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. ***
