Spectroscopic characterization of intermediates in the urate oxidase reaction

被引:94
作者
Kahn, K [1 ]
Tipton, PA [1 ]
机构
[1] Univ Missouri, Dept Biochem, Columbia, MO 65211 USA
关键词
D O I
10.1021/bi980446g
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
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 O-2 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 degrees C; 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).
引用
收藏
页码:11651 / 11659
页数:9
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