Enzymatic Properties

Cysteine, all peptides containing cysteine, and proteins with free sulfhydryl groups are all excellent substrates for mammalian membrane sulfhydryl oxidase (Table 1). Unlike the soluble flavoprotein microbial enzymes, this enzyme is not active with other simple thiol compounds such as DTT. Activity is completely lost upon treatment with EDTA (1) or reagents that react with sulfhydryl groups such as iodoacetate (24). Substrate inhibition is observed at concentrations > 5-10 Km (1, 5, 28). With GSH as the substrate, a pH optimum of 6.8-7.0 and a temperature optimum of 35°C have been established (1). The stoichiometry of sulfhydryl oxi-dase-catalyzed oxidation of GSH has been confirmed by quantitation of the disappearance of GSH and O2 and the appearance of H2O2 and GSSG (1, 5, 25). A number of observations, including kinetic studies (5), have indicated that catalysis follows a Bi Uni Uni Uni Ping-Pong mechanism as shown by the Cleland diagram below.

In addition to analysis of initial rate data (5), a substituted enzyme mechanism is supported by the specific covalent interaction with cysteinylsuccinami-dopropyl glass and its release by reducing agents such as GSH or DTT (25). Binding of GSH prior to release of H2O2 is supported by the requirement of a sulfhydryl oxidase substrate for the observed oxidation of horseradish peroxidase (HRP) in the presence of the enzyme (29). Enhancement of sulfhydryl oxidase-cat-alyzed oxidation of GSH in the presence of HRP is thought to occur by direct transfer of peroxide from sulfhydryl oxidase to HRP, thus speeding up the rate-limiting dissociation of peroxide from the enzyme (29).

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