title: Reasoning enantioselectivity and kinetics of seleno-subtilisin from the subtilisin template.
abstract: The active-site serine (Ser221) of subtilisin Carlsberg(from Bacillus 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 and Km values 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.
authors.affiliation: Food Chemistry, University of Würzburg, Am Hubland, Würzburg, 97074, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Häring
authors.forename: B
authors.initials: B
authors.lastname: Hubert
authors.forename: E
authors.initials: E
authors.lastname: Schüler
authors.forename: P
authors.initials: P
authors.lastname: Schreier
mesh_terms: D001407:Bacillus / Q000201:enzymology; D007700:Kinetics; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000737:chemistry*; D013698:Templates, Genetic
chemical_list: D013381:Subtilisins; C080590:selenosubtilisin