title: Characterization of 2-oxo-3-pentynoate as an active-site-directed inactivator of flavoprotein oxidases: identification of active-site peptides in tryptophan 2-monooxygenase.
abstract: 2-oxo-3-pentynoate has been characterized as an active-site-directed inhibitor of selected flavoprotein oxidases. Tryptophan 2-monooxygenase is irreversibly inactivated in an active-site-directed fashion. The addition of FAD affords no protection from inactivation, whereas the competitive inhibitor indole-3-acetamide fully protects the enzyme from inactivation. The inactivation follows first-order kinetics for at least five half-lives. The rate of inactivation shows saturation kinetics, consistent with the formation of a reversible complex between the alkylating agent and the enzyme before inactivation occurs. Values of 0.017 +/- 0.0005 min-1 and 44 +/- 7 microM were determined for the limiting rate of inactivation and the apparent dissociation constant for 2-oxo-3-pentynoate, respectively. Tryptic maps of tryptophan 2-monooxygenase treated with 2-oxo-3-pentynoate show that two peptides are alkylated in the absence of indole-3-acetamide but not in its presence. The two peptides were identified by mass spectrometry as residues 333-349 and 503-536. Based upon sequence analysis, cysteine 511 and either cysteine 339 or histidine 338 are the likely sites of modification. In contrast, incubation of D-amino acid oxidase or nitroalkane oxidase with 2-oxo-3-pentynoate results in a loss of 55% or 100%, respectively, of the initial activity. In neither case does a competitive inhibitor affect the rate of inactivation, suggesting that the effect is not due to modification of active-site residues.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
authors.forename: G
authors.initials: G
authors.lastname: Gadda
authors.forename: L J
authors.initials: LJ
authors.lastname: Dangott
authors.forename: W H
authors.initials: WH
authors.lastname: Johnson
authors.forename: C P
authors.initials: CP
authors.lastname: Whitman
authors.forename: P F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors; D049308:Dioxygenases; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005231:Fatty Acids, Unsaturated / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000037:antagonists & inhibitors; D010446:Peptide Fragments / Q000737:chemistry*; D011549:Pseudomonas / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C109822:2-oxo-3-pentynoic acid; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D010446:Peptide Fragments; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase; C023204:tryptophan 2-monooxygenase; D003605:D-Amino-Acid Oxidase