Studies on the mechanism of inhibition of xanthine oxidase by 5-diazoimidazole-4-carboxamide and related thioazoimidazole carboxamides

Abstract

The mechanism of inhibition of milk xanthine oxidase by 5-diazoimidazole-4-carboxamide (diazo-ICA) and by five related thioazoimidazole carboxamides (thioazo-ICAs) was studied. The extent of inhibition of xanthine oxidase by diazo-ICA and thioazo-ICAs decreased greatly when these compounds were preincubated in a buffer before the addition of substrate and enzyme. In 0.1 M Tris-HCl buffer, pH 7.5, thioazo-ICAs were converted to 2-azahypoxanthine, a cyclized product of diazo-ICA, which inhibits xanthine oxidase slightly. The inhibition of xanthine oxidase by thioazo-ICAs is probably due to this diazo-ICA. With xanthine as a variable substrate, diazo-ICA caused uncompetitive, irreversible inhibition. The inhibition of xanthine oxidase by diazo-ICA was reduced by simultaneous addition of a sulfhydryl compound, such as cysteine, cysteamine or reduced glutathione, but not other amino acids. Diazo-ICA inactivated the enzyme more significantly and rapidly than other sulfhydryl reagents. The inhibitory activity of diazo-ICA was potentiated strongly by Fe 2+, Mn 2+, Co 2+ and Cu 2+, and slightly by Mo 5+. Treatment of milk xanthine oxidase with diazo-ICA changed the absorption spectrum of the enzyme.