N-methyltryptophan oxidase from Escherichia coli: reaction kinetics with N-methyl amino acid and carbinolamine substrates.
N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of N-methyl-L-tryptophan (k(cat) = 4600 min(-1)). Other secondary amino acids (e.g., sarcosine) are oxidized at a slower rate. We have identified carbinolamines as a new class of alternate substrate. MTOX oxidation of the carbinolamine formed with L-tryptophan and formaldehyde yields N-formyl-L-tryptophan in a relatively slow reaction that does not compete with turnover of MTOX with N-methyl-L-tryptophan. Double reciprocal plots with N-methyl-L-tryptophan as the varied substrate are nearly parallel, but the slopes show a small, systematic variation depending on the oxygen concentration. N-Benzylglycine, a dead-end competitive inhibitor with respect to N-methyl-L-tryptophan, acts as a noncompetitive inhibitor with respect to oxygen. The results are consistent with a modified ping pong mechanism where oxygen binds to the reduced enzyme prior to dissociation of the imino acid product. MTOX is converted to a 2-electron reduced form upon anaerobic reaction with N-methyl-L-tryptophan, sarcosine, or the carbinolamine formed with L-tryptophan and formaldehyde. No evidence for a detectable intermediate was obtained by monitoring the spectral course of the latter two reactions. MTOX reduction with thioglycolate does, however, proceed via a readily detectable anionic, flavin radical intermediate. The reductive half-reaction with sarcosine at 4 degrees C exhibits saturation kinetics (k(lim) = 6.8 min(-1), K = 39 mM) and other features consistent with a mechanism in which a nearly irreversible reduction step (E(ox).S --> E(red).P) (k(lim)) is preceded by a rapidly attained equilibrium (K) between free E and the E.S complex. The 21 degrees C temperature difference can reasonably account for the 3.6-fold lower value obtained for k(lim) as compared with turnover at 25 degrees C (k(cat) = 24.5 min(-1)), suggesting that sarcosine is oxidized at a kinetically significant rate under anaerobic conditions and the reductive half-reaction is rate-limiting during turnover. These conclusions are, however, difficult to reconcile with steady-state kinetic patterns obtained with sarcosine that are consistent with a rapid equilibrium ordered mechanism with oxygen as the first substrate. The basis for the apparent stability of the MTOX.oxygen complex (K(d) = 72 microM) is unknown.
Khanna
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P
Department of Biochemistry, MCP Hahnemann School of Medicine, Philadelphia, Pennsylvania 19129, USA.
Schuman Jorns
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M
D000588:Amines / Q000737:chemistry; D000693:Anaerobiosis; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005557:Formaldehyde / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007097:Imines / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000737:chemistry*; D019163:Reducing Agents / Q000737:chemistry; D012521:Sarcosine / Q000737:chemistry; D013379:Substrate Specificity; D013864:Thioglycolates / Q000737:chemistry; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*
D000588:Amines; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D007097:Imines; D019163:Reducing Agents; D013864:Thioglycolates; D005557:Formaldehyde; C028129:tryptophan methyl ester; D014364:Tryptophan; C423606:N-benzylglycine; C102761:SolA protein, E coli; D010089:Oxidoreductases, N-Demethylating; D005998:Glycine; D012521:Sarcosine