J. Biol. Chem., Vol. 265, Issue 23, 13540-13546, Aug, 1990
Xanthine oxidase-catalyzed reductive debromination of 6-(bromomethyl)- 9H-purine with concomitant covalent modification of the FAD prosthetic group
DJ Porter
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Bovine milk xanthine oxidase was potently inhibited by 6-(bromomethyl)-
9H-purine in a time-dependent process with O2 as the electron acceptor. If
the enzyme were assayed with phenazene ethosulfate as an electron acceptor,
6-(bromomethyl)-9H-purine was not an inhibitor. The rate of formation of
inhibited enzyme increased with increasing concentrations of
6-(halomethyl)-9H-purine, decreased with increasing concentrations of O2,
and increased in the presence of xanthine. The inhibited enzyme regained
activity nonactinically at pH 7 with a t1/2 of 31 h. The optical difference
spectrum between native enzyme and inhibited enzyme suggested that the
enzyme-bound FAD was modified. This conclusion was confirmed by
demonstrating that activity was restored to the inhibited enzyme if the
enzyme-bound flavin was removed by treatment with CaCl2 and the resulting
apoenzyme was reconstituted with FAD. Aerobically, 6-
(bromomethyl)-9H-purine was oxidized by the enzyme to a species having a UV
spectrum consistent with hydroxylation of the purine ring to form a urate
analogue. Anaerobically, the enzyme reduced 6-(bromomethyl)-9H- purine to
6-methylpurine with 1 mol of enzyme being completely inhibited after
reduction of 23 mol of 6-(bromomethyl)-9H-purine. Thus,
6-(bromomethyl)-9H-purine was not only oxidized by xanthine oxidase but was
also reduced by the enzyme in a reaction that partitioned between formation
of 6-methylpurine and inhibition of the enzyme by modification of the
enzyme-bound flavin. Similar results were found when
6-(chloromethyl)-9H-purine was the inhibitor.
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