A Mechanistic Study of Trichoderma reesei Cel7B Catalyzed Glycosidic Bond Cleavage

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• 作者：Yu Zhang ; Shihai Yan ; Lishan Yao
• 刊名：The Journal of Physical Chemistry B
• 出版年：2013
• 出版时间：July 25, 2013
• 年：2013
• 卷：117
• 期：29
• 页码：8714-8722
• 全文大小：436K
• 年卷期：v.117,no.29(July 25, 2013)
• ISSN：1520-5207

文摘

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 k_cat 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鈥?000-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(鈭?).