SEQUENCE AND FUNCTIONAL ANALYSIS OF TRANSCARBOXYLASE

该提案中描述的研究以多亚单位酶为中心， 谢尔曼丙酸杆菌转羧酶。转羧酶是一种 由30个生物素组成的复杂生物素酶具有良好的特性 3种不同类型的多肽：12个1.3SE生物素亚基，6个5SE 二聚体外层酮酸中心亚基和112SH六聚体中心辅酶A 酯基亚基。每个亚基催化的反应是可以理解的 亚基可以解离和重组，形成活性酶。 虽然对催化和催化的机理有一个普遍的理解 亚基相互作用，细节刚刚成为实验性的 平易近人。已知1.3Se亚基的氨基酸序列 已经允许分离出几个小肽，它们模仿一些 体内亚基相互作用的性质。这些多肽已经牵连到 1.3SE亚基AS的末端和与生物细胞蛋白相邻的区域 参与外亚单位和中央亚基的结合。 使生物素定向，使其可以作为羧基载体。我们已经克隆了 1.3SE亚基和5SE单体的基因已经表达 前者在大肠杆菌中含量较高。表达的1.3SE亚基为 由大肠杆菌生物素合成酶生物标记，具有生物活性。 我们将使用这个克隆来产生一些变异的亚基，其 将对其进行生化检查。此外，我们还将推导出 ITS DNA中5SE单体的氨基酸序列及其克隆和测序 12SH单体基因。 我们将构建五个1.3SE亚单位变体，以测试一些 多肽研究衍生的有关亚基功能的假说。这个 这种方法的优点是可以在以下情况下完成更改 保持亚基的大部分或全部全长。具体来说， 我们将利用1.3SE基因内的限制性内切酶位点来生产 亚基在每个末端被截断，Bal 31生成一系列短 末端缺失和定点突变技术介绍 生物细胞素附近的点突变。每一种变体都将是 测试它的生物标记能力，结合外部和中央亚基， 起羧基载体的作用。根据这些研究的结果， 变异基因的进一步修饰将产生，表达在 并对其进行了检测。这样的突变蛋白质将更紧密地描绘出 1.3SE多肽与亚基相互作用的重要区域 和适当的酶功能。

The research described in this proposal centers on the multisubunit enzyme, transcarboxylase from Propionibacterium shermanii. Transcarboxylase is a well characterized complex biotin-containing enzyme composed of 30 polypeptides of 3 different types; 12 1.3SE biotinyl subunits, 6 5SE dimeric outer keto acid site subunits and 1 12SH hexameric central CoA ester site subunit. The reaction catalyzed by each subunit is understood and the subunits can be dissociated and reassembled to form active enzyme. Although there is a general understanding of the mechanism of catalysis and subunit interaction, the details are just now becoming experimentally approachable. The amino acid sequence of the 1.3SE subunit is known which has permitted the isolation of several small peptides which mimic some properties of in vivo subunit interactions. These peptides have implicated the termini and the region adjacent to the biocytin of the 1.3SE subunit as being involved in binding of the outer and central subunits together and in orienting the biotin so it can serve as a carboxyl carrier. We have cloned the genes for the 1.3SE subunit and for the 5SE monomer and have expressed the former at high levels in E. coli. The expressed 1.3SE subunit is biotinated by the E. coli biotinyl synthetase and is biologically active. We will use this clone to produce a number of variant subunits whose properties will be biochemically examined. In addition, we will deduce the amino acid sequence of the 5SE monomer from its DNA and clone and sequence the 12SH monomer gene. We will construct five 1.3SE subunit variants in order to test some of the hypotheses concerning subunit function derived from peptide studies. The advantage of this approach is that alterations can be accomplished while maintaining most or all of the full length of the subunit. Specifically, we will use restriction endonuclease sites within the 1.3SE gene to produce subunits truncated on each terminus, Bal 31 to generate a series of short terminal deletions and site directed mutagenesis techniques to introduce point mutations in the vicinity of the biocytin. Each variant will be tested for its ability to be biotinated, bind outer and central subunits, function as a carboxyl carrier. Depending on the results of these studies, further modifications of the variant genes will be generated, expressed in E. coli and tested. Such mutant proteins will more closely delineate the regions of the 1.3SE peptide which are important for subunit interactions and proper enzymatic function.