MOLECULAR CHARACTERIZATION OF ACYL-COA DEHYDROGENASES

酰基辅酶A转移酶（acyl-CoA dish ases，ACD）是一个进化上相关的酶家族， 参与脂肪酸β-氧化第一步的酶 以及亮氨酸、异亮氨酸和缬氨酸的中间代谢。 这些酶的缺陷越来越被认为是 人类遗传性代谢缺陷的重要原因。长 本申请的范围目标是研究重要的结构/ ACD基因家族在分子水平上的功能关系，以及 将这些信息与导致临床缺陷的突变联系起来 这些酶。本建议的具体目标包括：（1）分析 异戊酰辅酶A脱氢酶（IVD）成熟途径， 这个基因家族的，重点是四聚体的过程，2） 缺乏症患者突变型IVD等位基因的表达分析 IVD作为天然发生的功能突变的来源 重要性，以及3）结构/功能关系的表征 在IVD四聚体和底物测定中重要性 其他ACD家族成员的特异性。IVD成熟的途径 将通过代谢标记组织中过表达的IVD进行研究 通过真核表达载体培养细胞， 线粒体加工实验细胞分级分离研究将 用于确定这一过程的亚细胞定位， 从体外转运实验中过滤线粒体提取物 将识别线粒体内的中间体。体外诱变将 用于产生四聚体缺陷的IVD突变体， 鉴定对该过程至关重要的氨基酸基序和残基。突变体 来自IVD缺陷患者的等位基因将通过这些研究 同样的技术，以表征的功能性质， 这些病人细胞产生的异常酶蛋白。最后， 在这个基因家族的结构/功能关系将被剖析 在一个更严格的方式使用PCR技术在体外网站- 特异性诱变和结构域转换。这些调查将集中在 控制测定的氨基酸残基的性质 各种ACD的底物特异性。正常短链和长链 酰基-CoA脱氢酶将在原核表达中表达 系统，和SCAD三级和四级结构将研究x- 射线晶体学域切换技术将用于 合成具有改变的底物结合特异性的杂合ACD，和 底物特异性将与存在的ACD序列相关 以确定氨基酸序列的重要性， 特色这些研究将使我们更全面地了解 ACD基因家族，以及诊断和治疗能力的提高， 治疗患有这些疾病的患者。

The acyl-CoA dehydrogenases (ACDs) are a family of evolutionarily related enzymes involved in the first step of the beta-oxidation of fatty acids and in the intermediate metabolism of leucine, isoleucine and valine. Deficiencies of these enzymes are increasingly being recognized as important causes of inherited defects of metabolism in humans. The long range objective of this application is to investigate important structure/ function relationships in the ACD gene family at the molecular level, and relate this information to mutations responsible for clinical deficiencies of these enzymes. Specific aims of this proposal include 1) analysis of the pathway of maturation of isovaleryl-CoA dehydrogenase (IVD), a member of this gene family, focusing on the process of tetramerization, 2) expression analysis of mutant IVD alleles from patients with deficiencies of IVD as a source of naturally occurring mutations of functional significance, and 3) characterization of structure/function relationships important in tetramerization of IVD and determination of substrate specificity of other ACD family members. The pathway of IVD maturation will be studied by metabolic labeling of IVD overexpressed in tissue culture cells via a eukaryotic expression vector, and by in vitro mitochondrial processing experiments. Cell fractionation studies will be used to identify the subcellular localization of this process, and gel filtration of mitochondrial extracts from in vitro transport experiments will identify intra-mitochondrial intermediates. In vitro mutagenesis will be used to produce IVD mutants deficient in tetramerization in order to identify amino acid motifs and residues critical to this process. Mutant alleles from patients with deficiencies of IVD will be studied via these same techniques in order to characterize the functional nature of the abnormal enzyme proteins produced by these patients' cells. Finally, structure/function relationships within this gene family will be dissected in a more rigorous fashion using the techniques of PCR in vitro site- specific mutagenesis and domain switching. These investigations will focus on the nature of the amino acid residues controlling the determination of substrate specificity of the various ACDs. Normal short and long chain acyl-CoA dehydrogenases will be expressed in a prokaryotic expression systems, and SCAD tertiary and quarternery structure will be studied by x- ray crystallography. Domain switching techniques will be used to synthesize hybrid ACDs with altered substrate binding specificities, and the substrate specificity will be correlated to the ACD sequences present to identify amino acid sequences important in determining this characteristic. These studies will lead to a more complete understanding of the ACD gene family, and an improvement in the ability to diagnose and treat patients with these disorders.