FLAVOPROTEINS IN FATTY ACID METABOLISM

这项研究的长期目标是获得更深层次的 了解其结构、催化机理和 与脂肪酸有关的黄素蛋白的代谢调控 氧化。三种蛋白质构成了这项提案的主要焦点： 中链酰辅酶A脱氢酶及其生理活性 电子受体，电子转移黄素蛋白(ETF) 猪肾线粒体和过氧酶体酰辅酶A 酵母菌中的氧化酶。 中链所表现出的链长判别 脱氢酶将与线粒体的脱氢酶进行比较。 短链酶，看看特异性是否由 相似的特征。辅酶A亲和介质，其中辅酶是 连接到不同长度的凝胶碳氢化合物间隔物，将是 对短链、中链和长链的提纯进行了评价 酵素。对酰辅酶A脱氢酶的抑制作用 将研究反式-3-烯基-CoA衍生物，因为这些 硫代酯是不饱和脂肪酸β-氧化反应的中间体。 脂肪酸。还原中链的反应性 分子氧的脱氢酶和酰基辅酶A氧化酶 将通过快速反应技术在现场或 缺乏各种CoA衍生品来评估 络合作用对再氧化速率的影响。类似地， 促进介质还原ETF的Enoyl-CoA产品 链脱氢酶将用氧化还原失活进行研究 硫醚类似物。将使用几种方法来定位 异二聚体电子载体ETF中的FAD结合部位。 将尝试确定生理调节因子 ETF活动。脱氢酶与ETF的复合体 将通过使用黄素类似物的静态滴定进行研究 分光光度探头。FAD类比还将用于 将参与者保持在定义的氧化还原状态。2-和3- 烷基-辅酶A衍生物将用于标记活性中心 氧化型酰辅酶A脱氢酶和酰辅酶A中的多肽 氧化物酶。这些修改的目标残留物(S)将是 ，并测定了它们的氨基酸序列 检查可能的同源性。稳定的共价亚麻素 还原的中间链失活时形成的加合物 带有2-辛基-辅酶A的酰基-辅酶A将被 特色化的。

The long term goal of this research is to gain a deeper understanding of the structure, catalytic mechanism, and metabolic control of flavoproteins involved in fatty acid oxidation. Three proteins form the main focus of this proposal: the medium chain acyl-CoA dehydrogenase and its physiological electron acceptor, electron transferring flavoprotein (ETF) from pig kidney mitochondria, together with the peroxisomal acyl-CoA oxidase from yeast. The chain length discrimination shown by the medium chain dehydrogenase will be compared to that of the mitochondrial short chain enzyme to see whether specificity is dictated by similar features. CoA affinity media, in which the coenzyme is attached to the gel hydrocarbon spacers of various lengths, will be evaluated in the purification of short, medium, and long chain enzymes. The inhibition of the acyl-CoA dehydrogenases by trans-3-enoyl-CoA derivatives will be studied because these thioesters are intermediates in the beta-oxidation of unsaturated fatty acids. The reactivity of the reduced medium chain dehydrogenase and acyl-CoA oxidase toward molecular oxygen will be compared by rapid reaction techniques in the presence or absence of various CoA derivatives to assess the influence of complexation on the rate of reoxidation. Similarly, the role of enoyl-CoA product in facilitating reduction of ETF by the medium chain dehydrogenase will be investigated using redox-inactive thioether analogs. Several approaches will be used to locate the FAD binding site in the heterodimeric electron carrier, ETF. Attempts will be made to identify physiological modulators of ETF activity. Complexes between the dehydrogenase and ETF will be studied by static titrations using flavin analogs as spectrophotometric probes. FAD analogs will also be used to maintain the participants in defined redox states. 2- and 3- alkynoyl-CoA derivatives will be used to label active sites peptides in the oxidized acyl-CoA dehydrogenases and acyl-CoA oxidase. The target residue(s) of these modifications will be identified, and the amino acid sequences of these peptides examined for possible homologies. The stable covalent flavin adduct formed upon inactivation of the reduced medium chain acyl-CoA dehydrogenase with 2-octynoyl-CoA will be characterized.