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Reference type: Journal
Authors: Rich JO, Dordick JS
Article Title: Controlling subtilisin activity and selectivity in organic media by imprinting with nucleophilic substrates.
Publication date: 1997
Journal: Journal of the American Chemical Society
Volume: 119
Issue: (14)
Page numbers: 3245-3252.
DOI: 10.1021/ja9637715

Abstract: The activity and substrate specificity of subtilisin-catalyzed acylation of nucleosides in organic solvents can be controlled by lyophilizing the enzyme from an aqueous solution containing the substrate. This ''molecular imprinting'' technique was examined using thymidine as a model nucleoside, and the resulting subtilisin preparation was up to 50-fold more reactive toward thymidine acylation in nearly anhydrous tetrahydrofuran than subtilisin lyophilized from aqueous buffer in the absence of the nucleoside. Although several compounds lyophilized with subtilisin, including thymine and ribose, improved the rate of thymidine acylation, the thymidine- imprinted enzyme was the most efficient catalyst for this reaction. Furthermore, it was possible to alter the substrate selectivity of subtilisin by lyophilizing the enzyme in the presence of a different nucleophilic substrate. For example, imprinting made possible the discrimination between structurally different (i.e., sucrose versus thymidine) as well as structurally similar (i.e., thymidine versus deoxyadenosine) nucleophiles. Molecular modeling studies of the interaction of thymidine or the unrelated sucrose with subtilisin revealed that structural changes upon imprinting in the serine protease's catalytic triad may be responsible for the observed activation and selectivity changes. Further use of molecular dynamics indicated that structural changes in the catalytic triad occur during imprinting, and that these changes may be the major factor that contributes to imprinting-induced substrate selectivity. This contrasts with the previously held notion that imprinting influences mainly substrate binding


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