CRYSTAL STRUCTURE OF YEAST NAD-SPECIFIC ISOCITRATE DEHYDROGENASE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The oxidative decarboxylation of isocitrate to alpha-ketoglutarate catalyzed by mitochondrial NAD-specific isocitrate dehydrogenases is a rate limiting step in the tricarboxylic acid (TCA) cycle. The affinity of the yeast enzyme (IDH) for isocitrate is allosterically regulated, positively by AMP and negatively by ATP and NADH. This allosteric control has been proposed to contribute to inverse regulation of rates of energy production by oxidative pathways and by glycolysis. Thus, under conditions of energy sufficiency, i.e. when relative cellular ratios of [ATP]/[AMP] and of [NADH]/[NAD] are high, flux through the TCA cycle would be attenuated at the level of IDH, rates of glycolysis would increase, and the tricarboxylic acids, citrate and isocitrate, would be diverted into biosynthetic pathways. Yeast IDH is an octamer composed of four IDH1 and four IDH2 subunits. IDH1 (M.W. = 38,001) and IDH2 (M.W. = 37,755) share 42% sequence identity. Results of targeted mutagenesis studies suggest that the IDH2 subunit contains catalytic isocitrate/Mg2+ and NAD binding sites, whereas homologous sites in the IDH1 subunit function in cooperative binding of isocitrate and in binding of the allosteric activator AMP. An understanding of the oligomeric structure of IDH would thus illuminate relationships between homologous catalytic and regulatory ligand binding sites. We have produced crystals of IDH that diffract to 3.2¿¿¿ on a Rigaku FR-D rotating anode X-ray source. We anticipate that the determination of the oligomeric arrangement of IDH in the crystal structure will reveal the mode of allosteric control of this complex enzyme. Additionally, the information provided by the structure will direct future experiments investigating IDH enzymology.