Path of Electron Transfer on Reaction of Xanthine Oxidase with 6-mercaptopurine and hypoxanthine

Abstract

Enzymes often rely on the coupling of electrons and protons to affect primary metabolic steps involving charge transport and catalysis. The present theoretical study is intend to explore the path of electron transfer from substrate to active site and to provide a plausible route of electron transfer in the enzymatic catalysis from 6-MP or hypoxanthine to active site. Density functional theory (DFT)/B3LYP method were used to probe the path of electron or proton transfer mechanism from Mullikan charge. The Mullikan atomic charge of Mo decreased from 0.624793 to 0.398834, C_RH bound equatorial oxygen of active site is increased by 0.16132, H_RH decreased by 0.146701 that is around two fold increase in electron density this may be due to removal of electron as hydride toward sulfido terminal. Terminal sulfido (S_Mo-H_RH) decreased to -0.0107, O_eq of active site decreased to -0.43539 when it bound to C2 of substrate. The electronegativity of O_eq more than oxo group by 0.05154 hence O_eq is a better nucleophile. Therefore the catalytically labile site should be the metal-coordinated hydroxide (O_eq) rather than the apical oxo group (Mo=O). Sulfido terminal (S_Mo) decreased in electronegativity due to delocalization of electron density to active site Mo that facilitates the transfer of electron up on the attack of hydride from hydrogen bond substrate. Electron densities on S_front and S_back increased in (-0.2233 & -0.20266) respectively which may facilitate the movement of equatorial hydroxide towards C2 of substrates (C_RH). Charge distribution on C2 is (0.143402 and 0.139523) and on C8 is (0.140514 and 0.133494) respectively for 6-MP and hypoxanthine. This implies in both cases C2 shows electron deficiency and hence it is more electrophilic relative to C8. Charge density of H_RH bound C2 is (0.171649 and 0.15786) and H_RH bound C8 is (0.08821 and 0.080245) respectively for 6-MP and hypoxanthine hence hydrogen bound C2 is labile for hydride transfer due to high electronegative nature which indicate C2 position is more susceptible to nucleophilic attack by hydroxide of active site. Therefore it can be generalized that oxidation of 6-mercaptopurine or hypoxanthine by XO proceed through abstraction of proton by Glu₁₂₂₆ from equatorial hydroxide of active site followed by nucleophilic attach on C2 of substrate and hydride was transferred through concomitant release of oxidized substrate.

Key words:hypoxanthine, 6-mercaptopurine, Density functional theory, nucleophile