SUBUNITS AND QUATERNARY STRUCTURE OF TRANSCARBOXYLASE

转羧酶的亚基和四级结构

• 批准号: 3298713
• 负责人: DAVID SAMOLS
• 金额: $ 35.78万
• 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 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298713
• 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.

转羧酶是一种含生物素的酶， 细菌，谢氏丙酸杆菌（Propionibacterium shermanii）。 这只是众多例子之一 生物素酶广泛存在于生物体中。 它们发生在 哺乳动物和催化的重要步骤， 脂肪生成和氨基酸代谢。 在原核生物中， 被发现能产生质子梯度。 一 在婴儿遗传性疾病中缺乏这些酶， 往往是致命的。 转羧酶是最好的特点，所有的 生物素酶 它由3种不同类型的亚基组成， 每种催化剂都有不同的催化功能， 在整个反应中。 这些亚基可以被分离出来， 在部分反应中测量的单个活性。 此外，本发明还提供了一种方法， 完整的活性酶可以从个体重构 亚单位，并充分发挥作用。 每个亚基都已克隆， 氨基酸序列测定。 以克隆人为工具， 定点诱变，结构将在 单个亚基的特定位点。 总体目标是 解剖每个亚单位的结构，并确定 其结构与其催化功能的关系， 完整酶的作用。 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 是围绕生物素的保守的瓦尔/Ala Met Bct Met（Bct是 生物素基赖氨酸）。 这种保护作用在数百万年的 进化几乎可以肯定地表明，这一序列是必不可少的， 生物素酶的催化作用，或者它提供了 一种信号，通知合成酶将生物素连接到 赖氨酸，这是要被生物素化的特定赖氨酸， 装腔作势。 一个目标将是改变这一点， 保守位点和其他生物素亚基，以确定 生物素作为羧基载体的确切要求， 以确定是什么将合成酶导向赖氨酸， 被生物素化 还将进行定点诱变， 确定其他两个亚基的功能域。 到 提供有关结构和功能的进一步信息， 一个目标是确定 用X射线和电子显微镜观察亚基和亚基突变体 他们的晶体显微镜，从而关联的变化， 三级结构与功能的变化，观察到的网站- 定向诱变