Insights into the mechanism of flavoprotein-catalyzed amine oxidation from nitrogen isotope effects on the reaction of N-methyltryptophan oxidase.
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.
Ralph
Erik C
EC
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
Hirschi
Jennifer S
JS
Anderson
Mark A
MA
Cleland
W Wallace
WW
Singleton
Daniel A
DA
Fitzpatrick
Paul F
PF
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*
D000588:Amines; D009587:Nitrogen Isotopes; D005182:Flavin-Adenine Dinucleotide; D010089:Oxidoreductases, N-Demethylating