As a classic example for allosteric regulation by adenine nucleotides, yeast NAD(+) specific isocitrate dehydrogenase (IDH) is purported to be responsive to cellular energy levels and, as an enzyme in the tricarboxylic acid cycle, IDH in turn modulates these energy levels by controlling flux through oxidative metabolism. More recently, IDH has also been found to be an example of a metabolic enzyme with dual functions. The enzyme binds with significant affinity to specific sites in the 5'-untranslated regions of yeast mitochondrial mRNAs. This binding by IDH to mRNAs that encode subunits of inner-membrane respiratory complexes is hypothesized to transiently inhibit translation to prevent premature synthesis of these hydrophobic proteins in the mitochondrial matrix. We have shown that binding of mitochondrial mRNA also dramatically inhibits IDH activity, and that binding and inhibition by mRNA are alleviated by the allosteric activator of IDH, AMP. These observations suggest a novel mechanism for coordinate control of oxidative energy production and of mitochondrial gene expression through allosteric regulation of IDH. Proposed research will test hypotheses related to this mechanism. Yeast IDH is an octamer containing two types of homologous subunits. We have shown that the IDH2 subunit contains catalytic isocitrate/Mg(2+)-and NAD(+)-binding sites, whereas similar sites in the IDH1 subunit have evolved for regulatory binding of isocitrate and AMP. Proposed research seeks to clarify three-dimensional and quaternary structures of IDH to provide a novel model for co-evolution of homologous catalytic and regulatory subunits, and to elucidate the organization that structurally facilitates allosteric communication between these subunits. Concomitantly, mutant IDH enzymes with well-defined defects in specific catalytic and regulatory properties will be used in in vitro studies and to replace the wild-type enzyme in vivo to investigate the phenomena described above, i.e., allosteric control of oxidative metabolism and of mitochondrial gene expression at the level of translation.
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