Protein Engineering of Citric Acid Cycle Enzymes

9603656 Banaszak随着蛋白质结构和功能之间联系的加强，可以通过结合使用X射线晶体学和重组DNA方法来回答的基本问题的数量增加了。 PI提出的具体目标分为三个不同的领域，统一的主题是理解细胞产生代谢能量的分子机制，以及它们用于组装和控制这些途径的原理。 首先，PI和他的学生将完成来自E.杆菌 这些结构数据对于任何试图将真正的基质构建到当前的晶体学模型中的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E.大肠杆菌中的酶是来自酵母的酶的平行分析。 在这种情况下的目标是不相关的活性位点立体化学，而是对细胞器的目标信息的收集。 在这第二次奋进中，PI已经设定了确定一种或多种前体蛋白的分子结构的目标，这些前体蛋白将它们靶向线粒体。 最终的目标是与丝氨酸控制磷酸甘油酸脱氢酶的机制有关。 使用重组技术，结构和生物化学研究的监管结构域将进行和没有丝氨酸。 现代生物技术的核心是利用酶作为化学试剂。 这一领域的新发展将取决于改变这些蛋白质催化剂中常见的几种性质。 这些包括稳定性、控制和特异性。 该资助的主要研究者在他的实验室中使用了两种主要技术来研究和改变柠檬酸循环酶的特性。 一种是用X射线晶体学研究分子结构，另一种是用克隆技术来细微地改变分子并测试所产生的产物。 柠檬酸循环是一系列酶促反应，是几乎所有生物体的主要能量来源。 由NSF资助的PI和学生将尝试设计一种或多种酶的变化，以了解它们的催化机制和特异性。 在研究的另一个阶段，PI将制备大量的前体形式的酶，并通过X射线晶体学检查其分子结构，以研究靶向过程如何工作。

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.