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.

转羧酸酶是一种来自生物素的酶 细菌，谢尔马尼丙肽。 只是众多 生物素酶在生物体中很普遍。 它们发生在 哺乳动物和催化糖异生的重要步骤， 脂肪生成和氨基酸代替。 在procaryotes中，他们有 发现可以在跨成员之间产生质子梯度。 一个 这些酶在婴儿遗传疾病中的缺乏是 经常致命。 经羧基酶是所有的最佳特征 生物素酶。 它由3种不同类型的亚基组成， 每个都有不同的催化功能，这是必需的 在整体反应中。 这些亚基可以孤立，它们 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人中重组 亚基，完全活跃。 每个亚基都被克隆到 确定的氨基酸序列。 将克隆作为工具，通过 定点诱变，结构将在 单个亚基的特定站点。 总体目的是 剖析每个亚基的结构，并确定 相对于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 生物素酶和所有生物素酶中的羧基载体 是一个保守的Val/Ala Met Met Bct围绕生物素的BCT（BCT为 生物素赖氨酸）。 在数百万年内的这种保护 进化几乎可以肯定表明此顺序对于 一般来说，生物素酶的催化 通知合成酶的信号将生物素连接到 赖氨酸，这是要生物的特定赖氨酸 翻译后。 一个目的是对此进行更改 保守的部位和其他生物素亚基，以确定 确切的要求，生物素充当羧基载体和 确定是什么将合成酶引导到赖氨酸 生物为生物。 位置定向的诱变也将进行 确定其他两个亚基的功能域。 到 提供与结构和功能有关的更多信息， 一个目的是确定三维结构 X射线和电子的亚基的亚基和突变体 其晶体的显微镜，从而将变化相关 三级结构随着位点观察到的功能变化 定向诱变。