Role of Enzyme and Active Site Conformational Dynamics in the Catalysis by α-Amylase Explored with QM/MM Molecular Dynamics

[Image: see text] We assessed enzyme:substrate conformational dynamics and the rate-limiting glycosylation step of a human pancreatic α-amylase:maltopentose complex. Microsecond molecular dynamics simulations suggested that the distance of the catalytic Asp197 nucleophile to the anomeric carbon of the buried glucoside is responsible for most of the enzyme active site fluctuations and that both Asp197 and Asp300 interact the most with the buried glucoside unit. The buried glucoside binds either in a (4)C(1) chair or (2)S(O) skew conformations, both of which can change to TS-like conformations characteristic of retaining glucosidases. Starting from four distinct enzyme:substrate complexes, umbrella sampling quantum mechanics/molecular mechanics simulations (converged within less than 1 kcal·mol(–1) within a total simulation time of 1.6 ns) indicated that the reaction occurrs with a Gibbs barrier of 13.9 kcal·mol (–1), in one asynchronous concerted step encompassing an acid–base reaction with Glu233 followed by a loose S(N)2-like nucleophilic substitution by the Asp197. The transition state is characterized by a (2)H(3) half-chair conformation of the buried glucoside that quickly changes to the E(3) envelope conformation preceding the attack of the anomeric carbon by the Asp197 nucleophile. Thermodynamic analysis of the reaction supported that a water molecule tightly hydrogen bonded to the glycosidic oxygen of the substrate at the reactant state (∼1.6 Å) forms a short hydrogen bond with Glu233 at the transition state (∼1.7 Å) and lowers the Gibbs barrier in over 5 kcal·mol(–1). The resulting Asp197-glycosyl was mostly found in the (4)C(1) conformation, although the more endergonic B(3,O) conformation was also observed. Altogether, the combination of short distances for the acid–base reaction with the Glu233 and for the nucleophilic attack by the Asp197 nucleophile and the availability of water within hydrogen bonding distance of the glycosidic oxygen provides a reliable criteria to identify reactive conformations of α-amylase complexes.