Characterization of 2-oxo-3-pentynoate as an active-site-directed inactivator of flavoprotein oxidases: Identification of active-site peptides in tryptophan 2-monooxygenase

2-Oxo-3-pentynoate has been characterized as an active-site-directed inhibi tor of selected flavoprotein oxidases. Tryptophan 2-monooxygenase is irreve rsibly 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 inactiv ation follows first-order kinetics for at least five half-lives. The rate o f inactivation shows saturation kinetics, consistent with the formation of a reversible complex between the alkylating agent and the enzyme before ina ctivation occurs. Values of 0.017 +/- 0.0005 min(-1) and 44 +/- 7 mu M were determined for the limiting rate of inactivation and the apparent dissocia tion constant for 2-oxo-3-pentynoate, respectively. Tryptic maps of tryptop han 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, in cubation of D-amino acid oxidase or nitroalkane oxidase with 2-oxo-3-pentyn oate results in a loss of 55% or 100%, respectively, of the initial activit y. In neither case does a competitive inhibitor affect the rate of inactiva tion, suggesting that the effect is not due to modification of active-site residues.