UC Riverside

Xanthine oxidoreductase is a molybdenum-containing enzyme which catalyzes the hydroxylation on sp2 hybridized carbon centers of a broad family of substrates including purines, aldehydes and various other heterocycles. It catalyzes the sequential hydroxylation of physiological substrate hypoxanthine to uric acid. Deposition of uric acid crystals in human joints with accompanying inflammation is the major cause of gout. The production of reactive oxygen species by xanthine oxidase is implicated in the pathology of various inflammatory and cardiovascular diseases.

The current study mainly involves: (1) X-ray crystallography to elucidate the orientations of various substrates at the active site of bovine xanthine oxidase. Our observation of a single dominant productive orientation of xanthine, alternative orientations of hypoxanthine, a single nonproductive orientation of guanine and the dominant nonproductive orientation of indole-3-aldehyde correlates well with different catalytic activities of xanthine oxidase with these substrates and suggests the existence of dynamic sampling of substrate orientations at enzyme active site. (2) X-ray crystallography to reveal the orientations of inhibitors arsenite and quercetin at the active site of xanthine oxidase. The binding modes of these inhibitors provide structural basis for the mechanism of inhibition and insights into inhibitor design for potential therapeutics. (3) UV-visible spectroscopy to quantitatively characterize the kinetics and catalytic specificity in the reactions of sequential hydroxylation of hypoxanthine to uric acid. We conclude that the hydroxylation of hypoxanthine by xanthine oxidase is strictly specific toward C-2 over C-8, although 6,8-dihydroxypurine is an effective substrate as xanthine both of which can be converted to uric acid by xanthine oxidase. (4) Primary deuterium kinetic isotopic effect (KIE) study on the wild-type enzyme and Gln197 mutants of R. capsulatus xanthine dehydrogenase. The small apparent KIE on kcat suggests that hydrogen transfer step is neither rate-limiting for the wild-type enzyme nor for the mutants. We identify gain-of-function Q197A and loss-of-function Q197E mutants. (5) Computation of pKa of Glu802/232 of xanthine oxidoreductase and kinetic characterization of E232Q mutant. Our data suggest that Glu802/232 has an acidic pKa upon binding of xanthine or hypoxanthine which supports its catalytic role in facilitating proton tautomerization.