Nucleophile specificity in alpha-chymotrypsin- and subtilisin-(Bacillus subtilis strain 72) catalyzed reactions

Nucleophilic properties of amino-acid amides were studied systematically in acyl-transfer reactions catalyzed by alpha-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) using Mal-L-Ala-L-Ala-L-PheOMe as the acyl-group donor. In alpha-chymotrypsin-catalyzed reactions, the nucleophile reactivities increase in the following order: D-AlaNH2 < GlyNH2 < L-AlaNH2 < L-SerNH2 < L-ThrNH2 < L-HisNH2 < L-ValNH2 < L-LeuNH2 < L-TrpNH2 < L-MetNH2 < L-NvaNH2 < L-PheNH2 < L-IleNH2 < L-TyrNH2 < L-ArgNH2. In reactions catalyzed by subtilisin 72, the reactivities increase as follows: L-LeuNH2 < L-IleNH2 < L-ThrNH2 < L-ArgNH2 < L-TrpNH2 < L-NvaNH2 < L-ValNH2 < L-MetNH2 < L-AlaNH2 < L-SerNH2 < D-AlaNH2 < GlyNH2. In alpha-chymotrypsin-catalyzed reactions, hydrophobic interactions are entirely responsible for the differences between the reactivity of the nucleophiles for amides of all the amino-acids tested with the exception of D-AlaNH2, L-ArgNH2 and L-TyrNH2. In reactions catalyzed by subtilisin 72, amino-acid side-chain characteristics and the nucleophile reactivities are not related. The data obtained show the low selectivity of the S1' subsite of subtilisin 72 and high specificity of this subsite in alpha-chymotrypsin.