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

Nucleophilic properties of amino-acids were studied systematically in acyl-transfer reactions catalyzed by α-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) using Mal-l-Ala-l-Ala-l-PheOMe as the acyl-group donor. In α-chymotrypsin-catalyzed reactions, the nucleophile reactivities increase in the following order: d-AlaNH₂ < GlyNH₂ < l-AlaNH₂ < l-ScrNH₂ < l-ThrNH₂ < l-HisNH₂ < l-ValNH₂ < l-LcuNH₂ < 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-MctNH₂ < l-AlaNH₂ < l-ScrNH₂ < d-AlaNH₂ < GlyNH₂. In α-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 the 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 specify of this subsite in α-chymotrypsin.