INACTIVATORS OF LYPOXYGENASE

氧合酶灭活剂

• 批准号: 3292583
• 负责人: CHARLES H CLAPP
• 金额: $ 6.82万
• 依托单位: BUCKNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292583
• 关键词: chemical structure function; chemical synthesis; deuterium; enzyme inhibitors; enzyme mechanism; lipid peroxides; lipoxygenase; molecular site; oxidation; radionuclides; radiotracer; soybeans; stoichiometry

This project concerns soybean lipoxygenase, a nonheme iron protein that catalyzes the oxygenation of linoleic acid to 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD). The goals of the work are to understand the chemical properties and catalytic mechanism of this enzyme and to develop strategies that can be applied to the design of inhibitors of several physiologically and pharmaceutically interesting mammalian lipoxygenases. We have been studying the action of soybean lipoxygenase on 9-octadecenoic acid derivatives with substituents at position 12. 12-Iodo-cis-9-octadecenoic acid (12-IODE) and 12-mercapto-cis-9-octadecenoic acid (12-HSODE) are both irreversible inactivators of this enzyme. 12-BrODE does not inactivate the enzyme but is slowly converted to 9,11-octadecadienoic acid (9,11-ODE). Our working hypothesis is that ferric lipoxygenase can react with these compounds by two pathways: an elimination reaction to produce 9,11-ODE (Pathway 1) and a second pathway that is initiated by one-electron oxidation of X (Pathway 2). We propose that 12-BrODE reacts by Pathway 1, 12-HSODE reacts by Pathway 2, and 12-IODE reacts by both pathways. Pathway 2 leads to inactivation; in the case of 12-IODE there is evidence that inactivation occurs by a radical mechanism. During the period covered by this proposal we will test the hypothesis that 12-IODE reacts by both pathways by studying the action of soybean lipoxygenase on 11,11-dideuterio-12-IODE. If our hypothesis is correct, introduction of deuterium at position 11 should increase the flux through Pathway 2 due to a primary isotope effect on Pathway 1. We will also synthesize the R- and S- enantiomers of 12-BrODE to determine the stereospecificity of Pathway 1. In addition, we will use 1-14C-12-HSODE to determine whether inactivation by 12-HSODE is accompanied by covalent incorporation of the carbon skeleton of the inactivator into the protein. We will also use this labelled material to facilitate the isolation of the products from the action of lipoxygenase on 12-HSODE, and we will identify these products. Finally, the elimination reactions that we have observed provide evidence for a basic residue at the active site. We propose to synthesize and test 11-bromo-cis-9-octadecenoic acid and 11,12-oxido-9-octadecenoic acid as possible active-site directed-labelling reagents for this base.

该项目涉及大豆脂氧酶，这是一种非血红素铁蛋白 催化亚油酸的氧合为 13-氢氧基-9,11-二十二烯酸（13-HPOD）。 目标的目标 工作是了解化学特性和催化机制 这种酶并制定可应用于设计的策略 几种生理和药物有趣的抑制剂 哺乳动物脂氧酶。 我们一直在研究大豆脂氧酶对 9-二十二乙烯酸衍生物，具有取代基的位置12。 12- iodo-cis-9-octadecenoic酸（12-iode）和 12-Mercapto-CIS-9-二十二烯酸（12-Hsode）都是不可逆的 该酶的灭活剂。 12-树不会使酶失活，而是 缓慢转化为9,11-二十二烷二烯酸（9,11-ode）。 我们的工作 假设是脂氧酶可以与这些化合物反应 两种途径：消除反应产生9,11-ode（途径1）和 由x的一电子氧化引发的第二个途径 （路径2）。 我们提出，12个树枝通过途径1，12-hsode反应 通过途径2反应，而12个IODE通过两个途径反应。 途径2 导致失活；在12个IODE的情况下，有证据表明 灭活是通过一种自由基机制发生的。 在本提案所涵盖的期间，我们将检验假设 这12个IODE通过研究大豆的作用都通过两种途径做出反应 11,11-Dideuterio-12-iode上的脂氧合酶。 如果我们的假设正确， 在位置11引入氘应增加通道 途径2是由于对途径1的主要同位素效应而引起的。我们也将 合成12个brode的R-和S-剂，以确定 途径的立体特定性1。此外，我们将使用1-14c-12-hsode 确定12-hsode失活是否伴随着共价 将灭活剂的碳骨架掺入蛋白质中。 我们还将使用这种标记的材料来促进 Lipoxygoson在12-Hsode的作用中的产品，我们将 识别这些产品。 最后，我们有消除反应 观察到的有效位点的基本残留物为证据提供了证据。 我们 提议合成和测试11-溴-CIS-CIS-9-OCTADECENOIC酸和 11,12-氧化-9-二十多甲酸可能有效位点 该碱基的定向试剂。