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.