STRUCTURES AND MECHANISMS OF CITRATE ENZYMES

柠檬酸酶的结构和机制

• 批准号: 3224775
• 负责人: PAUL A SRERE
• 金额: $ 13.11万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-01-01 至 1993-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3224775
• 关键词: Escherichia coli; X ray crystallography; chemical structure function; citrate synthase; complementary DNA; conformation; enzyme mechanism; enzyme structure; genetic manipulation; molecular cloning; molecular site; point mutation; protein engineering; protein sequence; radiotracer; structural genes; swine

The goal of the research is to examine the structure and function of citrate synthase (CS) from pig and E. coli by using site-directed mutagenesis. Citrate synthase is an excellent choice to examine enzyme structure and function by using such molecular biological techniques. It is a key enzyme in aerobic energy production and metabolite interconversions. It is an important example of stereospecificity in enzyme reactions, and two distinct enzyme conformations participate during catalysis. In addition, pig citrate synthase (PCS) and E. coli citrate synthase (ECCS) have been crystallized and the three dimensional structure of PCS determined. Therefore, CS is an attractive enzyme to study by site-directed mutagenesis because of the possibility of obtaining the DNA, the protein crystals, and an in depth mechanism for the mammalian and bacterial forms of the enzyme. This additional comparative aspect between the CS from a eucaryote and a procaryote will enhance our ability to choose sites for mutagenesis, and also will explain the two different reaction mechanisms, protein structures, and regulatory behaviors of these divergently related proteins. To define in detail the reaction mechanism, structure, and biological function of CS, the cDNA encoding PCS will be isolated and sequenced. Site-directed mutagenesis of the PCS and ECCS DNAs will used to alter codons for catalytic and structural amino acid residues. The mutated and control DNAs will be expressed in vitro, and the synthesized proteins will be purified and crystallized. The partial enzyme reactions catalyzed by the control and mutated PCS and ECCS proteins will be determined and compared. The three dimensional structures of the control and mutated PCS and ECCS proteins will be compared to the known X-ray structure of PCS. In addition, the gene for an E. coli mutant that codes for the dimeric (eucaryotic) form of the enzyme will be isolated and sequenced. The amino acid sequence for the mutant ECCS will be compared to the PCS and non-mutant ECCS and may identify particualr amino acid residues that are important to subunit assembly or enzymatic function.

本研究的目的是检查结构和功能 猪柠檬酸合成酶（CS）和大肠杆菌E.大肠杆菌中进行定点 诱变 柠檬酸合酶是一个很好的选择， 酶的结构和功能，通过使用这种分子生物学 技术. 它是有氧能量产生的关键酶， 代谢物相互转化 这是一个重要的例子， 酶反应中立体专一性，两种不同的酶 构象参与催化过程。 此外，猪 柠檬酸合成酶（PCS）和E.大肠杆菌柠檬酸合酶（ECCS）具有 PCS的结晶和三维结构 测定 因此，CS是一种有吸引力的酶， 定点诱变，因为获得 DNA，蛋白质晶体，以及一个深入的机制， 哺乳动物和细菌形式的酶。 该附加 来自真核生物的CS和来自 原核生物将增强我们选择网站的能力， 诱变，也将解释两种不同的反应 机制，蛋白质结构和这些的调节行为， 不同相关的蛋白质。 为了详细定义反应， CS的分子机制、结构和生物学功能， 编码PCS将被分离和测序。 定点 PCS和ECCS DNA的诱变将用于改变密码子 催化和结构氨基酸残基。 突变和 对照DNA将在体外表达，合成的 蛋白质将被纯化和结晶。 部分酶 由对照和突变的PCS和ECCS催化的反应 将测定和比较蛋白质。 三维 对照和突变的PCS和ECCS蛋白的结构将 与已知的PCS的X射线结构进行比较。 此外，本发明还提供了一种方法， E的基因大肠杆菌突变体编码的二聚体 将酶的（真核）形式分离并测序。 将比较突变ECCS的氨基酸序列 与PCS和非突变型ECCS相关，并可识别特定的 对亚基组装重要的氨基酸残基，或 酶的功能