Insights into the Mechanism of Flavoprotein-Catalyzed Amine Oxidation from Nitrogen Isotope Effects on the Reaction of N-Methyltryptophan Oxidase

摘要

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(kcat/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 (DFT) 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.