Kinetic mechanism and specificity of bovine milk sulphydryl oxidase.
Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.
D000111:Acetylcysteine / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003538:Cystamine / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008892:Milk / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism*; D013379:Substrate Specificity; D050862:Oxidoreductases Acting on Sulfur Group Donors
D000111:Acetylcysteine; D003538:Cystamine; D003545:Cysteine; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors