ELECTRON TRANSFER FLAVOPROTEIN AND GLUTARIC ACIDEMIA II

这项研究计划的长期目标一直是将基本的 电子转移黄素蛋白(ETF)的生物化学及电子转移 黄素蛋白-泛醌氧化还原酶(ETF-Q0)导致人类先天错误 代谢，戊二酸血症II型(GA2)，并获得较好的 从缺陷研究中对正常蛋白质的认识 这些蛋白质。GA2会导致脂肪酸的氧化缺陷和 氨基酸，往往是致命的。拟议研究的目标是 在核苷酸和蛋白质水平上建立分子基础， ETF缺乏症。我们将(A)确定GA2患者的突变 ETF缺乏症；(B)在大肠杆菌中表达选定突变；(C) 研究突变体的动力学、氧化还原和结构性质 ETF和(D)解决了人类ETF的晶体结构。我们开发了两个 在大肠杆菌中表达ETF的系统。第一个表示Paracoccus 脱氮ETF。这只细菌ETF的亚基总体上有72% 与人类的序列相似性(约60%的序列同一性) 亚单位。该系统将主要用于光谱分析 我们已经承诺进行的调查。第二个，最近 利用表达载体表达人ETF的系统开发 最初是为细菌蛋白质而开发的，将用于 表现出人类的突变。其他基于基因的定点突变 用光亲和其他FAD类似物修饰人ETF 与脱辅基蛋白共价结合将扩展这些研究 ETF的基础生物化学超出了自然发生的范围 突变。这些研究的目的是确定FAD结合位点和 黄素蛋白脱氢酶与电子转移的对接位置 黄素蛋白氧化还原酶。突变体的动力学和氧化还原行为 将对ETF进行调查，以确定突变是否会影响利率 通过蛋白质的电子传递，或者它们是否影响黄素 氧化还原电位，因此通过蛋白质的电子转移成为 热力学上是不利的。正常和突变的结构研究 ETF将包括13C，15N和31P核磁共振，圆二色谱和 荧光光谱研究，并将由 结晶学研究也在进行中。

The long term aims of this research program have been to relate the basic biochemistry of electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Q0) to a human inborn error of metabolism, glutaric acidemia type II (GA2), and to obtain a better understanding of the normal proteins from investigations of defects in these proteins. GA2 results in defective oxidation of fatty acids and amino acids and is often fatal. The objective of the proposed research is to establish the molecular bases, at the nucleotide and protein levels, of ETF deficiency. We will (a) identify mutations in patients with GA2 due to ETF deficiency; (b) express selected mutations in E. coli; (c) investigate the kinetic, redox and structural properties of the mutant ETFs and (d) solve the crystal structure of human ETF. We developed two systems to express ETFs in E. coli. The first expresses Paracoccus denitrificans ETF. Subunits of this bacterial ETF have 72% overall sequence similarity (approximately 60% sequence identity) with the human subunits. This system will be used primarily for spectroscopic investigations to which we have already committed. The second, recently developed system expresses human ETF using the expression vector originally developed for the bacterial protein, and will be used to express human mutations. Other site-directed mutations based on modification of human ETF with photoaffinity and other FAD analogs that bind covalently to the apoprotein will extend these investigations of the basic biochemistry of ETF beyond those indicated by naturally occurring mutations. These investigations aim to define the FAD binding site and of docking sites for the flavoprotein dehydrogenases and electron transfer flavoprotein oxidoreductase. The kinetic and redox behavior of mutant ETFs will be investigated to determine whether mutations affect the rate of electron transfer through the protein or whether they affect the flavin redox potential, so that electron transfer through the protein becomes thermodynamically unfavorable. Structural studies on normal and mutant ETFs will include 13C, 15N and 31P NMR, circular dichroism and fluorescence spectroscopic investigations and will be complemented by crystallographic investigations which are also in progress.