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| - Rationalization of the Enantioselectivity of Subtilisin in DMF
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| - Herein we examine the origin of enantioselectivity in the serine protease subtilisin in DMF throughthe use of molecular dynamics (MD) and free energy perturbation (FEP) simulations. In particular, we areinterested in the resolution of a racemic mixture of sec-phenethyl alcohol by a transesterification reaction withthe acylating agent vinyl acetate, catalyzed by subtilisin in anhydrous dimethylformamide (DMF). To studythe enantioselectivity in this case, we examined the tetrahedral intermediate as a model of the enzyme transitionstate (as has been done in the past). A critical aspect of this study was the determination of the charge distributionof the two (R and S) tetrahedral intermediates through the use of a combined quantum mechanical/molecularmechanical electrostatic potential fitting methodology. In designing the active site charge model, we foundthat the R and S tetrahedral intermediates have significantly different charge distributions due to the presenceof the stereodifferentiating environment presented by the enzyme. In contrast the charge distribution obtainedfor models of the tetrahedral intermediate in the gas phase have similar charge distributions. From MDsimulations we find that both steric and electrostatic complimentarity plays a role in the enantioselectivity ofthis enzyme-catalyzed reaction. Through the use of FEP simulations we obtained a free energy difference thatis in good accord with experiment, which quantitatively supports the accuracy of our model and suggests thatall-atom molecular simulations are capable of providing accurate qualitative and quantitative insights intoenzyme catalysis in nonaqueous environments.
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