Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase THE SEQUENTIAL HYDROXYLATION OF HYPOXANTHINE TO URIC ACID

被引:101
作者
Cao, Hongnan [1 ]
Pauff, James M. [1 ,2 ]
Hille, Russ [1 ]
机构
[1] Univ Calif Riverside, Dept Biochem, Riverside, CA 92521 USA
[2] Ohio State Univ, Med Scientist Program, Columbus, OH 43210 USA
基金
美国能源部;
关键词
MACROMOLECULAR STRUCTURES; MOLYBDENUM; MECHANISM; DEHYDROGENASE; REFINEMENT; RESIDUES; ENZYME; SITE;
D O I
10.1074/jbc.M110.128561
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 1.8A resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6A 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.
引用
收藏
页码:28044 / 28053
页数:10
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