INHIBITION OF LIPOXYGENASE BY 12-IDO-9-OCTADECENOATE

12-IDO-9-十八碳烯酸酯对脂氧合酶的抑制

• 批准号: 3292580
• 负责人: CHARLES H CLAPP
• 金额: $ 5.2万
• 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 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292580
• 关键词: chemical structure function; chemical synthesis; enzyme inhibitors; enzyme mechanism; iodine; lipoxygenase; molecular site; radiotracer

Soybean lipoxygenase is a nonheme iron protein that catalyzes the oxygenation of lineolic acid to 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD). We have found that 12-iodo-cis-9-octadecenoic acid (12-IODE) is a time-dependent (t1/2 = 4 min at 4 Mum 12-IODE) irreversible inhibitor of this enzyme. This result is interesting, since 12-IODE is a poor alkylating agent and soybean lipoxygenase is not inhibited by high concentrations of good alkylating agents, such as iodoacetamide. Furthermore, inhibition by 12-IODE requires O2 and a low concentration of 13-HPOD, which also stimulates the normal catalytic reaction. These results suggest that 12-IODE may be a suicide inhibitor, and the goal of this project is to elucidate the inhibition mechanism. The knowledge gained from this study should be applicable to the design of specific inhibitors of a mammalian lipoxygenase involved in anaphylaxis and others that may be involved in platelet aggregation and in regulation of the activity of natural killer cells. Our working hypothesis is that 12-IODE reacts with a ferryl intermediate in the lipoxygenase-catalyzed decomposition of 13-HPOD and is thereby converted to a highly reactive iodoso compound, which reacts with an active-site nucleophile. To test this hypothesis we will carry out the following experiments. (1) We will inhibit that enzyme with 125I-12-IODE and determine if the iodine is released as 125IO-, as predicted by our hypothesis. (2) We will determine whether lipoxygenase catalyzes the O2- and 13-HPOD-dependent oxidation of the sulfur atom in 12-mercapto-cis-9-octadecenoic acid. (3) We will inhibit the enzyme with 1-14C-12-IODE and determine if radioactivity is incorporated into the protein in a way that is consistent with our hypothesis. Our proposed synthesis of 1-14C-12-IODE may provide a general method of incorporating isotopic carbon into unsaturated fatty acids that is less cumbersome than methods now in use.

大豆脂氧酶是一种非血红素铁蛋白，可催化 线酸向13-氢氧基-9,11-二十二烯酸的氧合氧合 （13-HPOD）。 我们发现12- iodo-cis-9-otctadecenoic Acid（12-iode）是 时间依赖性（T1/2 = 4 mum 12-iode）不可逆的抑制剂 这种酶。 这个结果很有趣，因为12-iode很差 烷基化剂和大豆脂氧酶不会被高抑制 良好的烷基化剂的浓度，例如碘乙酰胺。 此外，由12个IODE抑制需要O2，低浓度 13-HPOD，还刺激了正常的催化反应。 这些 结果表明12-IODE可能是自杀抑制剂，目的是 该项目是为了阐明抑制机制。 知识 从这项研究中获得的应该适用于特定的设计 参与过敏反应和其他的哺乳动物脂氧合酶的抑制剂 这可能参与血小板聚集和调节 天然杀手细胞的活性。 我们的工作假设是12个IODE与轮渡中间体反应 13-HPOD的脂氧合酶催化的分解，因此 转换为高反应性的iodoso化合物，该化合物与 活性位点亲核试剂。 为了检验这一假设，我们将执行 以下实验。 （1）我们将用125i-12-iode抑制该酶 并确定碘是否以125io释放，如我们的预测 假设。 （2）我们将确定脂氧合酶是否催化O2- 和13-Hpod依赖性硫原子中的氧化 12-羟基-CIS-9-二十二烯酸。 （3）我们将用 1-14c-12-iode并确定放射性是否纳入 蛋白质以与我们的假设一致的方式。 我们提出的 1-14C-12-iode的合成可能提供一种通用的方法 同位素碳成不饱和脂肪酸，比 现在使用的方法。