N-Methyltryptophan Oxidase from Escherichia coli:  Reaction Kinetics with N-Methyl Amino Acid and Carbinolamine Substrates

Abstract

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.

Pleiotropic Impact of a Single Lysine Mutation on Biosynthesis of and Catalysis by N-Methyltryptophan Oxidase

“…The apparent k cat observed with wild-type MTOX under these conditions (k cat app = 21.4 ± 0.6 s −1 ) is about 30% of the maximal turnover rate observed at saturating concentrations of NMT and oxygen (k cat = 77 ± 15 s-1) (2). Mutation of Lys259 to Gln, Ala or Met results in a 1300-, 2500- or 10,000-fold decrease, respectively, in turnover rate, as judged by the values obtained for k cat app (Table 1).…”

“…Other N-methyl amino acids, such as sarcosine, and carbinolamines, are poor alternate substrates. Steady-state kinetic studies indicate that NMT oxidation occurs via a ternary complex mechanism in which oxygen reacts with a reduced enzyme•NMT imine complex (2). …”

Rapid Method Using Two Microbial Enzymes for Detection of l -Abrine in Food as a Marker for the Toxic Protein Abrin

“…The pH of some of the tested food and beverage samples was Ͻ4.5 (apple juice, orange juice, and yogurt), but the reported optimum pH for tryptophanase from E. coli B ranged from 7.2 to 8.8 (18) and reported MTOX activity assays have been done at pHs of Ն8.0 (16,17,24). Therefore, the performance of the modified MeO-DMABA reagent was evaluated in samples when the pH was raised to 8.0.…”

“…1). The physiological function of MTOX is unknown, but L-abrine was shown to be the optimal substrate among those tested (15,17).…”

“…1). The physiological function of MTOX is unknown, but L-abrine was shown to be the optimal substrate among those tested (15,17).Tryptophan indole-lyase (tryptophanase; EC 4.1.99.1) catalyzes the reversible hydrolytic cleavage of L-tryptophan to yield indole, ammonia, and pyruvate (18) (Fig. 1).…”