Protein Engineering of Citric Acid Cycle Enzymes

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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.