STRUCTURE AND MECHANISM OF A FLAVOPROTEIN DEHYDROGENASE

黄素蛋白脱氢酶的结构和机制

• 批准号: 3276547
• 负责人: JUNG JA P. KIM
• 金额: $ 17.83万
• 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/3276547
• 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; 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.

该建议是扩展中链酰基的结构研究 猪肝脏的COA脱氢酶（MCAD）获得了更详细的图片 这种酶并确定相关酶的结构，人类MCAD， 猪短链酰基COA脱氢酶（SCAD）和类似的酶 来自MA细菌。 这些是执行酶家族的成员 脂肪酸分解代谢的第一步，主要 许多器官的燃料，包括肝，肾脏，心脏和骨骼肌肉。 饮食，生理状态和 疾病，以饥饿，妊娠和糖尿病为例。 关键 这些酶在代谢中的作用被人类的严重程度说明了 归因于三种遗传缺陷的疾病 脱氢酶。 猪肝线粒体的MCAD最近是 深入研究并取得了进展，以阐明 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 将化学功能与蛋白质的结构相关联并定义 催化机制。 该酶的分子量为172,000，并且 包含四个相同的亚基，每个亚基都包含一个等效的黄素 腺嘌呤二核苷酸（FAD）。 酶的三维X射线分析 在3A时，分辨率揭示了FAD的位置和方向 整个肽链的构象。 多肽折叠附近 FAD结合位点与其他结构不同 黄蛋蛋白。 建议将结构分析扩展到2a 分辨率并阐明酰基辅酶A氧化的详细机制 硫代和电子转移到电子转移黄蛋蛋白， 线粒体中HE脱氢酶的生理氧化剂。 也是 提议启动有关酶学的酶学和晶体学研究 人类MCAD；该酶已被克隆，研究将两者都采用 由定点诱变产生的天然蛋白质和改变的蛋白质。 晶体学研究也将在猪肝的SCAD上进行 以及M. elsdenii的丁酰基-COA脱氢酶 以适合X射线分析的形式结晶。 这些比较 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及其特征 确定每个酰基-COA脱氢酶的特异性。