The goal of the research is to examine the structure and function of citrate synthase (CS) from pig and E. coli by using site-directed mutagenesis. Citrate synthase is an excellent choice to examine enzyme structure and function by using such molecular biological techniques. It is a key enzyme in aerobic energy production and metabolite interconversions. It is an important example of stereospecificity in enzyme reactions, and two distinct enzyme conformations participate during catalysis. In addition, pig citrate synthase (PCS) and E. coli citrate synthase (ECCS) have been crystallized and the three dimensional structure of PCS determined. Therefore, CS is an attractive enzyme to study by site-directed mutagenesis because of the possibility of obtaining the DNA, the protein crystals, and an in depth mechanism for the mammalian and bacterial forms of the enzyme. This additional comparative aspect between the CS from a eucaryote and a procaryote will enhance our ability to choose sites for mutagenesis, and also will explain the two different reaction mechanisms, protein structures, and regulatory behaviors of these divergently related proteins. To define in detail the reaction mechanism, structure, and biological function of CS, the cDNA encoding PCS will be isolated and sequenced. Site-directed mutagenesis of the PCS and ECCS DNAs will used to alter codons for catalytic and structural amino acid residues. The mutated and control DNAs will be expressed in vitro, and the synthesized proteins will be purified and crystallized. The partial enzyme reactions catalyzed by the control and mutated PCS and ECCS proteins will be determined and compared. The three dimensional structures of the control and mutated PCS and ECCS proteins will be compared to the known X-ray structure of PCS. In addition, the gene for an E. coli mutant that codes for the dimeric (eucaryotic) form of the enzyme will be isolated and sequenced. The amino acid sequence for the mutant ECCS will be compared to the PCS and non-mutant ECCS and may identify particualr amino acid residues that are important to subunit assembly or enzymatic function.
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