pH and kinetic isotope effects on sarcosine oxidation by N-methyltryptophan oxidase.
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/K(O)()2 value is independent of the sarcosine concentration at all accessible concentrations of oxygen. The k(cat)/K(sarc) pH profile is bell-shaped, with pK(a) values of 8.8 and about 10; the latter value matches the pK(a) value of the substrate nitrogen. The k(cat) pH profile exhibits a single pK(a) value of 9.1 for a group that must be unprotonated for catalysis. There is no significant solvent isotope effect on the k(cat)/K(sarc) value. With N-methyl-(2)H(3)-glycine as the substrate, there is a pH-independent kinetic isotope effect on k(cat), k(cat)/K(sarc), 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.
Ralph
Erik C
EC
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Fitzpatrick
Paul F
PF
D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012521:Sarcosine / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D017931:DNA Primers; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C102761:SolA protein, E coli; D010089:Oxidoreductases, N-Demethylating; D012521:Sarcosine
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