This research will use genetic engineering techniques to examine an important point for control of metabolism in eucaryotic cells. Communication between central metabolic pathways in the cytoplasm (glycolysis and gluconeogenesis) and in mitochondria (TCA cycle) is accomplished through the shuttle of a select number of metabolites across the mitochondrial membrane. Our goal is to examine the role of compartmentalized isozymes of malate dehydrogenase in regulating the exchange of oxaloacetate and NAD(H) through the shuttle of malate. This proposal focuses on regulation of the shuttle cycle and of the TCA cycle by the mitochondrial isozyme. Structural analyses of the yeast (Saccharomyces cerevisiae) gene encoding mitochondrial malate dehydrogenase and of the wild type enzyme will be completed. The physiological importance of this enzyme will be examined through detailed study of expression under various metabolic conditions and through genetic construction of mutant forms of the enzyme to test in vivo function. Specific mutant forms to be constructed are enzymes with defects in catalysis or in structural features important for interaction with other mitochondrial proteins. Methods for testing different mitochondrial functions and interactions of malate dehydrogenase will be developed. These experiments will test the hypothesis that metabolic flux is controlled by alternate physical interactions of malate dehydrogenase with enzymes in the TCA cycle or in the malate/aspartate shuttle. Other goals of this proposal are to initiate study of the structure and expression of yeast cytoplasmic malate dehydrogenase and to continue studies of the function and expression of the single form of malate dehydrogenase in Escherichia coli.
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