J. Biol. Chem., Vol. 269, Issue 45, 27932-27940, 11, 1994
Reaction of 5-ethynyluracil with rat liver xanthine oxidase
DJ Porter
Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
5-Ethynyluracil is a time-dependent and tight binding inhibitor of xanthine
oxidase. The maximal value of the first-order rate constant for onset of
inhibition is 0.01 s-1, and the concentration of 5- ethynyluracil which
gives one-half of this value is 190 microM. Because the t1/2 for formation
of active enzyme from inhibited enzyme is greater than 30 h in the absence
of NADH, inhibition of xanthine oxidase by 5-ethynyluracil is functionally
irreversible. One equivalent of 5-[2-14C]ethynyluracil/equivalent of active
enzyme is required for complete inhibition. Allopurinol (100 microM), a
potent inhibitor of xanthine oxidase, and cyanide (5 mM), an inactivator of
the enzyme, do not abolish the binding of 5-[2-14C]ethynyluracil to the
enzyme. Because radiolabel is released from 5-[2-14C]ethynyluracil-treated
enzyme by treatment with 6 M guanidine HCl, a stable covalent bond is not
formed between the inhibitor and the enzyme. However, the radiolabel
released from inhibited enzyme is not 5-ethynyluracil. Moreover, NADH
restores catalytic activity to the inhibited enzyme and displaces the
radiolabel as 5-acetyluracil. Thermal denaturation of 5-
ethynyluracil-inhibited xanthine oxidase results in the release of
approximately equal amounts of 5-acetyluracil and a more hydrophilic
product. Consequently, the 5-ethynyluracil-xanthine oxidase complex yields
different degradation products of 5-ethynyluracil under different
denaturation conditions. Seven uracil analogues with 5- substituents were
tested as time-dependent inhibitors of xanthine oxidase. 5-Ethynyluracil is
the only uracil analogue that potently inhibited xanthine oxidase. The
reactivity of these uracil derivatives with sulfite was also determined.
5-Ethynyluracil is many fold more susceptible to nonenzymatic nucleophilic
addition of sulfite than are the other analogues. Thus, the potency of
these uracil analogues as inhibitors of xanthine oxidase is related to the
nonenzymatic reactivity of the analogues with sulfite.
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