Insights into the mechanism of flavoprotein-catalyzed amine oxidation from nitrogen isotope effects on the reaction of N-methyltryptophan oxidase

Erik C. Ralph, Jennifer S. Hirschi, Mark A. Anderson, W. Wallace Cleland, Daniel A. Singleton, Paul F. Fitzpatrick

Research output: Contribution to journalArticle

57 Scopus citations

Abstract

The mechanism of N-methyltryptophan oxidase, a flavin-dependent amine oxidase from Escherichia coli, was studied using a combination of kinetic isotope effects and theoretical calculations. The 15(k cat/Km) kinetic isotope effect for sarcosine oxidation is pH-dependent with a limiting value of 0.994-0.995 at high pH. Density functional theory calculations on model systems were used to interpret these isotope effects. The isotope effects are inconsistent with proposed mechanisms involving covalent amine-flavin adducts but cannot by themselves conclusively distinguish between some discrete electron-transfer mechanisms and a direct hydride-transfer mechanism, although the latter mechanism is more consistent with the energetics of the reaction.

Original languageEnglish (US)
Pages (from-to)7655-7664
Number of pages10
JournalBiochemistry
Volume46
Issue number25
DOIs
StatePublished - Jun 26 2007

ASJC Scopus subject areas

  • Biochemistry

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