title: 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 kcat/Kala 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.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
authors.forename: Erik C
authors.initials: EC
authors.lastname: Ralph
authors.forename: Mark A
authors.initials: MA
authors.lastname: Anderson
authors.forename: W Wallace
authors.initials: WW
authors.lastname: Cleland
authors.forename: Paul F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D000409:Alanine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007210:Indoleacetic Acids / Q000378:metabolism; D007700:Kinetics; D050830:L-Amino Acid Oxidase / Q000737:chemistry* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000737:chemistry; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005420:Flavoproteins; D007210:Indoleacetic Acids; D009587:Nitrogen Isotopes; C030737:indoleacetic acid; D006899:Mixed Function Oxygenases; C023204:tryptophan 2-monooxygenase; D050830:L-Amino Acid Oxidase; D000409:Alanine