MECHANISMS THAT REGULATE ENZYMES THAT METABOLIZE CIS UNSATURATED FATTY ACIDS

工作总结：目标是开发一个 对生物化学机制的理解 花生四烯酸和亚油酸通过这些酶的代谢。 据报道，一氧化氮(NO)可促进前列腺素的形成 通过提高PGHS的活性。我们发现NO是一种底物 对于PGHS-1和-2的过氧化物酶活性，但不能增强 体外环氧合酶活性。在巨噬细胞中，NO不起作用 刺激PGHS的表达或改变内毒素依赖的PGHS-2 表情。此外，NO不能增加前列腺素 在完整的细胞中形成。我们提出了过亚硝酸根的一种反应 NO和超氧化物的产物是罪魁祸首。其他研究 与PGHS-1一起表明形成了一种酪氨酸自由基，它可以 是引发花生四烯酸的活性中间体 氧合作用。最近，我们已经证明了NO与 酪氨酸基转化为一个新的特征根 经ESR分析为亚氨氧基。亚胺氧基自由基衰变 到硝基酪氨酸，导致酪氨酸部分硝化 小灵通。此外，它形成的硝基酪氨酸经常被用作 氧化应激的指标。我们已经确定了具体的 NO与酪氨酸反应硝化PHS中的酪氨酸部分 自由基如酪氨酸-385。这表明酪氨酸-385被氧化了 在催化过程中生成酪氨酸基，并表明该残基是 可能的活性部位或者是酶失活的部位。理解 调节酶活性和活性的生化机制 这些酶的表达将为我们提供对 潜在地控制或预防这些疾病状况。

Summary of Work: The goal is to develop an understanding of the biochemical mechanisms responsible for the metabolism of arachidonic acid and linoleic acid by these enzymes. Nitric oxide (NO) is reported to enhance prostaglandin formation by enhancement of PGHS activity. We found that NO is a substrate for the peroxidase of PGHS-1 and -2, but it does not enhance the cyclooxygenase activity in vitro. In macrophages, NO does not stimulate the expression of PGHS or alter LPS dependent PGHS-2 expression. Furthermore, NO did not enhance prostaglandin formation in intact cells. We propose htat peroxynitrite, a reaction product of NO and superoxide oxide is responsible. Other studies with PGHS-1 indicate that a tyrosyl radical is formed which could be the reactive intermediate that initiates arachidonic acid oxygenation. Recently, we have shown that NO reacts with the tyrosyl radical converting the radical to a new radical characterized by ESR analysis as an iminoxyl radical. The iminoxyl radical decays to nitrotyrosine resulting in the nitration of the tyrosine moiety on PHS. In addition, its formation of nitrotyrosine is often used as an indicator of oxidative stress. We have identified the specific tyrosine moiety in PHS nitrated by the reaction of NO with tyrosyl radical as Tyrosine-385. This indicates that tyrosine-385 is oxidized to tyrosyl radical during catalysis and suggests this residue is a likely active site or is the site of enzyme inactivation. Understanding the biochemical mechanisms which regulate the activities and expression of these enzymes will provide new insights into potentially controlling or preventing these disease conditions.