FLAVOPROTEINS IN FATTY ACID METABOLISM

描述：该提案主要关注酰基辅酶A脱氢酶 和代谢相关的酶。 这些黄素蛋白假定 在脂肪酸遗传缺陷中具有相当大的代谢重要性 在人体氧化和细胞毒性脂肪酸的生物活化中。 酰基辅酶A脱氢酶催化的三个基本方面将是 处理。 首先，氢键在极化中的作用， 将使用FAD和底物探测酰基-CoA硫酯的羰基 类似物 这些研究还将讨论氢键在生物化学中的重要性。 稳定烯醇化物类。 第二，去溶剂化的作用， 质子化状态和静电相互作用的活性位点内的 通过静态和快速反应检测酰基辅酶A脱氢酶 使用天然和突变蛋白质的研究。 氧气的调制 人们对黄素蛋白的反应性知之甚少，因此第三个目标是 使用定点突变和随机突变研究来评估 酰基辅酶A脱氢酶可通过增加溶剂而转化为氧化酶 FAD辅助因子的可访问性。 相应地，TRP166在以下方面的作用： 脱氢酶与电子转移黄素蛋白的相互作用 将被探测。 第四个目的是解决细胞毒性药物的作用方式。 卤代烃的4-硫代-脂肪酸衍生物， 三氯乙烯 相应的CoA硫酯被 酰基辅酶A脱氢酶释放高活性 α-卤代硫醇盐种类。 表征这些化合物的反应性 化合物的细胞毒性和致突变性的理解是重要的 这些物种。 最后，分泌的二硫键形成的最终氧化剂 蛋白质还不清楚。 从鸡蛋中分离的一种新的黄素氧化酶 将表征白色及其在二硫化物生成中的潜在作用 将被研究。

DESCRIPTION: This proposal focuses mainly on the acyl-CoA dehydrogenases and metabolically related enzymes. These flavoproteins have assumed considerable metabolic importance in genetic deficiencies of fatty acid oxidation in humans and in the bioactivation of cytotoxic fatty acids. Three fundamental aspects of acyl-CoA dehydrogenase catalysis will be addressed. First, the role of hydrogen bonding in the polarization of the carbonyl group of acyl-CoA thioesters will be probed using FAD and substrate analogs. These studies will also address the importance of H-bonds in the stabilization of enolate species. Second, the role of desolvation, protonation state and electrostatic interactions within the active site of the acyl-CoA dehydrogenase will be examined by static and rapid reaction studies using native and mutant proteins. The modulation of the oxygen reactivity of flavoproteins is very poorly understood, and so a third goal is to use site directed and random mutagenesis studies to assess whether the acyl-CoA dehydrogenase can be converted to an oxidase by increasing solvent accessibility to the FAD cofactor. Correspondingly, the role of TRP166 in the interaction between the dehydrogenase and electron transfer flavoprotein will be probed. A fourth aim addresses the mode of action of cytotoxic 4-thia-fatty acid derivatives of halogenated hydrocarbons such as trichloroethylene. The corresponding CoA thioesters are activated by the acyl-CoA dehydrogenase with the release of highly reactive alpha-halo-thiolate species. Characterization of the reactivity of these compounds is important in understanding the cytotoxicity and mutagenicity of these species. Finally, the ultimate oxidant for disulfide bond formation in secreted proteins is still unclear. A new flavoenzyme oxidase isolated from egg white will be characterized and its potential role in disulfide generation will be studied.