STRUCTURE OF BRANCHED CHAIN KETO ACID DEHYDROGENASE

支链酮酸脱氢酶是一种多酶 所有三个分支的新陈代谢所共有的复合体 假单胞菌和哺乳动物中的链状氨基酸。在哺乳动物身上 它调节代谢产物从这些氨基酸流向 能量代谢并转化为糖异生，是 新陈代谢先天错误簇的突变称为 枫糖浆尿病。哺乳动物和细菌 络合物已得到提纯，由四个 多肽，E1(α和β亚基)，E2和E3(脂酰胺) 脱氢酶)。哺乳动物复合体受 E1α亚基的磷酸化，细菌复合体是 受L调节，是一种积极的效应物质--缬氨酸。长期的 本研究的目的是了解该基因的结构、功能 和调节细菌复合体的活性，这是一种 这是理解这两种络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亚单位 将从携带带有 这些亚基的结构基因。这些多肽将是 具有特征的，用作抗原以制备特定的抗血清和 溴化氰多肽将被部分测序。DNA 将确定结构基因的序列，以便 确定丙酮酸和2-丙酮酸的任何进化关系 大肠杆菌的酮戊二酸脱氢酶。一个类似的学习计划是 对E2亚基的结构和功能提出了建议 将与丙酮酸和2-亚基的E2亚基进行比较 大肠杆菌的酮戊二酸脱氢酶。 (2)研究支链酮的基因表达调控 酸脱氢酶操纵子。这些研究的目标将是 以确定操纵子是正向调控还是负向调控。 调控基因和结构基因将通过 结构基因的亚克隆和表达将被 在存在和不存在调控基因的情况下进行研究。 (3)丙酮酸和2-酮戊二酸的克隆和测序 恶臭假单胞菌脱氢酶结构基因。这两个都是 将复合体克隆到宿主范围广的pKT230中， DNA序列将被确定并与 支链酮酸脱氢酶以及与丙酮酸 和大肠杆菌的2-酮戊二酸脱氢酶。

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.