Substrate orientation and catalytic specificity in the action of xanthine oxidase: the sequential hydroxylation of hypoxanthine to uric acid.

TitleSubstrate orientation and catalytic specificity in the action of xanthine oxidase: the sequential hydroxylation of hypoxanthine to uric acid.
Publication TypeJournal Article
Year of Publication2010
AuthorsCao, H, Pauff JM, Hille R
JournalThe Journal of biological chemistry
Volume285
Pagination28044-53
Date Published2010 Sep 03
Abstract

Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp(2)-hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 A resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 A resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg(880) and Glu(802), previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.

10.1074/jbc.M110.128561
Pub Med Link

http://www.ncbi.nlm.nih.gov/pubmed/20615869?dopt=Abstract

Alternate JournalJ. Biol. Chem.
Citekey20615869