Spectroscopic characterization of intermediates in the urate oxidase reaction.
The oxidation of urate catalyzed by soybean urate oxidase was studied under single-turnover conditions using stopped-flow absorbance and fluorescence spectrophotometry. Two discrete enzyme-bound intermediates were observed; the first intermediate to form had an absorbance maximum at 295 nm and was assigned to a urate dianion species; the second intermediate had an absorbance maximum at 298 nm and is believed to be urate hydroperoxide. These data are consistent with a catalytic mechanism that involves formation of urate hydroperoxide from O2 and the urate dianion, collapse of the peroxide to form dehydrourate, and hydration of dehydrourate to form the observed product, 5-hydroxyisourate. The rate of formation of the first intermediate was too fast to measure accurately at 20 degreesC; the second intermediate formed with a rate constant of 32 s-1 and decayed with a rate constant of 6.6 s-1. The product of the reaction, 5-hydroxyisourate, is fluorescent, and its release from the active site occurred with a rate constant of 31 s-1.
Kahn
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K
Department of Biochemistry, University of Missouri-Columbia 65211, USA.
Tipton
P A
PA
D004735:Energy Transfer; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013025:Glycine max / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D014503:Urate Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014527:Uric Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D014527:Uric Acid; D014503:Urate Oxidase