Protein Engineering of Citric Acid Cycle Enzymes

柠檬酸循环酶的蛋白质工程

• 批准号: 9603656
• 负责人: Leonard Banaszak
• 金额: $ 39万
• 依托单位: University of Minnesota-Twin Cities
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9603656&HistoricalAwards=false
• 关键词: Protein; Engineering; Citric; Acid; Cycle

9603656 Banaszak As the link between protein structure and function strengthens, the number of fundamental questions which can be answered through combined use of x-ray crystallography and recombinant DNA methods increases. The specific objectives proposed by the PI fall into three diverse areas, having the unifying theme of understanding the molecular mechanisms by which cells produce metabolic energy, and the principles they use to assemble and control these pathways. First, the PI and his students will complete the x-ray crystallographic studies of fumarase from E. coli. This structural data will be critical for any attempts to build the true substrates into the current crystallographic model. Sidechains affecting the biding and electrostatics of a catalytic water molecule will be studied by mutagenesis. Closely linked to the x-ray crystallographic studies of fumarase from E. coli are parallel analyses of the enzyme from yeast. The objectives in this case are not related to active site stereochemistry but rather toward the gathering of information on organelle targeting. In this second endeavor, the PI has set the goal of determining the molecular structure of one or more of the precursor proteins targeting them to mitochondria. The final objective is related to mechanisms for serine control of an enzyme called phosphoglycerate dehydrogenase. Using recombinant technology, structural and biochemical studies of the regulatory domain will be done with and without serine. Modern biotechnology centers on the use of enzymes as chemical reagents. New developments in this area will depend on changing several properties commonly found in these protein catalysts. These include stability, control and specificity. The PI for this grant has used two major techniques in his laboratory to study and alter the properties of enzymes of the citric acid cycle. One involves studies of molecular structure using x-ray crystallography; the other employs cloning techniques to change the molecule sli ghtly and test the resulting products. The citric acid cycle is a sequence of enzymatic reactions that is the principle source of energy for nearly every living organism. The PI and students supported by this NSF grant will attempt to engineer changes in one or more of these enzymes in order to understand their catalytic mechanism and specificity. In another phase of the studies, the PI will prepare quantities of the precursor form of fumarase and examine its molecular structure by x-ray crystallography to study how the targeting process works.

9603656随着蛋白质结构和功能之间的联系加强，可以通过结合使用X射线结晶学和重组dna方法回答的基本问题的数量增加。PI提出的具体目标分为三个不同的领域，具有统一的主题，即了解细胞产生新陈代谢能量的分子机制，以及它们用来组装和控制这些途径的原则。首先，PI和他的学生将完成从大肠杆菌中提取富马酸酶的X射线结晶学研究。这些结构数据对于将真实的底物构建到当前的晶体模型中的任何尝试都是至关重要的。影响催化水分子的结合和静电的侧链将通过诱变来研究。与大肠杆菌富马酸酶的X射线结晶学研究密切相关的是对酵母富马酸酶的平行分析。这种情况下的目标与活性部位立体化学无关，而是收集细胞器靶向的信息。在第二项努力中，PI设定了确定一个或多个以线粒体为靶点的前体蛋白的分子结构的目标。最终的目标是与一种名为磷酸甘油酸脱氢酶的酶的丝氨酸控制机制有关。利用重组技术，将在有丝氨酸和不有丝氨酸的情况下进行调节域的结构和生化研究。现代生物技术的核心是将酶用作化学试剂。这一领域的新进展将取决于这些蛋白质催化剂中常见的几种性质的改变。这些因素包括稳定性、可控性和特异性。这项资助的PI在他的实验室中使用了两项主要技术来研究和改变柠檬酸循环中的酶的特性。一种涉及使用X射线结晶学研究分子结构；另一种使用克隆技术来细微地改变分子并测试结果。柠檬酸循环是一系列的酶反应，几乎是所有生物的主要能量来源。PI和NSF资助的学生将尝试改变这些酶中的一个或多个，以了解它们的催化机制和特异性。在另一阶段的研究中，PI将制备大量富马酸酶的前体形式，并通过X射线结晶学检查其分子结构，以研究靶向过程是如何工作的。