LAUSR

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 requi...