The inhibition of xanthine oxidase by 8-bromoxanthine.
The interaction of xanthine oxidase with the substrate analog 8-bromoxanthine has been examined in an effort to determine the nature of interaction of purines with the active site of the enzyme. It is found that 8-bromoxanthine is an inhibitor of xanthine oxidase with a Ki of approximately 400 microM; inhibition is uncompetitive with respect to xanthine and noncompetitive with respect to molecular oxygen. While 8-bromoxanthine has only a slight effect on the reaction of reduced enzyme with oxygen, it dramatically slows the rate of enzyme reduction by xanthine, suggesting that inhibition does involve the interaction of 8-bromoxanthine with the molybdenum center of the enzyme. KD determinations for binding of 8-bromoxanthine to oxidized and reduced xanthine oxidase indicate that the inhibitor binds preferentially to the fully reduced form of the molybdenum center (MoIV), with dissociation constants of 1.5 mM and 18 microM for oxidized and reduced enzyme, respectively. This preferential binding to the reduced form of the enzyme is manifested in a significant increase in the oxidation-reduction potentials of the molybdenum center as determined by potentiometric titrations with 8-bromoxanthine complexed with xanthine oxidase. The shape of the Mov EPR signal observed in the course of these titrations as well as a comparison with results of reductive titrations and KD determinations with uric acid and xanthine indicate that 8-bromoxanthine interacts with the molybdenum center of xanthine oxidase in a way that is typical of purine substrates and products, despite the presence of the bulky Br group. The inhibitor thus has a potential as a probe of enzyme-substrate interactions, particularly using the technique of x-ray absorption spectroscopy.
D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D007700:Kinetics; D008892:Milk / Q000201:enzymology; D010084:Oxidation-Reduction; D010100:Oxygen; D011199:Potentiometry; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors*; D014970:Xanthines / Q000494:pharmacology*
D014970:Xanthines; C040350:8-bromoxanthine; D014969:Xanthine Oxidase; D010100:Oxygen