SUBUNITS AND QUATERNARY STRUCTURE OF TRANSCARBOXYLASE

转羧酶的亚基和四级结构

• 批准号: 3298714
• 负责人: DAVID SAMOLS
• 金额: $ 37.42万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298714
• 关键词: Escherichia coli; Propionibacterium; X ray crystallography; biotin; chemical binding; chemical group; chemical structure function; conformation; electron microscopy; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; high performance liquid chromatography; ligase; lysine; molecular cloning; nuclear magnetic resonance spectroscopy; oligopeptides; point mutation; protein engineering; protein sequence; site directed mutagenesis

Transcarboxylase is a biotin-containing enzymes from the bacterium, Propionibacterium shermanii. It is but one of many biotin enzymes which are widespread in organisms. They occur in mammals and catalyze important steps in gluconeogenesis, lipogenesis and amino acid metaboslim. In procaryotes, they have been found to generate proton gradients across members. A deficiency of these enzymes in genetic disorders of infants is often fatal. Transcarboxylase is the best characterized of all the biotin enzymes. It is made up of 3 different types of subunits, each of which has a different catalytic function, which is required in the overall reaction. These subunits can be isolated and their individual activity measured in the partial reactions. In addition, the intact active enzyme can be reconstituted from the individual subunits and is fully active. Each subunit has been cloned and its amino acid sequence determined. With the clones as tools, by site-directed mutagenesis, the structures will be altered at specific sites of the individual subunits. The overall aim is to dissect the structure of each subunit and determine the relationship of its structure to its catalytic function in relation to the action of the intact enzyme. The action of biotin is as the carboxyl carrier in biotin enzymes and in all biotin enzymes there is a conserved Val/Ala Met Bct Met surrounding the biotin (Bct is biotinyl lysine). This conservation during millions of years of evolution almost certainly indicates this sequence is essential for the catalysis of biotin enzymes in general, or that it provides the signal that informs the synthetase which attaches the biotin to the lysine, that this is the particular lysine that is to be biotinated posttranslationally. One aim will be to make changes at this conserved site and others of the biotinyl subunit to determine the exact requirement for the biotin to act as a carboxly carrier and to determine what directs the synthetase to the lysine that is to be biotinated. Site-directed mutagenesis will also be done to determine the functional domains of the other two subunits. To provide further information relating to structure and function, one aim will be to determine the three-dimensional structure of the subunits and mutants of the subunits by X-ray and electron microscopy of their crystals and thus correlate the changes in tertiary structure with changes in functions observed by site- directed mutagenesis.

转羧酶是一种含有生物素的酶，来自于 细菌，舍尔曼丙酸杆菌。它只是众多产品中的一个 生物素酶在生物体中广泛存在。它们发生在 并催化糖异生的重要步骤， 脂肪生成和氨基酸代谢。在原核生物中，它们有 已被发现在成员之间产生质子梯度。一个 这些酶在婴儿遗传性疾病中的缺陷 通常是致命的。转羧酶是最具特点的所有 生物素酶。它由3种不同类型的亚基组成， 每一种都有不同的催化功能，这是必需的 在整体反应中。这些亚基可以被分离出来，它们的 在部分反应中测量的个体活动。此外, 完整的活性酶可以从个体重组 子单元，并处于完全活动状态。每个亚基都被克隆了，它的 氨基酸序列测定。以克隆为工具，通过 定点突变，这些结构将在 个别亚基的特定位点。总体目标是 解剖每个亚基的结构，并确定 它的结构与其催化功能之间的关系 完整的酶的作用。生物素的作用是作为 生物素酶和所有生物素酶中的羧基载体 是生物素周围保守的Val/Ala Met BCT Met(BCT是 生物素赖氨酸)。在数百万年的时间里，这种保护 进化几乎可以肯定地表明，这个序列对于 生物素酶的催化作用，或者说它提供了 通知合成酶将生物素连接到 赖氨酸，这是要被生物标记的特定赖氨酸 翻译后的。一个目标将是在这一点上做出改变 生物素亚基的保守位点和其他来确定 生物素作为羧基载体的确切要求和 以确定是什么将合成酶定向到赖氨酸，即 被生物化了。定点突变也将被用来 确定其他两个亚基的功能结构域。至 提供与结构和功能有关的进一步信息， 其中一个目标是确定其三维结构 X射线和电子能谱分析亚基及其突变体 对它们的晶体进行显微镜观察，从而将它们在 三级结构与现场观测到的功能变化- 定向诱变。

项目成果

Effect of deletion from the carboxyl terminus of the 12 S subunit on activity of transcarboxylase.

12 S 亚基羧基末端缺失对转羧酶活性的影响。

• 发表时间: 1993
• 期刊: The Journal of biological chemistry
• 作者: Woo,SB;Shenoy,BC;Wood,HG;Magner,WJ;Kumar,GK;Beegen,H;Samols,D
• 通讯作者: Samols,D

Purification and characterization of the recombinant 5 S subunit of transcarboxylase from Escherichia coli.

大肠杆菌转羧酶重组 5S 亚基的纯化和表征。

• DOI: 10.1006/prep.1993.1060
• 发表时间: 1993
• 期刊: Protein expression and purification
• 影响因子: 1.6
• 作者: Xie,Y;Shenoy,BC;Magner,WJ;Hejlik,DP;Samols,D
• 通讯作者: Samols,D

The conserved methionines of the 1.3 S biotinyl subunit of transcarboxylase: effect of mutations on conformation and activity.

转羧酶 1.3 S 生物素亚基的保守蛋氨酸：突变对构象和活性的影响。

• DOI: 10.1006/abbi.1993.1362
• 发表时间: 1993
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: Shenoy,BC;Samols,D;Kumar,GK
• 通讯作者: Kumar,GK

Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence.

从基因组 DNA 序列推导出的转羧酶 5 S 亚基的一级结构。

• DOI: 10.1016/0014-5793(93)80271-u
• 发表时间: 1993
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Thornton,CG;Kumar,GK;Shenoy,BC;Haase,FC;Phillips,NF;Park,VM;Magner,WJ;Hejlik,DP;Wood,HG;Samols,D
• 通讯作者: Samols,D

The importance of methionine residues for the catalysis of the biotin enzyme, transcarboxylase. Analysis by site-directed mutagenesis.

蛋氨酸残基对于生物素酶转羧酶催化的重要性。

• 发表时间: 1992
• 期刊: The Journal of biological chemistry
• 作者: Shenoy,BC;Xie,Y;Park,VL;Kumar,GK;Beegen,H;Wood,HG;Samols,D
• 通讯作者: Samols,D