The Solvent Dependence of Enzymatic Selectivity

The exacting selectivity of enzymatic catalysis is the most valuable characteristic of enzymes to the synthetic chemist. Ironically, this strict specificity also limits the generality of enzymatic synthesis, because enzymes that catalyze the reaction of interest with the desired selectivity are not always available. Nonaqueous enzymology, and the discovery that enzymatic selectivity can be changed by the reaction medium, thus greatly enhances the utility of enzyme-catalyzed syntheses. To further exploit this solvent effect, we seek herein to mechanistically explain the dependence of enzymatic selectivity on the solvent. The substrate specificity of the serine protease subtilisin Carlsberg in the transesterification reaction of N-Ac-L-Ser-OEt and N-Ac-L-Phe-OEt with propanol has been examined in 20 anhydrous solvents. The serine substrate is strongly favored in some solvents, while the phenylalanine substrate is greatly preferred in others. An equation has been derived on the basis of the thermodynamics of substrate desolvation, which correctly predicts the substrate specificity as a function of the solvent-to-water partition coefficients of the substrates and the substrate specificity of the enzyme-catalyzed hydrolysis of the esters in water. This model is herein demonstrated to be independent of the enzyme and the substrate, so long as the latter is removed from the solvent in the transition state. Experimentally measured solvent-to-water partition coefficients are nonideal for use in the prediction of the solvent dependence of enzymatic selectivity for several reasons. First, partition coefficients cannot be readily measured for water-miscible solvents. Second, the mutual solubility of aqueous and organic phases influences the measured partition coefficients. Third, the effects of additives to the reaction medium, such as a second substrate, cannot be included. These problems have been overcome by calculating the partition coefficients from the substrate activity coefficients using UNIFAC. For the case of substrate specificity, the differential free energy of desolvation for two substrates is primarily driven by chemical differences in the substrates. In cases of stereoselectivity (e.g. enantioselectivity, prochiral selectivity, and regioselectivity), however , chemically identical substrates lead to the formation of multiple products. For such identical substrates, desolvation energy differences arise from differences in the solvation of the substrates in the transition states which lead to the various products. Our model has been expanded to account for partial transition state desolvation, which is assessed using molecular modeling based on the crystal structure of the enzyme. Using this methodology , we are able to quantitatively predict the solvent dependence of the enantioselectivity of cross-linked chymotrypsin …