Rationalization of the enantioselectivity of subtilisin in DMF

Herein we examine the origin of enantioselectivity in the serine protease s ubtilisin in DMF through the use of molecular dynamics (MD) and free energy perturbation (FEP) simulations. In particular, we are interested in the re solution of a racemic mixture of sec-phenethyl alcohol by a transesterifica tion reaction with the acylating agent vinyl acetate, catalyzed by subtilis in in anhydrous dimethylformamide (DMF). To study the enantioselectivity in this case, we examined the tetrahedral intermediate as a model of the enzy me transition state (as has been done in the past). A critical aspect of th is study was the determination of the charge distribution of the two (R and S) tetrahedral intermediates through the use of a combined quantum mechani cal/molecular mechanical electrostatic potential fitting methodology. In de signing the active site charge model, we found that the R and S tetrahedral intermediates have significantly different charge distributions due to the presence of the stereodifferentiating environment presented by the enzyme. In contrast the charge distribution obtained for models of the tetrahedral intermediate in the gas phase have similar charge distributions, From MD s imulations we find that both steric and electrostatic complimentarity plays a role in the enantioselectivity of this enzyme-catalyzed reaction. Throug h the use of FEP simulations we obtained a free energy difference that is i n good accord with experiment, which quantitatively supports the accuracy o f our model and suggests that all-atom molecular simulations are capable of providing accurate qualitative and quantitative insights into enzyme catal ysis in nonaqueous environments.