STRUCTURE AND MECHANISM OF A FLAVOPROTEIN DEHYDROGENASE

黄素蛋白脱氢酶的结构和机制

• 批准号: 3276553
• 负责人: JUNG JA P. KIM
• 金额: $ 19.7万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-03-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3276553
• 关键词: X ray crystallography; acidity /alkalinity; acyl coA; acyl coA dehydrogenases; chemical chain length; chemical structure function; computer simulation; conformation; crystallization; electron density; electron transport; enzyme inhibitors; enzyme mechanism; enzyme structure; flavin adenine dinucleotide; flavoproteins; genetic manipulation; liver; mitochondria; oxidoreductase; protein purification; protein sequence; protein structure function; site directed mutagenesis; swine; thioester

This proposal is to extend the structural studies on the medium chain acyl- CoA dehydrogenase (MCAD) of pig liver to obtain a more detailed picture of this enzyme and to determine structures of related enzymes, the human MCAD, the porcine short chain acyl CoA dehydrogenase (SCAD) and a similar enzyme from ma bacterium. These are members of a family of enzymes that carry out the first oxidative step in the catabolism of fatty acids, the principal fuel for many organs, including liver, kidney, heart and skeletal muscle. The rate o oxidation can be altered by diet, physiological state and disease, exemplified by starvation, pregnancy and diabetes. The critical role of these enzymes in metabolism is illustrated by the severity of human diseases attributed to inherited deficiencies of each of the three dehydrogenases. The MCAD from pig liver mitochondria has recently been studies intensively and progress has been made in elucidating the catalytic, structural and cellular properties of the enzyme. Detailed structural information from high resolution X-ray analysis will enable us to relate chemical functions to the structure of the protein and to define the catalytic mechanism. The enzyme has a molecular weight of 172,000 and contains four identical subunits, each containing one equivalent of flavin adenine dinucleotide (FAD). Three-dimensional X-ray analysis of the enzyme at 3A resolution has revealed the location and orientation of the FAD and the conformation of the entire peptide chain. The polypeptide folding near the FAD binding site is different from the structures seen in other flavoproteins. It is proposed to extend the structural analysis to 2A resolution and to elucidate the detailed mechanism of oxidation of acyl-CoA thioesters and transfer of electrons to the electron transfer flavoprotein, the physiological oxidant of he dehydrogenase in mitochondria. It is also proposed to initiate both enzymatic and crystallographic studies on the human MCAD; this enzyme has been cloned and the studies will employ both the native protein and altered proteins made by site-directed mutagenesis. Crystallographic studies will also be carried out on the SCAD of pig liver and on the butyryl-CoA dehydrogenase of M. elsdenii, which has already been crystallized in a form suitable for X-ray analysis. Comparisons of these structures will enable us to ascertain the structures involved in the general catalytic mechanisms of this group of enzymes and the features that determine the specificity of each acyl-CoA dehydrogenase.

该提案旨在扩展中链酰基的结构研究 猪肝脏 CoA 脱氢酶 (MCAD) 以获得更详细的图像 这种酶并确定相关酶的结构，人类 MCAD， 猪短链酰基辅酶A脱氢酶（SCAD）和类似的酶 来自ma细菌。 这些是酶家族的成员，它们执行 脂肪酸分解代谢的第一个氧化步骤， 许多器官的燃料，包括肝脏、肾脏、心脏和骨骼肌。 氧化速率可以通过饮食、生理状态和 疾病，例如饥饿、怀孕和糖尿病。 关键的 这些酶在新陈代谢中的作用可以通过人类的严重程度来说明 归因于这三种遗传缺陷的疾病 脱氢酶。 最近，来自猪肝线粒体的 MCAD 深入研究并在阐明问题方面取得了进展 酶的催化、结构和细胞特性。 详细的 高分辨率 X 射线分析的结构信息将使我们能够 将化学功能与蛋白质结构联系起来并定义 催化机制。 该酶的分子量为 172,000 包含四个相同的亚基，每个亚基含有一当量的黄素 腺嘌呤二核苷酸（FAD）。 酶的三维X射线分析 3A 分辨率揭示了 FAD 的位置和方向 整个肽链的构象。 多肽折叠近 FAD 结合位点与其他药物中看到的结构不同 黄素蛋白。 建议将结构分析扩展到2A 解析并阐明酰基辅酶A氧化的详细机制 硫酯和电子转移至电子转移黄素蛋白， 线粒体中脱氢酶的生理氧化剂。 这也是 建议启动酶学和晶体学研究 人类 MCAD；这种酶已被克隆，研究将同时使用这两种酶 天然蛋白质和通过定点诱变产生的改变的蛋白质。 还将对猪肝的 SCAD 进行晶体学研究 以及埃氏巨球菌的丁酰辅酶A脱氢酶，该酶已被 以适合X射线分析的形式结晶。 这些的比较 结构将使我们能够确定涉及的结构 这组酶的一般催化机制和特征 确定每种酰基辅酶A脱氢酶的特异性。