N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of secondary amino acids such as N-methyltryptophan or N-methylglycine (sarcosine). MTOX is one of several flavin-dependent amine oxidases whose chemical mechanism is still debated. The kinetic properties of MTOX with the slow substrate sarcosine were determined. Initial rate data are well-described by the equation for a ping-pong kinetic mechanism, in that the V/KO2 value is independent of the sarcosine concentration at all accessible concentrations of oxygen. The k cat/Ksarc pH profile is bell-shaped, with pKa values of 8.8 and about 10; the latter value matches the pKa value of the substrate nitrogen. The kcat pH profile exhibits a single pKa value of 9.1 for a group that must be unprotonated for catalysis. There is no significant solvent isotope effect on the kcat/K sarc value. With N-methyl-2H3-glycine as the substrate, there is a pH-independent kinetic isotope effect on kcat, kcat/Ksarc, and the rate constant for flavin reduction, with an average value of 7.2. Stopped-flow spectroscopy with both the protiated and deuterated substrate failed to detect any intermediates between the enzyme-substrate complex and the fully reduced enzyme. These results are used to evaluate proposed chemical mechanisms.
|Original language||English (US)|
|Number of pages||8|
|State||Published - Mar 1 2005|
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