Probing the active site chemistry of β-glucosidases along the hydrolysis reaction pathway.

β-Glucosidases (EC 3.2.1.21) can be found in all domains of living organisms, where they play essential roles in the removal of terminal glucosyl residues from nonreducing ends of saccharides and glycosides. Two active site amino acid residues, a nucleophile and a proton donor, play key roles in the hydrolytic mechanism. Besides these two highly conserved catalytic residues, there are other conserved amino acids in the active site of β-glucosidases that make direct hydrogen bonds to the glycosyl moiety at the -1 subsite. In this study, the catalytic mechanism of a GH1 β-glucosidase (BGlu1) is systematically studied. On the basis of the quantum mechanical studies, the side chain of Tyr315 in an interaction with both O5 of the glucose ring and the nucleophilic glutamate contributes significantly to the energy profile. Glu440 and the conserved Asn175 are the other residues in the polar interaction with -1 glucose with considerable influence on the free energy of the reaction. Gln29, His130, and Trp441, which also form hydrogen bonds to the glycosyl moiety, are found to have relatively a minor effect on the reaction. Different arrangements of active site residues in the high-level [quantum mechanics (QM)] and low-level [molecular mechanics (MM)] regions during the hybrid QM/MM calculations indicate that Tyr315 lowers the energy barrier in the deglycosylation step (by 11.95 kcal/mol) while Glu440 mainly reduces the energy barrier of the glycosylation step. Exclusion of either of these two residues from the QM region results in deviation of the geometric parameters of the enzyme-substrate complex from those expected for the preactivated distorted structure of the substrate.