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

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(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 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.

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

“…It is believed that amino acids in this position have a side-chain length adapted to the size of the substrate (7,12) so that substrate binding is adequately tight and associated with possible changes in geometry, thereby facilitating subsequent catalysis (glycolate oxidation). Active site preorganization has been presumed in a similar reaction catalyzed by choline oxidase, consisting in O-H stretching in the ␣-hydroxyl group to facilitate proton abstraction in subsequent steps (32,33).…”

Microbial Community Degradation of Widely Used Quaternary Ammonium Disinfectants

“…4B). Among the latter genes, a predicted amine oxidase showed about 15-fold greater relative transcript abundance at 0.5 h compared to levels at 0 and 48 h. Amine oxidases have been shown to carry out oxidative deamination reactions in addition to oxidative conversion of amines to aldehydes (28)(29)(30); thus, they might be responsible for the first dealkylation step (i.e., cleaving the C alkyl -N bond) of the biochemically characterized BAC degradation pathway.…”

Irreversible Inhibition of Monoamine Oxidase B by the Antiparkinsonian Medicines Rasagiline and Selegiline: A Computational Study

“…Common to all mechanisms is that the activating stage mentioned is performed by the N5 atom on the flavin (Scheme 1), which is also the site of the irreversible inhibitor attack. Erdem et al [28] assumed that the hydride mechanism is unlikely to take place, because hydride transfer is associated with a barrier too high to be readily crossed, [29] unlike in the amino acid oxidase [30][31][32][33] and alcohol oxidase [2,34,35] classes of flavoenzymes, for which hydride mechanisms are suggested. Studies by Miller and Edmondson [36] with benzylamine analogues showed that attachment of electron-withdrawing groups to the para-position of the benzylamine substrate increases the rate of the reaction in the case of MAO A, which provides strong experimental evidence that proton transfer, and not hydride anion abstraction, is an integral part of the rate-limiting step.…”