title: 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.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
authors.forename: Erik C
authors.initials: EC
authors.lastname: Ralph
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Hirschi
authors.forename: Mark A
authors.initials: MA
authors.lastname: Anderson
authors.forename: W Wallace
authors.initials: WW
authors.lastname: Cleland
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Singleton
authors.forename: Paul F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D000588:Amines / Q000737:chemistry*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D007700:Kinetics; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000588:Amines; D009587:Nitrogen Isotopes; D005182:Flavin-Adenine Dinucleotide; D010089:Oxidoreductases, N-Demethylating