Mechanistic studies on xanthine oxidoreductase enzymes

In humans, the xanthine oxidoreductase enzymes are known to catalyze the final two steps of purine metabolism by converting hypoxanthine successively to xanthine and uric acid. Xanthine oxidoreductase also metabolizes a wide variety of drugs in vivo, and activates a number of antiviral prodrugs of clinical importance. Therefore, understanding the reaction mechanism of this enzyme is of prime importance in furthering our understanding of enzyme-drug interactions. In order to provide a greater understanding on the reaction mechanism, theoretical and enzymatic spectroscopic experimental approaches have been utilized. The theoretical approaches were used to elucidate the electronic structure and geometry of the reductive-half reaction. During the initial stage of catalysis, the substrate bound tetrahedral complex was expected to be transformed to the product bound intermediate by passing through the tetrahedral transition state. The transition state structures were modeled and characterized by one imaginary negative frequency that were stabilized by energies ranging between 0.33 - 19.0 kcal/mol. The Mulliken atomic charge and Mayer bond order profiles were provided, respectively, for selected atoms and the bonds associated with them. Based on the electronic structure and bonding descriptions, the re-allocation of an electron on Mo-center was proposed to take place through an inner-sphere mechanism, with concomitant transfer of a proton or formal hydride transfer from the substrate carbon to the active site sulfido terminal. The formation of stable intermediate, in the presence of lumazine and bovine milk xanthine oxidase, was described by spectral bands centered at 650 nm. Similar spectral bands were also detected, in the presence of an electron acceptor (2, 6 — dichlorophenolindophenol, DCIP-), when the enzymes (bmXOR, wild type RcXDH, or RcXDH-Glu232Ala mutant) were reacted with lumazine. Finally, the enzymes were shown to exhibit variable activities and steady-state kinetic parameters when the reactions between the same substrates and enzymes were probed using the Amplex/H2O2 and DCIP-/O2•- assay methods. The variation in activities and steady-state kinetic parameters were then proposed to be due to the factors that affected the affinity and product release stages of the catalytic cycle