GENE STRUCTURE OF CARBAMYL PHOSPHATE SYNTHETASE

氨甲酰磷酸合成酶的基因结构

• 批准号: 3273356
• 负责人: CAROL J LUSTY
• 金额: $ 26.6万
• 依托单位: PUBLIC HEALTH RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3273356
• 关键词: Escherichia coli; Saccharomyces; adenosine triphosphate; allosteric site; arginine; carbamoylphosphate synthase; chemical binding; chemical structure function; enzyme mechanism; enzyme structure; enzyme substrate; gene deletion mutation; gene expression; genetic manipulation; genetic mapping; genetic recombination; glutamate ammonia ligase; glutaminase; laboratory rat; molecular cloning; mutant; nucleic acid sequence; ornithine; protein engineering; protein sequence; transposon /insertion element; uridine monophosphate

Carbamyl phosphate synthetase is an important housekeeping enzyme that provides carbamyl phosphate for arginine and pyrimidine biosynthesis. Our previous studies on the carbamyl phosphate synthetase genes of E. coli, yeast and rat indicated that despite differences in the substrate specificity, cofactor requirements, and subunit compositions of this phylogenetically diverse group of enzymes, they all have a common evolutionary origin. The present day arginine-specific synthetases are chimeric proteins composed of smaller modular units that endow the enzyme with a number of catalytic functions, including amide-nitrogen transfer and ATP dependent formation of C-N and C-O bonds. There are also compelling reasons to think that in some instances catalytic domains may have been modified and as a result acquired regulatory functions. In the present proposal, we intend to continue our studies of the arginine-specific carbamyl phosphate synthetases, the end objective being to describe the molecular mechanisms by which the catalytic and regulatory attributes of an evolutionarily and functionally related group of enzymes are modified and adapted in response to metabolic circumstances. Both genetic and recombinant DNA approaches will be used to achieve a dissection of the catalytic and regulatory sites of the enzyme. Most of these studies will employ mutants of E. coli impaired in carbamyl phosphate synthetase. A collection comprising 361 independent mutants has been isolated and is currently being analyzed. Carbamyl phosphate synthetases purified from strains expressing different growth requirements will be studied for their catalytic and regulatory properties. Mutations describing novel phenotypes will be mapped and sequenced to identify the catalytic and regulatory domains. Combined with in vitro mutagenesis of specific residues in the protein, this approach is anticipated to provide information about the regions that catalyze carbamate synthesis, convert carbamate to carbamyl phosphate, and bind the allosteric modifiers. A second set of studies will strive to identify the acetylglutamate regulatory domain in the rat enzyme. The coding sequence will De modified by deletions in the 5' coding region and expressed either in E. coli or in yeast. These experiments will test the hypothesis that a former glutaminase site has been converted to an acetylglutamate regulatory site.

氨基甲酰磷酸合成酶是重要的管家酶 为精氨酸和嘧啶提供了磷酸氨基甲酰基 生物合成。 我们先前关于磷酸氨基甲酰基的研究 大肠杆菌，酵母和大鼠的合成酶基因表明，尽管 底物特异性，辅因子要求的差异， 和该系统发育多样的组的亚基组成 酶，它们都有共同的进化起源。 现在 天精氨酸特异性合成酶是组成的嵌合蛋白 赋予酶的较小模块化单元 催化功能，包括酰胺氮转移和ATP C-N和C-O键的依赖形成。 也有 令人信服的理由认为在某些情况下催化 域可能已经修改，因此获得了调节 功能。 在本提案中，我们打算继续我们的 精氨酸特异性氨基甲酰磷酸合成酶的研究， 最终目标是描述分子机制 进化的催化和调节属性 并修改了与功能相关的酶组 应对代谢情况。 遗传和重组DNA方法都将用于实现 酶的催化和调节位点的解剖。 这些研究中的大多数将采用大肠杆菌突变体受损 氨基甲酰磷酸合成酶。 包含361的系列 独立突变体已被孤立，目前正在 分析。 从菌株中纯化的氨基甲酰磷酸合成酶 表达不同的增长要求将研究其 催化和调节性能。 描述小说的突变 表型将被映射和测序以鉴定催化 和监管域。 结合体外诱变 蛋白质中的特定残基，预计该方法将 提供有关催化氨基甲酸酯区域的信息 合成，将氨基甲酸酯转换为氨基甲酰磷酸盐，并结合 变构修饰符。 第二组研究将努力 识别大鼠酶中的乙酰谷氨酸调节结构域。 编码序列将通过5'编码中的删除进行修改 区域，在大肠杆菌或酵母中表达。 这些 实验将检验以前的谷氨酰胺酶部位的假设 已转化为乙酰谷氨酸调节部位。