Sulfhydryl oxidase-catalyzed formation of disulfide bonds in reduced ribonuclease.
Sulfhydryl oxidase isolated from bovine skim milk membrane vesicles catalyzes de novo formation of disulfide bonds with the substrates cysteine, cysteine-containing peptides, and reduced proteins using molecular oxygen as the electron acceptor. Initial rates for sulfhydryl oxidase-catalyzed oxidation of reduced ribonuclease exhibited typical Michaelis-Menten kinetics at low substrate concentrations. Substrate inhibition of the oxidative activity was observed at ribonuclease concentrations greater than 40 microM, similar to that observed with reduced glutathione or other small thiol substrates. The inhibition was more pronounced when ribonuclease activity was used to monitor the rates, presumably due to concentration-dependent formation of nonnative disulfide bonds. Thus, a maximum in the rate of regain of ribonuclease activity was observed at a 40 microM concentration, while optimum recovery was observed at 30 microM. The Michaelis constant obtained with reduced ribonuclease is 17.4 microM which corresponds to a sulfhydryl concentration of 0.14 mM, a value that compares favorably with the best small thiol substrate, reduced glutathione. Disulfide-containing intermediates in the oxidation pathway, as determined by ion-exchange chromatography of alkylated reaction mixtures, appeared to be similar for air oxidation and enzyme-catalyzed oxidation of the protein. The pH optimum, tissue location, and kinetic characteristics of sulfhydryl oxidase are compatible with a suggested physiological function of direct catalysis of disulfide bond formation in secretory proteins or indirect participation through provision of oxidized glutathione for protein disulfide-isomerase-catalyzed thiol/disulfide interchange.
Janolino
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VG
Department of Food Science, North Carolina State University, Raleigh 27695-7624.
Swaisgood
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HE
D000818:Animals; D002852:Chromatography, Ion Exchange; D004220:Disulfides / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008892:Milk / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism* / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism / Q000494:pharmacology; D050862:Oxidoreductases Acting on Sulfur Group Donors
D004220:Disulfides; D005978:Glutathione; D010088:Oxidoreductases; D012259:Ribonuclease, Pancreatic; D013438:Sulfhydryl Compounds; D050862:Oxidoreductases Acting on Sulfur Group Donors