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 15 N 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 15 V/K alanine value was 1.0145 ± 0.0007 at pH 8. NMR analyses give an equilibrium 15 N isotope effect for deprotonation of the alanine amino group of 1.0233 ± 0.0004, allowing calculation of the 15 N 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.The flavoenzyme tryptophan 2-monooxygenase (TMO1) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of L-tryptophan (Scheme 1) in the first step of a twostep biosynthetic pathway for the plant hormone indoleacetic acid (10-12). The kinetic mechanism of TMO has been determined with its fastest substrate L-tryptophan (13), and can be divided into two half-reactions (Scheme 2). The reductive half-reaction involves cleavage of the α-CH bond of the amino acid (AA) and transfer of a hydride equivalent to the FAD to form the enzyme-bound imino acid. This is identical to the reaction of the flavoprotein L-amino acid oxidases and similar to the general reaction of flavoprotein amine oxidases. In the oxidative half-reaction of TMO, the reduced cofactor reacts with oxygen to produce hydrogen peroxide (14). Decarboxylation of the imine acid to the amide is thought to occur through the reaction of the hydrogen peroxide with the imino acid still bound to the enzyme (pathway a), analogously to the mechanism proposed by Lockridge et al. (15) for the decarboxylation of pyruvate by lactate oxidase. Although indoleacetamide is the only product of tryptophan turnover by wild-type TMO, amino acid oxidation can be uncoupled from decarboxylation to yield a keto acid in mutant enzymes (pathway b) (16,17).Despite their ubiquity and functional diversity, all flavin-dependent amine oxidases have thus far fallen into two structural families. One family includes D-amino acid oxidase (18), monomeric sarcosine oxidase (19) and glycine oxidase (20), while monoamine oxidase (21), *Address correspondence to:Paul F. Fitzpatrick,.Department of Biochemistry and Biophysics, 2128 TAMU, College Station, TX 77843-2128, Ph: 979-845-5487, Fax: 979-845-4946, Email: fitzpat@tamu.edu † This work was supported by NIH grants R01 GM58698 (PFF), R01 GM18938 (WWC), and T32 GM08523 (ECR) NIH Public Access (22), and L-amino acid oxidase (23) represent a separate f...