Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and 15N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

Abstract

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 k_cat/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.