PEROXIDE DEPENDENCE OF THE SEMISYNTHETIC ENZYME SELENOSUBTILISIN

Selenosubtilisin, a semisynthetic enzyme produced by chemical modifica tion of subtilisin's catalytic serine, mimics the antioxidant enzyme g lutathione peroxidase, catalyzing the reduction of hydroperoxides by 3 -carboxy-4-nitrobenzenethiol. In analogy with the natural peroxidase, a variety of hydroperoxides are accepted as substrates for the semisyn thetic enzyme, whereas the dialkyl compound tert-butyl peroxide is not . Kinetic investigations reveal that k(max) is dependent upon the natu re of the hydroperoxide, indicating that peroxide-mediated oxidation o f the enzymic selenolate is at least partially rate-limiting. Experime nts with the radical trap 2,6-di-tert-butyl-4-methylphenol suggest tha t, while the nonenzymic reaction between tert-butyl hydroperoxide and thiol involves free radicals, the same reaction catalyzed by selenosub tilisin does not. The studies described here support the enzyme's prop osed ping-pong mechanism and are consistent with previous mechanistic observations.