Reasoning Enantioselectivity and Kinetics of Seleno-Subtilisin from the Subtilisin Template

Abstract

The active-site serine (Ser221) of subtilisin Carlsberg(fromBacillus licheniformis) and subtilisin BPN′ (fromBacillus amyloliquefaciens) was chemically converted into a selenocystein. Contrary to subtilisin's protease activity the semisynthetic seleno-subtilisin catalyzed the reduction of hydroperoxides. Enantioselectivity and kinetics of this reaction were studied by kinetic resolution of five racemic alkyl aryl hydroperoxides catalyzed by the seleno-subtilisin variants. Due to the identical tertiary structure of subtilisin and seleno-subtilisin, the enzymes have comparable substrate binding properties. Thus, a rational screening for suitable peroxidase substrates featuring structural characteristics of known subtilisin substrates was enabled. The enantioselective recognition of (S)-configured alkyl aryl hydroperoxides by seleno-subtilisin was comprehensible by subtilisin's preference for comparable (S)-alkyl aryl amines or alcohols. The analysis of chiral products by multidimensional gas chromatography revealed enantiomeric excesses up to 98%. Kinetics of seleno-subtilisin were rationalized on the basis of the established substrate–catalyst interactions of the subtilisin framework. The Carlsberg and BPN′ peroxidase variants revealed typical differences in turnover numbers (kcat) and Michaelis–Menten affinity constants (Km) already known from subtilisin variants. Turnover numbers of seleno-subtilisin BPN′ were lower andKmvalues were higher in comparison to Carlsberg variant. Substrate affinity of several substituted 1-arylethyl hydroperoxides to seleno-subtilisin was reasonable in comparison to corresponding aryl boronic acid inhibitors of subtilisin.