The structure of the Asn102 mutant of trypsin was determined in order to distinguish whether the reduced activity of the mutant at neutral pH results from an altered active site conformation or from an inability to stabilize a positive charge on the active site histidine. The active site structure of the Asn102 mutant of trypsin is identical to the native enzyme with respect to the specificity pocket, the oxyanion hole, and the orientation of the nucleophilic serine. The observed decrease in rate results from the loss of nucleophilicity of the active site serine. This decreased nucleophilicity may result from stabilization of a His57 tautomer that is unable to accept the serine hydroxyl proton.
Subtilisin from B. am:z;loliguefaciens has been used as a model system to prote1n engineer altered physicochemical characteristics by site-directed mutagenesis. This method has been successful in altering the enzyme's stability, specificity and activity. At several sites that had been identified as being important to enzyme function, the naturally occurring residue was substituted by all nineteen other amino acid types resulting in a wide variety of physicochemical changes. We have chosen a subset of site-directed mutants to study crystallographically aimed at developing relationships between observed structural changes and altered properties.
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