Structural and Kinetic Studies of Xanthine Oxidase and the Xanthine Oxidase Family of Enzymes

Xanthine dehydrogenase/oxidase (XDH/XO) is a molybdenum-containing enzyme which is involved with hydroxylation a number of sp2-hybridized centers including purines, heterocycles, and aldehydes. Its main role in the cell is to convert hypoxanthine to xanthine and xanthine to uric acid. Although xanthine oxidase has been studied for decades, details of its mechanism and how the active site allows for substrate specificity and catalysis are still not completely known. In the present work, several methods have been used to investigate the mechanism of xanthine oxidase and the roles of the active site residues towards substrate binding and catalysis. 1.) The kinetic rates of bovine xanthine oxidase and variants of the homologous Rhodobacter capsulatus xanthine dehydrogenase toward various substrates have been observed. Investigation of the E232Q variant of R. capsulatus xanthine dehydrogenase revealed that removal of the ionizable Glutamate 232 resulted in a dramatic loss in activity at higher pH, as compared to wild-type enzyme, providing insight into the role of Glu 232. 2.) We used X-ray crystallography to investigate interactions of the enzyme with the slow substrate indole-3-aldehyde and the non-substrate guanine. The dominant nonproductive orientations of the molecules correlate with the observed kinetic rates. 3.) The effects of active site residues on the chemical step of the reaction were investigated utilizing kinetic isotope effect studies. With the primary deuterium isotope effects, previously described by Dr. Cao, and intrinsic isotope effects Dk, derived from the tritium isotope effect studies conducted for bovine xanthine oxidase and bacterial xanthine dehydrogenase, the extent that the chemical step is rate-limiting was calculated for each. Comparison of the enzymes with amino acid substitution variants allowed for insight into the role of the active site residues by the monitoring of changes (or lack of change) in the rate of the chemical step and it's c