On catalytic preorganization in oxyanion holes: highlighting the problems with the gas-phase modeling of oxyanion holes and illustrating the need for complete enzyme models.

Oxyanion holes play a major role in catalyzing enzymatic reactions, yet the corresponding energetics is frequently misunderstood. The main problem may be associated with the nontrivial nature of the electrostatic preorganization effect, without following the relevant formulation. That is, although the energetics of oxyanion holes have been fully quantified in early studies (which include both the enzymatic and reference solution reactions), the findings of these studies are sometimes overlooked, and, in some cases, it is assumed that gas-phase calculations with a fixed model of an oxyanion hole are sufficient for assessing the corresponding effect in the protein. Herein, we present a systematic analysis of this issue, clarifying the problems associated with modeling oxyanions by means of two fixed water molecules (or related constructs). We then re-emphasize the point that the effect of the oxyanion hole is mainly due to the fact that the relevant dipoles are already set in an orientation that stabilizes the TS charges, whereas the corresponding dipoles in solution are randomly oriented, resulting in the need to pay a very large reorganization energy. Simply calculating interaction energies with relatively fixed species cannot capture this crucial point, and considering it may help in advancing rational enzyme design.