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J. Biol. Chem., Vol. 259, Issue 3, 1570-1576, Feb, 1984
R Hille and RC Stewart
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.
The inhibition of xanthine oxidase by 8-bromoxanthine
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