STRUCTURE OF BRANCHED CHAIN KETO ACID DEHYDROGENASE

支链酮酸脱氢酶是一种多酶 复合体，这是共同的代谢所有三个分支 哺乳动物中的氨基酸链。 在哺乳动物中 它调节代谢物从这些氨基酸流向 能量代谢，并转化为再生，是网站的 遗传性代谢缺陷群中的一种突变， 枫糖浆尿病 哺乳动物和细菌 复合物已被纯化，并由四个 多肽，E1（α和β亚基），E2和E3（硫辛酰胺 脱氢酶）。 哺乳动物复合体由 通过E1 α亚基的磷酸化，细菌复合物被 由正效应子L-缬氨酸调节。 长期 本研究的目的是了解其结构、功能 和调节细菌复合体的活性， 理解两种复合物活性的良好模型。 本研究的具体目的是：（1）研究 E1和E2亚基的结构和功能。 E1亚基 将从E中纯化。大肠杆菌TB 1，其携带质粒， 这些亚基的结构基因。 这些多肽将是 其特征在于，用作抗原以制备特异性抗血清， 溴化氰肽将被部分测序。 的DNA 将确定结构基因的序列，以便 确定丙酮酸和2- 酮戊二酸脱氢酶杆菌 类似的研究计划是 E2亚基的结构和功能， 将与丙酮酸的E2亚基和2- 酮戊二酸脱氢酶杆菌 (2)支链酮基因表达调控的研究 酸性脱氢酶操纵子 这些研究的目的是 以确定操纵子是正调节还是负调节。 调控基因和结构基因将被分离， 亚克隆和结构基因的表达将是 在调节基因存在和不存在的情况下进行研究。 (3)丙酮酸和2-酮戊二酸的克隆和测序 恶臭假单胞菌的脱氢酶结构基因。 这两 将复合物克隆到pKT 230中，pKT 230是一种广泛宿主范围的质粒， DNA序列将被确定并与 支链酮酸脱氢酶以及丙酮酸 和2-酮戊二酸脱氢酶。杆菌

Branched chain keto acid dehydrogenase is a multienzyme complex which is common to the metabolism of all three branched chain amino acids in pseudomonas and in mammals. In mammals it regulates the flow of metabolites from these amino acids into energy metabolism and into gluconeogenesis and is the site of the mutation in the cluster of inborn errors of metabolism known as maple syrup urine disease. Both the mammalian and bacterial complexes have been purified and are composed of four polypeptides, E1 (alpha and beta subunits), E2 and E3 (lipoamide dehydrogenase). The mammalian complex is regulated by phosphorylation of the E1 alpha subunit, the bacterial complex is regulated by L-valine which is a positive effector. The long-term objective of this research is to understand the structure, function and regulation of activity of the bacterial complex, which is a good model for understanding activity of both complexes. The Specific Aims of the present research are to: (1) Study the structure and function of the E1 and E2 subunits. The E1 subunits will be purified from E. coli TB1 carrying a plasmid with the structural genes for these subunits. These polypeptides will be characterized, used as antigens to prepare specific antisera and cyanogen bromide peptides will be partially sequenced. The DNA sequence of the structural gene will be determined in order to identify any evolutionary relationship to pyruvate and 2- ketoglutarate dehydrogenase of E. coli. A similar plan of study is proposed for the structure and function of the E2 subunit which will be compared with the E2 subunits of pyruvate and 2- ketoglutarate dehydrogenases of E. coli. (2) Study the regulation of gene expression of branched chain keto acid dehydrogenase operon. The objective of these studies will be to determine if the operon is positively or negatively regulated. The regulatory and structural genes will be separated by subcloning and the expression of the structural genes will be studied in the presence and absence of the regulatory gene. (3) Clone and sequence the pyruvate and 2-ketoglutarate dehydrogenase structural genes of P. putida. Both of these complexes will be cloned into pKT230, a broad host range plasmid, and the DNA sequence will be determined and compared with branched chain keto acid dehydrogenase as well as with pyruvate and 2-ketoglutarate dehydrogenase of E. coli.