SEQUENCE AND FUNCTIONAL ANALYSIS OF TRANSCARBOXYLASE

转羧酶的序列和功能分析

• 批准号: 3280630
• 负责人: DAVID SAMOLS
• 金额: $ 9.26万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280630
• 关键词: Escherichia coli; Propionibacteriaceae; biotin; enzyme complex; enzyme structure; gel electrophoresis; gene expression; genetic manipulation; molecular cloning; point mutation

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.

该提案中描述的研究集中在多亚基酶上， 来自谢氏丙酸杆菌的转羧酶。 转羧酶是一种 一种充分表征的含生物素的复合酶，由30 3种不同类型的多肽; 12个1.3SE生物素亚基，6个5SE 二聚体外部酮酸位点亚基和1 12 SH六聚体中央CoA 酯位点亚基 每个亚基催化的反应被理解为 并且亚基可以解离和重新组装以形成活性酶。 虽然对催化作用的机理有了一般的了解， 亚基相互作用，细节现在只是成为实验 平易近人 已知1.3SE亚基的氨基酸序列， 已经允许分离几种小肽， 体内亚基相互作用的性质。 这些肽与 1.3SE亚基的末端和与生物胞素相邻的区域为 参与外部和中心亚基的结合， 对生物素进行定向，使其可以作为羧基载体。 我们已经克隆 1.3SE亚基和5SE单体的基因，并已表达 前者在E.杆菌 表达的1.3SE亚基是 E.大肠杆菌生物素合成酶，具有生物活性。 我们将用这个克隆产生一些变异亚基， 将进行生化检查。 此外，我们还将推出 来自其DNA的5SE单体的氨基酸序列以及克隆和序列 12 SH单体基因。 我们将构建五个1.3SE亚基变体，以测试一些1.3SE亚基变体。 来自肽研究的关于亚基功能的假设。 的 这种方法的优点是可以实现改变， 保持亚基的大部分或全部全长。 具体地说， 我们将使用1.3SE基因内的限制性内切酶位点来产生 在每个末端截短的亚基，Bal 31，以产生一系列短的 末端缺失和定点诱变技术来引入 在生物胞素附近的点突变。 每种变体都将 测试其被生物素化、结合外部和中央亚基的能力， 作为羧基载体。 根据这些研究的结果， 变异基因的进一步修饰将被产生、表达， E.大肠杆菌，并进行了测试。 这种突变蛋白将更紧密地描绘出 1.3SE肽中对亚基相互作用重要的区域 和适当的酶功能。