the reaction proceeds in a concerted way, meaning the nucleophilic attack and the glycosidic bond cleavage occur simultaneously. The glycosylation step is rate limiting with a barrier of 18.9 kcal/mol, comparable to the experimental value derived from the kcat measured in this work. The function of four residues R108, Y146, Y170, and D172, which form a hydrogen-bond network involving the substrate, is studied by conservative mutations. The mutants, including R108K, Y146F, Y170F, and D172N, decrease the enzyme activity by about 150-8000-fold. Molecular dynamics simulations show that the mutations disrupt the hydrogen-bond network, cause the substrate to deviate from active binding and hinder either the proton transfer from E201 to O4(+1) or the nucleophilic attack from E196 to C1(-1).