BIOCHEMICAL AND MOLECULAR BASIS OF ETF-QO DEFICIENCY

II型戊二酸血症（GA2）是一种由电子转移黄蛋白（ETF）或电子转移黄蛋白-泛醌氧化还原酶（ETF- qo）缺乏引起的遗传性代谢性疾病。因为ETF是（至少）九种黄蛋白脱氢酶的电子受体，缺乏这些蛋白质中的任何一种都会损害线粒体氧化途径。本提案侧重于ETF- qo及其与ETF的反应。使用最近建立的ETF-QO基因结构来扩增单个外显子，将鉴定免疫和酶学上确定的ETF-GO缺乏症GA2患者的ETF-QO突变。突变型和野生型etf与ETF-QO之间的相互作用将在动力学实验中进行研究。这些实验采用了两个区域的位点导向突变体，基于几种etf的晶体结构的比较（与Kim博士合作确定），预测这些突变体会参与复杂的形成。要研究的位点定向突变的基本原理也基于先前的实验，这些实验涉及ETF与ETF- qo相互作用中β -ETF亚基的特定赖氨酸含区。ETF-QO的三维结构将通过x射线晶体学来确定，以更好地了解突变的影响和野生型蛋白的功能。最后，我们将建立表达系统，研究选定的致病突变对该整体膜蛋白黄素和铁硫氧化还原中心的影响。

Glutaric acidemia type II (GA2) is an inherited metabolic disease resulting from deficiency of electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). Because ETF is the electron acceptor for (at least) nine flavoprotein dehydrogenases, deficiency of either of these proteins impairs mitochondrial oxidation pathway. This proposal focuses on ETF-QO, and on its reaction with ETF. Mutations in ETF-QO from GA2 patients with immunologically and enzymatically established ETF-GO deficiency will be identified, using the recently established structure of the ETF-QO gene to amplify individual exons. The interactions between mutant and wild- type ETFs and ETF-QO will be investigated in kinetic experiments. These experiments employ site-directed mutants in two regions that, based on comparisons of the crystal structures of several ETFs (determined in collaboration with Dr. Kim), are predicted to participate in complex formation. The rationale for the site-directed mutations to be studied is also based on previous experiments that implicate specific lysine-containing regions of the beta-ETF subunit in the interaction of ETF with ETF-QO. The three-dimensional structure of ETF-QO will be determined by x-ray crystallography, to better understand the effects of mutations and the functioning of the wild-type protein. Finally, we will establish expression systems to investigate the effects of selected disease- causing mutations on the flavin and iron sulfur redox centers of this integral membrane protein.