We analyze the role of solvation
for enzymatic catalysis in two
distinct, artificially designed Kemp Eliminases, KE07 and KE70, and
mutated variants that were optimized by laboratory directed evolution.
Using a spatially resolved analysis of hydration patterns, intermolecular
vibrations, and local solvent entropies, we identify distinct classes
of hydration water and follow their changes upon substrate binding
and transition state formation for the designed KE07 and KE70 enzymes
and their evolved variants. We observe that differences in hydration
of the enzymatic systems are concentrated in the active site and undergo
significant changes during substrate recruitment. For KE07, directed
evolution reduces variations in the hydration of the polar catalytic
center upon substrate binding, preserving strong protein–water
interactions, while the evolved enzyme variant of KE70 features a
more hydrophobic reaction center for which the expulsion of low-entropy
water molecules upon substrate binding is substantially enhanced.
While our analysis indicates a system-dependent role of solvation
for the substrate binding process, we identify more subtle changes
in solvation for the transition state formation, which are less affected
by directed evolution.