Protein Engineering of Citric Acid Cycle Enzymes

9318699 Banaszak柠檬酸由9个酶反应组成，它们共同构成所有生命系统中化学能的主要来源。 来自脂肪、碳水化合物和/或蛋白质的双碳化合物以称为乙酰辅酶A的代谢物的形式进入这个循环，并且在氧气的存在下被分解以产生ATP。 一种重要的化学能储存形式。循环中的九个化学反应中的每一个都由酶催化。在真核细胞（细菌和病毒以外的生物体）中，酶位于一个称为细胞器的细胞器中。 这些酶由核基因编码，在细胞质中生物合成。 如果在真核细胞中，柠檬酸循环酶在胞质溶胶中产生，但在线粒体中起作用，那么细胞必须有一种识别这些酶并将其转移到线粒体中的方法。 试图确定结构因子，这是重要的线粒体酶的靶向和易位是第一个问题，这将是在这个项目中进行研究。 在一个小容器中，蛋白质浓度非常高，并且其中的液体非常粘稠。 已经提出，柠檬酸循环中的连续酶可以形成弱复合物，促进代谢物的扩散。 对这种酶的可能结构的研究：酶复合物是该项目的第二个方面。 大多数柠檬酸循环酶的分子结构是已知的。 然而，催化4个碳的羧酸、L-苹果酸和脱氢酶之间的脱水水合反应的脱氢酶的结构是未知的。 该基金将支持从真核和原核细胞中确定酶结构的研究。 这些研究将使用X射线晶体学进行。最后，使用重组DNA技术，将在柠檬酸循环的两种酶中进行系统的改变，即脱氢酶和苹果酸，以探测它们的催化化学。拟议的研究的重点涉及两个柠檬酸循环酶，脱氢酶和苹果酸脱氢酶（MDH）的结构/功能关系的测定，使用定点突变和X射线晶体学分析的组合。 具体目标是：（1）完成了E.（2）从酵母中结晶出完整的脱氢酶前体，（3）截短我们的重组酵母脱氢酶DNA并重新克隆到胞质和线粒体脱氢酶的假定形式中，（4）使用晶体学研究得到的晶体学坐标和静态中间体来检查脱氢酶和MDH的催化机制，（5）启动对制备这种酶：酶复合物的结晶形式的研究，和（6）研究线粒体识别和转移酶和MDH的结构方面。 后者的重要性来自于这样的事实，即在真核细胞中，柠檬酸循环的反应发生在线粒体内膜基质内，但酶在环醛中合成。 这是可能的，因为真核生物形式的脱氢酶和MDH经历一系列的细胞凋亡， h）的 0* 0*0 识别/处理步骤。 独特的贡献，拟议的实验包括确定的分子结构的非含铁水合酶（水解酶），一类酶，目前没有晶体学信息。 此外，线粒体蛋白质进口和酶的结构数据：酶复合物识别应该是可能的，从合并晶体学和致突变研究。 其他重要贡献包括更好地理解酶和MDH的催化机制，以及从两种酶的工程特异性和动力学变化中识别关键原子。 ***

9318699 Banaszak The citric acid consists of nine enzymatic reactions which together form the major source of chemical energy in all living systems. Two carbon compounds derived from fats, carbohydrate and/or protein enter this cycle in the form of a metabolite called acetyl coenzyme A and in the presence of oxygen are broken down to generate and ATP an important storage form of chemical energy. Each of the nine chemical reactions in the cycle is catalyzed by an enzyme. In eukaryotic cells (organisms beyond bacteria and viruses), the enzymes are located in an organelle called a mitochondrion. The enzymes are coded for by nuclear genes and biosynthesized in the cytosol. If in eukaryotic cells, the citric acid cycle enzymes are made in the cytosol but operate in the mitochondria, the cell must have a way of identifying these enzymes and translocating them into mitochondria. Attempts to define structural factors which are important to both targeting and translocation of mitochondrial enzymes is the first problem which will be studied in this project. In a mitochondrion, the protein concentration is very high and the fluid within it is very viscous. It has been proposed that sequential enzymes in the citric acid cycle may form weak complexes facilitating the diffusion of metabolites. Studies of the possible structures for such enzyme:enzyme complexes is a second facet to the project. The molecular structures of most of the citric acid cycle enzymes are known. However, the structure of the enzyme fumarase which catalyzes a dehydration hydration reaction between the 4 carbon carboxylic acids, L malate and fumarase, is not known. The grant will support studies on the stru cture determination of the enzyme from both eukaryotic and prokaryotic cells. The studies will be carried out using x ray crystallography. Last of all using recombinant DNA techniques, systematic changes will be made in two of the enzymes of the citric acid cycle, fumarase and malate to probe their catalytic chemistry. %%% The focus of the proposed study involves the determination of the structure/function relationships of two citric acid cycle enzymes, fumarase and malate dehydrogenase (MDH) using a combination of site directed mutagenesis and x ray crystallographic analyses. The specific objectives are: (1) to complete the crystal structure of fumarase (fumc) from E. Coli, (2) to crystallize the intact precursor of fumarase from yeast, (3) to truncate our recombinant yeast fumarase DNA and to reclone in the putative forms of the cytosolic and mitochondrial fumarases, (4) to examine the catalytic mechanism of both fumarase and MDH using crystallographic coordinates and static intermediates derived from crystallographic studies, (5) to initiate studies on the preparation of crystalline forms of such enzyme:enzyme complexes, and (6) to study the structural aspects of mitochondrial recognition and translocation for fumarase and MDH. The significance of the latter derives from the fact that in eukaryotic cells, the reactions of the citric acid cycle occur within the mitochondrial inner membrane matrix but the enzymes are synthesized in the cylosal. This is possible because the eukaryotic forms of fumarase and MDH undergo a series of cell h) 0*0*0* recognition/processing steps. Unique contributions of the proposed experiments include the determination of the molecular structure of a non iron containing hydratase (fumarase), a class of enzymes for which no crystallographic information is currently available. In addition, structural data on mitochondrial protein import and enzyme:enzyme complex recognition should be possible from the combined crystallographic and mutagenic studies. Other significant contributions include a better understanding of the catalytic mechanism of fumarase and MDH and the identification of critical atoms from engineering specificity and kinetic changes in the two enzymes. ***