Tautomerization of the substrate heterocycle in the course of the reaction of xanthine oxidase

Abstract

Tautomerism in several forms of pteridines and purines relevant to the reaction of the molybdenum hydroxylase xanthine oxidase has been studied using ab inito Density Functional theory calculations in vacuo and the Kikwood/Onsager reaction field in solvent with high dielectric constant. The results indicate that during the oxidative hydroxylation catalyzed by the mononuclear molybdenum center of xanthine oxidase both classes of substrates undergo a proton shift from the pyrimidine subnucleus to the imidazole (for purines) or pyrazine (for pteridines) subnucleus. This shift has three principal effects; (i) compensation of the negative charge accumulating on the imidazole subnucleus of substrate; (ii) partial transfer of negative from the imidazole (pyrizine, in pteridines) to the pyrimidine dione; and (iii) lowering of the energy of intermediates formed in the course of the reaction. The results of our calculations are supported by the UV-Vis spectra of pteridinedione N-anion as well as the observed kinetic isotope effects and pH dependence of the apparent rate of the product formation and decay observed during oxidative hydroxylation of pteridine and purine substrates catalyzed by xanthine oxidase. The present results provide an explanation of the otherwise anomalous observation that methylation of N3 in xanthine, a site remote from the hydroxylation chemistry, results in nearly complete loss reactivity.