Epoxide hydrolysis as a model system for understanding flux through a branched reaction scheme.

The epoxide hydrolase StEH1 catalyzes the hydrolysis of -methylstyrene oxide to 1-phenyl-propane-1,2-diol. The (,)-epoxide is exclusively transformed into the (1,2)-diol, while hydrolysis of the (,)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure-activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (,)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (,)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the -(,) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.