A Mechanistic Study of Trichoderma reesei Cel7B Catalyzed Glycosidic Bond Cleavage

An ONIOM study is performed to illustrate the mechanism of Trichoderma reesei Cel7B catalyzed p-nitro-phenyl 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 150-8000-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 O-4(+1) or the nucleophilic attack from E196 to C-1(-1).