A comprehensive mechanistic study of the molybdoflavoenzyme xanthine oxidase is proposed focusing on two central aspects of the catalytic behavior of this important enzyme, which catalyzes the final two steps in purine catabolism. In the first aspect of the proposed work, the reductive half-reaction of the catalytic cycle will be comprehensively examined with the aim of establishing the detailed chemistry that occurs in the conversion of xanthine to uric acid. The results will be interpreted in terms of a specific chemical mechanisms proposed for the reductive half-reaction. The structure of the complex of alloxanthine with xanthine oxidase will specifically be examined in this aspect of the proposed work. This complex is thought to be a particularly stable analog to a specific catalytic intermediate, and is also of clinical significance in that its great stability is the basis for the remarkable clinical efficacy of allopurinol. This well-tolerated drug is used in the treatment of hyperuricemia associated with such diverse conditions as gout and gouty arthritis, and enzyme deficiencies in hypoxanthine-guanine phosphoribosyltransferase (Lesch-Nyhan syndrome) and glucose-6-phosphatase (von Gierke's disease). In the second aspect of the proposed work, the interaction of the four oxidation-reduction centers in xanthine oxidase will be quantitatively examined in an effort to determine the rates at which reducing equivalents equilibrate in the enzyme, and the extent to which the equilibration of reducing equivalents influences catalytic turnover. The presence in xanthine oxidase of multiple sites capable of reversibly accepting reducing equivalents offers the opportunity to examine the transfer of electrons from one biological center to another while being held at a fixed distance and orientation within a more or less rigid polypeptide matrix. This has become an area of great interest in biophysics, and the careful examination of the properties of xanthine oxidase is expectd to yield significant information regarding the principles governing electron transfer in biological systems. The mode of interaction of the various centers in xanthine oxidase may also be relevant in the production of superoxide by xanthine oxidase. This too is of clinical significance given that enzyme-generated superoxide has been proposed to play a significant role in ischemia-related tissue damage (in atherosclerosis and heart attack).

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
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Biochemistry Study Section (BIO)
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Ohio State University
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