Rationalization of the enantioselectivity of subtilisin in DMF

Herein we examine the origin of enantioselectivity in the serine
protease subtilisin in DMF through the use of molecular dynamics (MD)
and free energy perturbation (FEP) simulations. In particular, we are
interested in the resolution of a racemic mixture of sec-phenethyl
alcohol by a transesterification reaction with the acylating agent vinyl
acetate, catalyzed by subtilisin in anhydrous dimethylformamide (DMF).
To study the enantioselectivity in this case, we examined the
tetrahedral intermediate as a model of the enzyme transition state (as
has been done in the past). A critical aspect of this study was the
determination of the charge distribution of the two (R and S)
tetrahedral intermediates through the use of a combined quantum
mechanical/molecular mechanical electrostatic potential fitting
methodology. In designing 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 simulations we find that both
steric and electrostatic complimentarity plays a role in the
enantioselectivity of this enzyme-catalyzed reaction. Through the use of
FEP simulations we obtained a free energy difference that is in good
accord with experiment, which quantitatively supports the accuracy of
our model and suggests that all-atom molecular simulations are capable
of providing accurate qualitative and quantitative insights into enzyme
catalysis in nonaqueous environments.