title: Identification of Tyr413 as an active site residue in the flavoprotein tryptophan 2-monooxygenase and analysis of its contribution to catalysis.
abstract: The flavoenzyme tryptophan 2-monooxygenase catalyzes the oxidation of tryptophan to indoleacetamide, carbon dioxide, and water. The enzyme is a homologue of l-amino acid oxidase. In the structure of l-amino acid oxidase complexed with aminobenzoate, Tyr372 hydrogen bonds with the carboxylate of the inhibitor in the active site. All 10 conserved tyrosine residues in tryptophan 2-monooxygenase were mutated to phenylalanine; steady state kinetic characterization of the purified proteins identified Tyr413 as the residue homologous to Tyr372 of l-amino acid oxidase. Y413F and Y413A tryptophan 2-monooxygenase were characterized more completely with tryptophan as the substrate to probe the contribution of this residue to catalysis. Mutation of Tyr413 to phenylalanine results in a decrease in the value of the first-order rate constant for reduction of 35-fold and a decrease in the rate constant for oxidation of 11-fold. Mutation to alanine decreases the rate constant for reduction by 200-fold and that for oxidation by 33-fold. Both mutations increase the K(d) value for tryptophan and the K(i) values for the competitive inhibitors indoleacetamide and indole pyruvate by 5-10-fold. Both mutations convert the enzyme to an oxidase, in that the products of the catalytic reactions of both are indolepyruvate and hydrogen peroxide. The V/K(trp)-pH profiles for the Tyr413 mutant enzymes no longer show the pK(a) value of 9.9 seen in that for the wild-type enzyme, allowing identification of Tyr413 as the active site residue in the wild-type enzyme which must be protonated for catalysis. Substitution of Tyr413 abolishes the formation of the long wavelength charge transfer species observed in the wild-type enzyme. The data are consistent with the main role of Tyr413 being to maintain the correct orientation of tryptophan for effective hydride transfer and imino acid decarboxylation.
authors.affiliation: Departments of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
authors.forename: Pablo
authors.initials: P
authors.lastname: Sobrado
authors.forename: Paul F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D013053:Spectrophotometry; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics; D014757:Viper Venoms / Q000201:enzymology
chemical_list: D005420:Flavoproteins; D014757:Viper Venoms; D014443:Tyrosine; D010649:Phenylalanine; D006899:Mixed Function Oxygenases; C023204:tryptophan 2-monooxygenase; D000409:Alanine