2009
DOI: 10.1074/jbc.m804517200
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Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase

Abstract: Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the role of Arg-880 in catalysis. Here we report additional crystal structures of as-isolated, functional xanthine oxidase in the course of reacti… Show more

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Cited by 99 publications
(110 citation statements)
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“…The crystal structure of the complex of xanthine oxidase with 6-mercaptopurine was solved at 2.6 Å resolution, with final R factors R cryst of 21.7% and R free of 26.9% (Table 1). The overall structure of the homodimer in both current crystal structures is very similar to that seen previously for the oxidized bovine enzyme (11) and our own previously reported structures of the enzyme in complex with 2-hydroxy-6-methylpurine, xanthine, and lumazine (13,14). As seen previously (13,14), finite electron density for the C-terminal residues of one monomer of the single homodimer in the asymmetric unit was evident, and these residues were added during refinement (residues 1316 -1326 and 1316 -1325 for the structures in complex with hypoxanthine and 6-mercaptopurine, respectively).…”
Section: Methodssupporting
confidence: 67%
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“…The crystal structure of the complex of xanthine oxidase with 6-mercaptopurine was solved at 2.6 Å resolution, with final R factors R cryst of 21.7% and R free of 26.9% (Table 1). The overall structure of the homodimer in both current crystal structures is very similar to that seen previously for the oxidized bovine enzyme (11) and our own previously reported structures of the enzyme in complex with 2-hydroxy-6-methylpurine, xanthine, and lumazine (13,14). As seen previously (13,14), finite electron density for the C-terminal residues of one monomer of the single homodimer in the asymmetric unit was evident, and these residues were added during refinement (residues 1316 -1326 and 1316 -1325 for the structures in complex with hypoxanthine and 6-mercaptopurine, respectively).…”
Section: Methodssupporting
confidence: 67%
“…With each substrate, one orientation has C-2 positioned appropriately for hydroxylation by the molybdenum center, and the second has C-8 so positioned instead. Both of the orientations seen here have the exocyclic functional group at the C-6 position adjacent to a conserved arginine residue (Arg 880 in the bovine enzyme), consistent with our previous conclusions regarding the catalytic role of Arg 880 in stabilizing negative charge accumulation on the heterocycle at this position in the course of the reaction (12)(13)(14). In light of these results, we have undertaken further kinetic studies and confirm that xanthine is indeed the sole product of enzyme action on hypoxanthine; to within our detection limits 6,8-dihydroxypurine is not formed, despite the fact that hypoxanthine is able to bind in an orientation that permits such hydroxylation.…”
supporting
confidence: 77%
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“…Docking of esculetin (1) was performed with the program GOLD (version 5.1; Cambridge Crystallographic Data Centre, UK) [20], [21] and the X-ray crystal structure of the XO/lumazine complex (Protein Data Bank entry 3ETR) [22]. The protein structure was prepared in GOLD for docking by adding hydrogen atoms and deleting all water molecules and lumazine.…”
Section: Prediction Of Inhibitor Binding Poses By Computational LImentioning
confidence: 99%