TAUTOMERIZATION OF THE SUBSTRATE HETEROCYCLE IN THE COURSE OF THE REACTION OF XANTHINE-OXIDASE

Tautomerism in several forms of pteridines and purines relevant to the reaction mechanism of the molybdenum hydroxylase xanthine oxidase has been studied using ab initio Density Functional theory calculations i n vacuo and the Kirkwood/Onsager reaction field in solvent with high d ielectric constant. The results indicate that during the oxidative hyd roxylation catalyzed by the mononuclear molybdenum center of xanthine oxidase both classes of substrates undergo a proton shift from the pyr imidine subnucleus to the imidazole (for purines) or pyrazine (for pte ridines) subnucleus. This shift has three principal effects: (i) compe nsation of the negative charge accumulating on the imidazole subnucleu s of substrate; () partial transfer of negative charge from the imidaz ole ring (pyrazine, in pteridines) to the pyrimidine dione; and (iii) lowering of the energy of intermediates formed in the course of the re action. The results of our calculations are supported by the UV-Vis sp ectra of pteridinedione N-anion as well as the observed kinetic isotop e effects and pH dependence of the apparent rate of the product format ion and decay observed during oxidative hydroxylation of pteridine and purine substrates catalyzed by xanthine oxidase. The present results provide an explanation for the otherwise anomalous observation that me thylation of N3 in xanthine, a site remote from the hydroxylation chem istry, results in nearly complete loss of reactivity. (C) 1997 Elsevie r Science S.A.