MOLECULAR CHARACTERIZATION OF ACYL-COA DEHYDROGENASES

酰辅酶A脱氢酶(ACDs)是一个进化相关的家族 参与脂肪酸第一步β氧化的酶 在亮氨酸、异亮氨酸和缬氨酸的中间代谢中。 这些酶的缺陷越来越多地被认为是 人类遗传性代谢缺陷的重要原因。《长河》 此应用程序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 将这一信息与导致临床缺陷的突变联系起来 这些酶中。这项建议的具体目标包括1)分析 异戊酰辅酶A脱氢酶(IVD)的成熟途径 该基因家族的基因，侧重于四聚化过程，2) 缺乏症患者IVD突变等位基因的表达分析 IVD作为功能性自然发生突变的来源 意义，以及3)结构/功能关系的表征 IVD四聚反应及底物测定的重要性 ACD家族其他成员的特异性。IVD成熟的途径 将通过组织中过度表达的IVD的代谢标记来研究 通过真核表达载体培养细胞，并通过体外培养 线粒体加工实验。细胞分级研究将是 用于鉴定这一过程的亚细胞定位，以及凝胶 线粒体体外转运实验中提取液的筛选 将识别线粒体内的中间体。体外诱变将 用于产生缺乏四聚作用的IVD突变体，以便 确定对这一过程至关重要的氨基酸基序和残基。突变型 来自IVD缺陷患者的等位基因将通过这些研究 相同的技术，以表征 由这些患者的细胞产生的异常酶蛋白。最后， 我们将剖析该基因家族中的结构/功能关系 以更严格的方式使用体外聚合酶链式反应技术- 特异性突变和结构域转换。这些调查将集中于 关于控制测定的氨基酸残基性质的探讨 不同ACDs的底物特异性。正常短链和长链 酰辅酶A脱氢酶将在原核表达 系统，以及SCAD的三级和四级结构将通过x- 射线结晶学。域切换技术将用于 合成具有改变的底物结合特性的杂交ACD，以及 底物特异性将与存在的ACD序列相关联 确定在确定这一点上重要的氨基酸序列 很有特色。这些研究将导致对 ACD基因家族的一部分，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.