STRUCTURE AND MECHANISM OF A FLAVOPROTEIN DEHYDROGENASE

这一建议是对中链酰基- 猪肝脏的CoA脱氢酶（MCAD），以获得更详细的图片， 并确定相关酶，人MCAD， 猪短链酰基CoA脱氢酶（SCAD）和类似酶 来自细菌。 它们是一个酶家族的成员， 脂肪酸催化剂的第一个氧化步骤， 为许多器官提供燃料，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。 临界 这些酶在代谢中的作用通过人类疾病的严重性来说明。 疾病归因于三个遗传缺陷的每一个 - 是的 来自猪肝线粒体的MCAD最近被 研究深入，并在阐明 酶的催化、结构和细胞特性。 详细 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的结构联系起来， 催化机制。 该酶的分子量为172，000， 含有四个相同的亚基，每个亚基含有一个当量的黄素， 腺嘌呤二核苷酸（FAD）。 酶的三维X射线分析 3A分辨率显示了FAD的位置和方向， 整个肽链的构象。 多肽折叠近 FAD结合位点不同于在其他细胞中所见的结构， 黄素蛋白 建议将结构分析扩展到2A 分离并阐明酰基辅酶A氧化的详细机制 硫酯和电子转移到电子转移黄素蛋白， 线粒体中He脱氢酶的生理氧化剂。 也是 建议启动酶和晶体学研究， 人类MCAD;这种酶已经被克隆，研究将使用 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 以及对M. elsdenii，已经被 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和特点， 确定每种酰基辅酶A脱氢酶的特异性。

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.