Enzymes in organic synthesis: Use of subtilisin and a highly stable mutant derived from multiple site-specific mutations

Article abstract of DOI:10.1021/ja00159a006

A subtilisin mutant (subtilisin 8350) derived from subtilisin BPN' via six-specific mutations (Met50Phe, Gly169Ala, Asn76Asp, Gln206Cys, Tyr217Lys, and Asn218Ser) was found to be 100 times more stable than the wild-type enzyme in aqueous solution at room temperature and 50 times more stable than the wild type in anhydrous dimethylformamide. Kinetic studies using ester, thio ester, and amide substrates, and the transition-state analogue inhibitor Boc-Ala-Val-Phe-CF₃, indicate the both the wild-type and the mutant enzymes have very similar specificities and catalytic properties. The inhibition constant (K(i)) = 5.0 μM) for the wild-type enzyme is approximately 5 times that of the mutant enzyme (K(i)) = 1.1 μM), suggesting that the mutant enzyme binds the reaction transition state more strongly than the wild-type enzyme. This result is consistent with the observed rate constants for the corresponding ester and amide substrates; i.e. the k(cat)/k(m) values for the mutant are larger than those for hhe wild-type enzyme. Application of the mutant enzyme and the wild-type enzyme to organic synthesis has been demonstrated in the regioselective acylation of nucleosides in anhydrous dimethylformamide (with 65-100% regioselectivity at the 5'-position), in the enantioselective hydrolysis of N-protected and unprotected common and uncommon amino acid esters in water (with 85-98% enantioselectivity for the L-isomer), and in the synthesis of di- and oligopeptides via aminolysis of N-protected amino acid and peptide esters. The enzymatic peptide synthesis was carried out under high concentrations of DMF (~50%) to improve substrate solubility and to minimize enzymatic peptide cleavage. Low enantioselectivity was observed in the enzymatic transformation of non-amino acid alcohols and acids.