Peroxide dependence of the semisynthetic enzyme selenosubtilisin.
Selenosubtilisin, a semisynthetic enzyme produced by chemical modification of subtilisin's catalytic serine, mimics the antioxidant enzyme glutathione 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 semisynthetic enzyme, whereas the dialkyl compound tert-butyl peroxide is not. Kinetic investigations reveal that kmax is dependent upon the nature of the hydroperoxide, indicating that peroxide-mediated oxidation of the enzymic selenolate is at least partially rate-limiting. Experiments with the radical trap 2,6-di-tert-butyl-4-methylphenol suggest that, while the nonenzymic reaction between tert-butyl hydroperoxide and thiol involves free radicals, the same reaction catalyzed by selenosubtilisin does not. The studies described here support the enzyme's proposed ping-pong mechanism and are consistent with previous mechanistic observations.
Bell
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IM
Department of Chemistry, Scripps Research Institute, La Jolla, California 92037.
Hilvert
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D002384:Catalysis; D007700:Kinetics; D010545:Peroxides / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism*
D010545:Peroxides; D013381:Subtilisins; C080590:selenosubtilisin