Mechanism of glycoside hydrolysis: A comparative QM/MM molecular dynamics analysis for wild type and Y69F mutant retaining xylanases

Abstract

Computational simulations have been performed using hybrid quantum-mechanical/molecular-mechanical potentials to investigate the catalytic mechanism of the retaining endo-β-1, 4-xylanase (BCX) from B. circulans. Two-dimensional potential-of-mean-force calculations based upon molecular dynamics with the AM1/OPLS method for wild-type BCX with a p-nitrophenyl xylobioside substrate in water clearly indicates a stepwise mechanism for glycosylation: the rate-determining step is nucleophilic substitution by Glu78 to form the covalently bonded enzyme-substrate intermediate without protonation of the leaving group by Glu172. The geometrical configuration of the transition state for the enzymic reaction is essentially the same as found for a gas-phase model involving only the substrate and a propionate/propionic acid pair to represent the catalytic glutamate/glutamic acid groups. In addition to stabilizing the ^2,5B boat conformation of the proximal xylose in the non-covalent reactant complex of the substrate with BCX, Tyr69 lowers the free-energy barrier for glycosylation by 42 kJ mol⁻¹ relative to that calculated for the Y69F mutant, which lacks the oxygen atom O_Y. B3LYP/6-31+G* energy corrections reduce the absolute height of the barrier to reaction. In the oxacarbenium ion-like transition state O_Y approaches closer to the endocyclic oxygen O_ring of the sugar ring but donates its hydrogen bond not to O_ring but rather to the nucleophilic oxygen of Glu78. Comparison of the average atomic charge distributions for the wild-type and mutant indicates that charge separation along the bond between the anomeric carbon and O_ring is matched in the former by a complementary separation of charge along the O_Y–H_Y bond, corresponding to a pair of roughly antiparallel bond dipoles, which is not present in the latter.