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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Sobrado P,Fitzpatrick PFdoi
10.1021/bi035300isubject
Has Abstractpub_date
2003-12-02 00:00:00pages
13833-8issue
47eissn
0006-2960issn
1520-4995journal_volume
42pub_type
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