A Buried Water Molecule Influences Reactivity in α‑Amylase on a Subnanosecond Time Scale

The subset of catalytically competent conformations can be significantly small in comparison with the full conformational landscape of enzyme–substrate complexes. In some enzymes, the probability of finding a reactive conformation can account for up to 4 kcal/mol of activation barrier, even when the substrate remains tightly bound. In this study, we sampled conformations of human pancreatic α-amylase with bound substrate in a molecular dynamics (MD) simulation of over 100 ns and calculated energy profiles along the reaction coordinate. We found that reactive states require a hydrogen bond between a buried water molecule and E233, which is the general acid in the glycolysis mechanism. The effect of this single, nonreactive, intermolecular interaction is as important as the correct positioning and orientation of the reacting residues to achieve a competent energy barrier. This hydrogen bond increases the acidity of E233, facilitating proton transfer to the glycosidic oxygen. In the MD simulation, this required hydrogen bond was observed in more than half of the microstates, indicating that human pancreatic α-amylase is efficient at maintaining this important interaction in the reactant state. Furthermore, this hydrogen bond formed and vanished on a subnanosecond time scale. Interactions between the reacting groups also change on this time scale. All of these changes led to instantaneous activation energy oscillations from 9.3 to 28.3 kcal/mol on a much smaller time scale in comparison to the turnover rate. These results are in agreement with the observed kinetics being determined by a few transient conformations that require low energy barriers.