Copper inhibition of glutathione reductase and its reversal with gold thiolates, thiol, and disulfide compounds

Abstract

Yeast glutathione reductase was inhibited 50% by 1.0 μ m cupric sulfate. Pretreatment of the enzyme with reduced nicotinamide dinucleotide phosphate was required to obtain the inhibition, which indicated a requirement for reduced protein sulfhydryl groups. It was suggested that copper was complexed with vicinal protein sulfhydryl groups as previously described for attachment of arsenite to glutathione reductase; both copper and sodium arsenite promoted the diaphorase activity of the enzyme. A number of thiol compounds reversed the inhibition; the most effective was d-penicillamine, which gave 50% reversal at 60 μ m. Gold sodium thiomalate and gold thioglucose as well as two disulfide compounds [5,5′-dithiobis-(2-nitrobenzoic acid)] and [6,6′-dithiobis-(nicotinic acid)] also restored, in part, the activity of copper-inhibited enzyme. When the last two compounds were included in assay mixtures, the reaction product was not glutathione but was the mixed disulfide of glutathione with the corresponding thiol compound. In contrast to the mechanism of action of the thiol compounds, which involves complexing or reduction of the copper, evidence was presented that suggests that gold thiolates and disulfide compounds act as surrogate substrates for the inhibited enzyme, and in this way displace copper from it. That gold and disulfide compounds and d-penicillamine restore, in part, activity to copper-inhibited glutathione reductase was discussed in relationship to use of these compounds in treatment of rheumatoid arthritis.