Mechanism of Substrate and Inhibitor Binding of Rhodobacter capsulatus Xanthine Dehydrogenase

被引:39
作者
Dietzel, Uwe
Kuper, Jochen
Doebbler, Jennifer A. [3 ,4 ]
Schulte, Antje [4 ]
Truglio, James J. [4 ]
Leimkuehler, Silke [1 ]
Kisker, Caroline [2 ]
机构
[1] Univ Potsdam, Inst Biochem & Biol, D-14476 Potsdam, Germany
[2] Univ Wurzburg, Inst Biol Struct, Rudolf Virchow Ctr Expt Biomed, D-97078 Wurzburg, Germany
[3] SUNY Stony Brook, Dept Phys, Stony Brook, NY 11794 USA
[4] SUNY Stony Brook, Dept Pharmacol Sci, Stony Brook, NY 11794 USA
基金
美国国家卫生研究院;
关键词
CRYSTAL-STRUCTURE; ACTIVE-SITE; OXIDASE; CATALYSIS; PTERIN-6-ALDEHYDE; IDENTIFICATION; SPECIFICITY; INSERTION; RESIDUES; ENZYME;
D O I
10.1074/jbc.M808114200
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
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (alpha beta)(2) heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6-aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E(B)232Q) to prevent substrate turnover. The hypoxanthine-and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue GluB-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg(B)-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K-i of 103.57 +/- 18.96 nM for pterin-6-aldehyde.
引用
收藏
页码:8759 / 8767
页数:9
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