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首页> 外文期刊>Biochemistry >Characterization of 2-oxo-3-pentynoate as an active-site-directed inactivator of flavoprotein oxidases: identification of active-site peptides in tryptophan 2-monooxygenase.
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Characterization of 2-oxo-3-pentynoate as an active-site-directed inactivator of flavoprotein oxidases: identification of active-site peptides in tryptophan 2-monooxygenase.

机译：2-氧代-3-戊酸作为黄酮蛋白氧化酶的活性位点灭活剂的表征：色氨酸2-单加氧酶中活性位点肽的鉴定。

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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.
机译：2-氧代-3-戊酸已被表征为所选黄素蛋白氧化酶的活性位点导向抑制剂。色氨酸2-单加氧酶以活性位点导向的方式不可逆地失活。 FAD的添加不能防止灭活，而竞争性抑制剂吲哚-3-乙酰胺可以完全保护酶免受灭活。失活遵循一级动力学至少五个半衰期。失活速率显示出饱和动力学，与失活发生之前烷基化剂和酶之间可逆络合物的形成相一致。对于2-氧代-3-戊酸的灭活极限速率和表观解离常数分别确定为0.017 +/- 0.0005 min-1和44 +/- 7 microM。用2-氧代-3-戊酸处理的色氨酸2-单加氧酶的胰蛋白酶图谱表明，在不存在吲哚-3-乙酰胺但不存在的情况下，两个肽均被烷基化。通过质谱鉴定这两个肽为残基333-349和503-536。基于序列分析，半胱氨酸511和半胱氨酸339或组氨酸338是可能的修饰位点。相反，将D-氨基酸氧化酶或硝基烷氧化酶与2-氧代-3-戊酸一起温育分别导致初始活性损失55％或100％。在任何情况下，竞争性抑制剂均不会影响灭活速率，这表明该作用并非归因于活性位点残基的修饰。

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