PMID:15723552
| Citation |
Ralph, EC and Fitzpatrick, PF (2005) pH and kinetic isotope effects on sarcosine oxidation by N-methyltryptophan oxidase. Biochemistry 44:3074-81 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC1752209 Online version:10.1021/bi047716h |
| Keywords |
Base Sequence; DNA Primers; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Hydrogen-Ion Concentration; Kinetics; Oxidation-Reduction; Oxidoreductases, N-Demethylating/chemistry; Oxidoreductases, N-Demethylating/genetics; Oxidoreductases, N-Demethylating/metabolism; Oxygen Consumption; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Sarcosine/chemistry; Sarcosine/metabolism; Spectrophotometry; Substrate Specificity |
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