Stoichiometric Inhibition of Reduced Xanthine Oxidase by Hydroxypyrazolo[3,4-d]pyrimidines

The inhibition of reduced bovine milk xanthine oxidase by 4-hydroxypyrazolo[3,4-d]pyrimidine (4-HPP) and 4,6-dihydroxypyrazolo[3,4-d]pyrimidine (4,6-diHPP) is stoichiometric or titrating in type, i.e. the inhibition is virtually complete (g90%) at concentrations of inhibitor equimolar with that of the enzyme. The dissociation constant of the reduced enzyme-4,6-diHPP complex, determined by the method of Easson and Stedman (Proc. Roy. Soc. Ser. B Biol. Sci., 121, 142 (1936)), is 5.4 x 10-10 m. No decrease in the fractional inhibition is detectable on increasing the substrate concentration from 0.04 to 0.33 mm, or on 10-fold dilution of the enzyme-inhibitor complex. The inhibitory properties of 4-HPP are virtually identical with those of 4,6-diHPP; this identity in behavior is the result of the conversion of 4-HPP to 4,6-diHPP by the enzyme. The rate of development of 4,6-diHPP inhibition is markedly temperature-dependent, more than 2 hours being required to reach maximal inhibition at 2°, but less than 1 min at 40°. The time required to reach maximal inhibition is not decreased by increasing the 4,6-diHPP level 100-fold, from 28 mµm to 2.8 µm; the inhibition appears, therefore, to be caused, not by the enzyme-inhibitor complex initially formed, but to a subsequent internal rearrangement of the latter to a less readily dissociable form.