Protein Engineering of Citric Acid Cycle Enzymes

柠檬酸由9个酶促反应组成，它们共同构成了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/或蛋白质的两种碳化合物以一种叫做乙酰辅酶a的代谢物的形式进入这个循环，在氧气的存在下被分解，产生ATP一种重要的化学能储存形式。循环中的九种化学反应都是由一种酶催化的。在真核细胞（细菌和病毒之外的生物体）中，酶位于称为线粒体的细胞器中。这些酶由核基因编码并在细胞质中生物合成。如果在真核细胞中，柠檬酸循环酶是在细胞质中产生的，但在线粒体中起作用，细胞必须有一种方法来识别这些酶并将它们转运到线粒体中。试图定义对线粒体酶的靶向和易位都很重要的结构因素是本项目将研究的第一个问题。在线粒体中，蛋白质浓度非常高，其中的液体非常粘稠。有人提出，顺序酶在柠檬酸循环可能形成弱复合物促进代谢物的扩散。研究这种酶的可能结构：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碳羧酸、L苹果酸和延胡索酸酶之间脱水水化反应的延胡索酸酶的结构尚不清楚。该基金将支持真核和原核细胞中酶的结构测定研究。这些研究将使用x射线晶体学进行。最后，利用重组DNA技术，系统地改变柠檬酸循环中的两种酶，富马酸酶和苹果酸酶，以探测它们的催化化学性质。该研究的重点是利用位点定向诱变和x射线晶体学分析的结合，确定两种柠檬酸循环酶，富马酸酶和苹果酸脱氢酶（MDH）的结构/功能关系。具体目标是：(1)完成大肠杆菌中延胡索酶（fuma）的晶体结构，(2)结晶酵母中延胡索酶的完整前体，(3)截断重组酵母延胡索酶DNA，并以推测的胞质和线粒体延胡索酶的形式进行克隆，(4)利用晶体坐标和从晶体学研究中得到的静态中间体来检验延胡索酶和MDH的催化机制。(5)开始研究这种酶的结晶形式：酶复合物的制备；(6)研究延胡索酶和MDH的线粒体识别和易位的结构方面。后者的意义在于，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，而酶是在环胞中合成的。这是可能的，因为真核形式的延胡索酶和MDH经历了一系列细胞h) 0*0*0*的识别/处理步骤。提出的实验的独特贡献包括确定不含铁的水合酶（延胡索酸酶）的分子结构，这是一类目前没有晶体学信息的酶。此外，结合晶体学和诱变研究，线粒体蛋白质输入和酶：酶复合物识别的结构数据应该是可能的。其他重要贡献包括更好地理解富马酸酶和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鉴定关键原子。＊＊＊

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. ***