An Orbital Control of Substrate Reactivity on the Enzyme Xanthine Oxidase and the Substrate-Enzyme Binding Cofactor Reduction

Abstract

In humans, the xanthine oxidase enzyme plays an important role in physiological hydroxylation of hypoxanthine to xanthine, xanthine to. uric acid and it has been a target of drugs against disease such as gout and reperfusion injury. For this reason knowledge of detailed reaction mechanism by which the enzyme functions is desirable. In order to understand the reaction mechanism for the oxidation of lumazine by xanthine oxidase particularly the spectral behaviors associated with the formation of violapterin ( oxidation product of lumazine) and the spectral behaviors of the reduced enzyme bound to products, EREo-products such as; EREo-uric acid, EREo-alloxanthine, EREo-violapterin, EREo-formate, and ERE0-C02, computational approach is applied. In addition, electronic structure calculation is carried out to understand why the spectral band ( centered at 650 nm) is a unique signature of EREo-violapterin intermediate. Besides this, molecular orbital analysis for the constituent fragments are calculated to understand the unique spectral band at 650 nm. The spectral band at 650 nm has been assigned as metal to ligand charge transfer transition band. The metal to ligand charge transfer is due to the equilibrium between structure (d) i.e ((EREo)-[Mo 1 \-SMoH)-Oeq-CviolapterinD and structure (e) i.e v ((EREo)-[Mo (-SMoH)-Oeq-CviolapterinD· The charge transfer is assigned from Mo dxy Vl orbital to empty rr* orbital of violapterin. The data from electronic structure calculation was compared with experimental data in order to develop a plausible reaction mechanism for the product release stage. As a result, a reaction mechanism for the product release stage of the oxidation of lumazine by xanthine oxidase has been proposed. Our reaction mechanism for the product release stage involved a step wise mechanism. Accordingly, the formation of structure (d) i.e product bound or ((ERrn)-[Mo1v(-SM0H)-Ocq-CviolaptcrinD is energetically favorable by -50.36 K.cal/mol compared to the structures proposed to be formed during the product release stage. Structure (d) is proposed to undergo one electron oxidation to form structure (e). The formation of structure (e) is favorable by -14.67 K.cal/mol. Structure (e) is proposed to undergo one electron oxidation and loss of a proton simultaneously to give the product, violapterin and the enzyme as the ligand water enters. The energy for the one electron oxidation and loss of a proton is -559.52 K. cal / rnol. The spectral behaviors of EREo-product intermediates such as ER1: n-uric acid , ERuY alloxanthine, ER1:o-violapterin, ERED-formate, and ERED-C02 has been determined. It is found that the difference spectral behavior between the ERED-products such as: ERrn-uric acid, ERrn-alloxanthine, ERED-violapterin, ERrn-formate, and ERrn-C02 is due to difference in the functional group of their substrates and environment of their interaction site