Abstract
We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.
Highlights
Incub t i me folic acid is 10-fold more effective than allopurinol andhas beenshown toinhibitthe activity of that tetrahydrofolicacid is 4-fold more effective than allopu- thymidylatesynthetase (16, 17), and morerecently, it was rinol as inhibitors for the oxidatioonf xanthine by mammal- determined a noncompetitive inhibitor of human and other ian xanthine oxidase
If the observed low level of enzymic activity in vitro reflects a comparatively low level of the enzymic activity in uiuo, it mighbt e assumed that cellular levels of the coenzymemay suffice to effectively
Summary
Hypoxanthine to uric acid and is believed to be subject to a large degree of metabolic regulation and control (1). Similar studieswith folic acid and itscoenzyme derivatives showed no detectable enzyme utilization of these compounds as substrates.Folic acid and its coenzyme derivatives, were determined potent competitive inhibitors of xanthine oxidase activity in vitro. Massey et al ( 7 )have elegantly determined themechanism of the inactivationof xanthine oxidase by allopurinol These authors haveshown that allopurinol is a substrate for the enzyme and that its oxidation product alloxanthine is strongly bound at theactive site of the enzyme. Re- In this studyw, e have demonstrated thatfolate compounds versibility of the enzymic inhibition by folic acid was and the folate analog amethopterin are potent inhibitors of demonstrated. Most potent inibitorswhich have been determined to datefor 2-Amino-4-hydroxypteridine wasa poor substrate for mammalian xanthine oxidase.
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