Reaction of 5-ethynyluracil with rat liver xanthine oxidase.
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.
Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
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D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003486:Cyanides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008962:Models, Theoretical; D011485:Protein Binding; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D014498:Uracil / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification
D002250:Carbon Radioisotopes; D003486:Cyanides; C090390:5-(difluorosulfomethyl)uracil; C073482:eniluracil; D014498:Uracil; D000493:Allopurinol; D014969:Xanthine Oxidase