FLAVOPROTEINS IN FATTY ACID METABOLISM

脂肪酸代谢中的黄素蛋白

• 批准号: 3274046
• 负责人: Colin Thorpe
• 金额: $ 10.02万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3274046
• 关键词: acyl coA dehydrogenases; acyl group; affinity chromatography; chemical chain length; electron transport; enzyme mechanism; enzyme structure; enzyme substrate; fatty acid metabolism; flavin adenine dinucleotide; flavoproteins; gel filtration chromatography; kidney; mitochondria; oxidation reduction reaction; oxidoreductase; peroxisome; physical separation; spectrometry; stoichiometry; stop flow technique; swine; thioester; yeasts

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.

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， 参与脂肪酸的黄素蛋白的代谢控制 氧化 三种蛋白质构成了该提案的主要焦点： 中链酰基辅酶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，以及过氧化物酶体酰基辅酶A 酵母中的氧化酶 中链显示的链长判别 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。 CoA亲和介质，其中辅酶是 连接到不同长度的凝胶烃间隔物上， 在短链、中链和长链的纯化中进行评价 内切酶 抑制酰基-CoA脱氢酶， 将研究反式-3-烯酰基-CoA衍生物，因为这些 硫酯是β-氧化不饱和脂肪酸的中间体， 脂肪酸 还原中链的反应性 脱氢酶和酰基辅酶A氧化酶对分子氧 将通过快速反应技术在存在或 缺乏各种CoA衍生物来评估 络合作用对再氧化速率的影响。 同样， 烯酰辅酶A产物在促进培养基减少ETF中的作用 链脱氢酶将使用无氧化还原活性的 硫醚类似物。 将使用几种方法来定位 异二聚体电子载体中的FAD结合位点。 将尝试鉴定以下的生理调节剂： ETF活动 脱氢酶和ETF之间的复合物 将研究静态滴定使用黄素类似物， 分光光度探针 FAD类似物也将用于 保持参与者处于确定的氧化还原状态。 2-和3- 炔酰辅酶A衍生物将用于标记活性位点 氧化酰基辅酶A脱氢酶和酰基辅酶A 氧化酶。 这些修饰的靶残基将是 这些肽的氨基酸序列 检查可能的同源性。 稳定的共价黄素 还原中链失活后形成的加合物 酰基辅酶A脱氢酶与2-辛炔酰基辅酶A将被 表征了