FLAVOPROTEINS IN FATTY ACID METABOLISM

描述：本提案主要针对酰基辅酶A脱氢酶。 和代谢相关的酶。这些黄素蛋白假定 脂肪酸遗传缺陷中相当重要的代谢作用 人体内的氧化和细胞毒性脂肪酸的生物激活。 酰辅酶A脱氢酶催化的三个基本方面是 地址。首先，氢键在极化过程中的作用 将使用FAD和底物来探索酰基辅酶A硫代酯的羰基 类比。这些研究还将讨论氢键在 烯醇化物种的稳定。第二，荒凉的作用， 活性中心内的质子化状态和静电相互作用 酰辅酶A脱氢酶将通过静态和快速反应进行检测 使用天然蛋白和突变蛋白的研究。氧的调节 人们对黄素蛋白的反应性知之甚少，因此第三个目标 是使用定点定向和随机突变研究来评估 通过增加溶剂可以将酰基辅酶A脱氢酶转化为氧化酶 时尚辅因的可及性。相应地，TRP166在 脱氢酶与电子转移黄素蛋白的相互作用 将会被调查。第四个目标涉及细胞毒的作用模式 卤代烃的4-硫代脂肪酸衍生物，如 三氯乙烯。相应的CoA硫代酯由 酰辅酶A脱氢酶释放的高活性 阿尔法-晕-硫酸盐物种。这些化合物的反应性的表征 化合物对于理解细胞毒性和致突变性很重要。 这些物种。 最后，最终氧化剂在分泌物中形成二硫键 蛋白质仍不清楚。从鸡蛋中分离出一种新的黄素酶 白色将被表征及其在二硫化物生成中的潜在作用 将会被研究。

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.