Enzymatic enhancement of the catalytic rate of sulfhydryl oxidase.
The rate of oxidation of glutathione by solubilized sulfhydryl oxidase was significantly enhanced in the presence of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). This enhancement was proportional to the amount of active peroxidase in the assay, but could not be attributed solely to the oxidation of glutathione catalyzed by the peroxidase. A change in the Soret region of the horseradish peroxidase spectrum was observed when both glutathione and peroxidase were present. Moreover, addition of glutathione to a sulfhydryl oxidase/horseradish peroxidase mixture resulted in a rapid shift of the absorbance maximum from 403 nm to 417 nm. This shift indicates the oxidation of horseradish peroxidase. Spectra for three isozyme preparations of horseradish peroxidase, two acidic and one basic, all underwent this red-shift in the presence of sulfhydryl oxidase and glutathione. Cysteine and N-acetylcysteine could replace glutathione. Addition of catalase had no effect on the oxidation of peroxidase, indicating that the peroxide involved in the reaction was not derived from that released into the bulk solution by sulfhydryl oxidase-catalyzed thiol oxidation. Further evidence for a direct transfer of the hydrogen peroxide moiety was obtained by addition of glutaraldehyde to a sulfhydryl oxidase/horseradish peroxidase/N-acetylcysteine mixture. Size exclusion chromatography revealed the formation of a high-molecular-weight species with peroxidase activity, which was completely resolved from native horseradish peroxidase. Formation of this species was absolutely dependent on the presence of both the cysteine-containing substrate and sulfhydryl oxidase. The observed enhancement of sulfhydryl oxidase catalytic activity by the addition of horseradish peroxidase supports a bi uni ping-pong mechanism proposed previously for sulfhydryl oxidase.
D000111:Acetylcysteine / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D005976:Glutaral; D005978:Glutathione / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism / Q000494:pharmacology*; D007526:Isoelectric Point; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008892:Milk / Q000201:enzymology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010544:Peroxidases / Q000494:pharmacology*; D013053:Spectrophotometry; D050862:Oxidoreductases Acting on Sulfur Group Donors
D000111:Acetylcysteine; D003432:Cross-Linking Reagents; D005976:Glutaral; D005978:Glutathione; D006735:Horseradish Peroxidase; D007527:Isoenzymes; D010088:Oxidoreductases; D010544:Peroxidases; D050862:Oxidoreductases Acting on Sulfur Group Donors