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Relative Timing of Hydrogen and Proton Transfers in the Reaction of Flavin Oxidation Catalyzed by Choline Oxidase.
- Source :
-
Biochemistry . 2/19/2013, Vol. 52 Issue 7, p1221-1226. 6p. - Publication Year :
- 2013
-
Abstract
- The oxidation of the reduced flavin in choline oxidase was investigated with pH, solvent viscosity, and kinetic isotope effects (KIEs) in steady-state kinetics and time-resolved absorbance spectroscopy of the oxidative half-reaction in a stopped-flow spectrophotometer. Both the effects of isotopic substitution on the KIEs and the multiple KIEs suggest a mechanism for flavin oxidation in which the H atom from the reduced flavin and a H+ from the solvent or a solvent exchangeable site are transferred in the same kinetic step. Stopped-flow kinetic data demonstrate flavin oxidation without stabilization of flavin-derived species. Solvent viscosity effects establish an isomerization of the reduced enzyme. These results allow us to rule out mechanisms for flavin oxidation in which C4a-peroxy and -hydroperoxy flavin intermediates accumulate to detectable levels in the reaction of flavin oxidation catalyzed by choline oxidase. A mechanism of flavin oxidation that directly results in the formation of oxidized flavin and hydrogen peroxide without stabilization of reaction intermediates is consistent with the data presented. [ABSTRACT FROM AUTHOR]
- Subjects :
- *HYDROGEN
*PROTON transfer reactions
*FLAVINS
*CHOLINE
*KINETIC isotope effects
Subjects
Details
- Language :
- English
- ISSN :
- 00062960
- Volume :
- 52
- Issue :
- 7
- Database :
- Academic Search Index
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 86252331
- Full Text :
- https://doi.org/10.1021/bi3016235