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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2011)

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.

    Cover page: Crystallographic, Kinetic and Computational Studies on the Reaction Mechanism of Xanthine Oxidoreductase
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2014)
    The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.
      Cover page: The reductive half-reaction of xanthine dehydrogenase from Rhodobacter capsulatus: the role of Glu232 in catalysis.
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2019)
      The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.
        Cover page: Target highlights in CASP13: Experimental target structures through the eyes of their authorsCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2019)
        The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.
          Cover page: Target highlights in CASP13: Experimental target structures through the eyes of their authors
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