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Over the last several decades, biochemical research has helped elucidate the structural and functional characteristics of NADP+-dependent isocitrate dehydrogenase (ICDH), a citric acid cycle enzyme that catalyzes the oxidative decarboxylation of 2R,3S-isocitrate into α-ketoglutarate, releasing NADPH and two molecules of CO2. In 2001, John Ferguson and Valeri Thomson of Bard College sequenced two putative NADP+-dependent isocitrate dehydrogenase genes from the genome of eukaryotic ciliate Tetrahymena pyriformis: a mitochondrial targeted (ICD1) and a peroxismal targeted (ICD2) NADP+-dependent ICDH gene. Although the conceptual translation of ICD2 matched the amino acid sequence of the purified putative-peroxismal ICDH, the conceptual translation of ICD1 suggested a lack of identity with the amino acid sequence of the mitochondrial ICDH. In 2003, a Bard undergraduate, Juliet Morrison, expressed ICD1 from T. pyriformis on a recombinant Escherichia coli strain and successfully purified the active protein. In the current experiment, my goal is to perform a comprehensive kinetic study of the putative mitochondrial NADP+-dependent ICDH from T.pyriformis. Kinetic studies are essential to understanding reaction mechanisms catalyzed by enzymes and their regulation in physiological conditions. Isocitrate dehydrogenase is one of the rate controlling enzymes of the citric acid cycle and so its regulation is essential to controlling the flux of citric acid cycle intermediates. Furthermore, ICDH has been implicated in protecting the cell against oxidative damage through the supply of NADPH. Ultimately, a kinetic study of a novel mitochondrial NADP+-dependent isocitrate dehydrogenase from T. pyriformis may help elucidate the evolutionary conservation of its kinetic mechanism across diverse eukaryotic and prokaryotic taxa.
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Zota, Anshul, "A Kinetic Study of a Putative NADP+-dependent Isocitrate Dehydrogenase from Tetrahymena Pyriformis" (2011). Senior Projects Spring 2011. 108.