SEQUENCE AND FUNCTIONAL ANALYSIS OF TRANSCARBOXYLASE

转羧酶的序列和功能分析

基本信息

批准号：
3280632
负责人：
DAVID SAMOLS
金额：
$ 9.11万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
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/3280632
关键词：
Escherichia coli Propionibacteriaceae biotin enzyme complex enzyme structure gel electrophoresis gene expression genetic manipulation molecular cloning point mutation

项目摘要

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于多亚基酶， Shermanii丙菌群的转羧酸酶。转羧酸酶是a 含有复合生物素的含有良好特征的酶，由30个组成三种不同类型的多肽； 12 1.3se生物素亚基，6 5SE 二聚体外酮酸位点亚基和1个12SH六聚体中央COA 酯场地亚基。每个亚基催化的反应被理解并且可以解离并重新组装以形成活性酶。尽管对催化和亚基相互作用，细节刚刚开始实验平易近人。 1.3Se亚基的氨基酸序列已知已经允许分离几种模仿一些小肽体内亚基相互作用的特性。这些肽牵连末端和区域与1.3Se亚基的生物细胞相邻参与将外部和中央亚基的结合在一起以及参与定向生物素，以便可以用作羧基载体。我们已经克隆了 1.3SE亚基和5SE单体的基因，并已表达前者在大肠杆菌中高水平。表达的1.3se亚基是由大肠杆菌生物素合成酶生物生物，并且具有生物活性。我们将使用此克隆来产生许多变体亚基特性将在生化检查。此外，我们将推论从其DNA和克隆和序列的5SE单体的氨基酸序列 12sh单体基因。我们将构建五个1.3Se亚基变体，以测试一些关于从肽研究得出的亚基功能的假设。这这种方法的优势是可以实现更改维持子单元的大部分或全部长度。具体来说，我们将使用1.3SE基因内的限制性核酸内切酶位点生成在每个终端上截断的亚基，BAL 31，生成一系列短终端缺失和站点定向诱变技术引入生物细胞附近的点突变。每个变体将是测试了其生物培养，结合外部和中央亚基的能力，充当羧基载体。根据这些研究的结果，将生成变体基因的进一步修改大肠杆菌并测试。这种突变蛋白将更加紧密地描述 1.3SE肽的区域对于亚基相互作用很重要和适当的酶促功能。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mutagenesis affecting the carboxyl terminus of the biotinyl subunit of transcarboxylase. Effects on biotination.

影响转羧酶生物素亚基羧基末端的诱变。

DOI：
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Murtif,VL;Samols,D
通讯作者：
Samols,D

Effect of mutations at Met-88 and Met-90 on the biotination of Lys-89 of the apo 1.3S subunit of transcarboxylase.

Met-88 和 Met-90 突变对转羧酶 apo 1.3S 亚基 Lys-89 生物素化的影响。

DOI：
10.1096/fasebj.2.9.3131174
发表时间：
1988
期刊：
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
影响因子：
0
作者：
Shenoy,BC;Paranjape,S;Murtif,VL;Kumar,GK;Samols,D;Wood,HG
通讯作者：
Wood,HG

Evolutionary conservation among biotin enzymes.

生物素酶之间的进化保守性。