Spectroscopic characterization of intermediates in the urate oxidase reaction

Biochemistry. 1998 Aug 18;37(33):11651-9. doi: 10.1021/bi980446g.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Energy Transfer
  • Kinetics
  • Models, Chemical
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Soybeans / enzymology
  • Spectrometry, Fluorescence
  • Urate Oxidase / chemistry*
  • Urate Oxidase / genetics
  • Urate Oxidase / metabolism
  • Uric Acid / analogs & derivatives
  • Uric Acid / chemistry
  • Uric Acid / metabolism

Substances

  • Recombinant Proteins
  • Uric Acid
  • Urate Oxidase