Tryptophan 2-monooxygenase (TMO) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of L-tryptophan during the biosynthesis of indoleacetic acid. Structurally and mechanistically, the enzyme is a member of the family of L-amino acid oxidases. Deuterium and 15N kinetic isotope effects were used to probe the chemical mechanism of L-alanine oxidation by TMO. The primary deuterium kinetic isotope effect was pH independent over the pH range 6.5-10, with an average value of 6.0 ± 0.5, consistent with this being the intrinsic value. The deuterium isotope effect on the rate constant for flavin reduction by alanine was 6.3 ± 0.9; no intermediate flavin species were observed during flavin reduction. The kcat/K ala value was 1.0145 ± 0.0007 at pH 8. NMR analyses gave an equilibrium 15N isotope effect for deprotonation of the alanine amino group of 1.0233 ± 0.0004, allowing calculation of the 15N isotope effect on the CH bond cleavage step of 0.9917 ± 0.0006. The results are consistent with TMO oxidation of alanine occurring through a hydride transfer mechanism.
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