Abstract
The flavoenzyme tryptophan 2-monooxygenase catalyzes the oxidation of tryptophan to indole acetamide, 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 Kd value for tryptophan and the Ki values for the competitive inhibitors indole acetamide 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 indole pyruvate and hydrogen peroxide. The V/Ktrp-pH profiles for the Tyr413 mutant enzymes no longer show the pKa 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 amino acid decarboxylation.
Recommended Citation
P. Sobrado and P. F. Fitzpatrick, "Identification of Tyr413 as an Active Site Residue in the Flavoprotein Tryptophan 2-Monooxygenase and Analysis of its Contribution to Catalysis," Biochemistry, vol. 42, no. 47, pp. 13833 - 13838, American Chemical Society, Dec 2003.
The definitive version is available at https://doi.org/10.1021/bi035300i
Department(s)
Chemistry
International Standard Serial Number (ISSN)
0006-2960
Document Type
Article - Journal
Document Version
Citation
File Type
text
Language(s)
English
Rights
© 2024 American Chemical Society, All rights reserved.
Publication Date
02 Dec 2003
PubMed ID
14636050
Comments
National Institute of General Medical Sciences, Grant R01GM058698