| Abstract
| - Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an allosterically regulated tricarboxylicacid cycle enzyme that has been shown to bind specifically and with high affinity to 5‘-untranslatedregions of yeast mitochondrial mRNAs. The absence of IDH has been shown to result in reduced expressionof mitochondrial translation products, leading to the suggestion that this macromolecular interaction maycontribute to regulating rates of translation. The interaction with mitochondrial mRNAs also produces adramatic inhibition of IDH catalytic activity that is specifically alleviated by AMP, the primary allostericactivator of IDH. Using mutant forms of IDH with defined catalytic or regulatory kinetic defects, wefound that residue changes altering ligand binding in the catalytic site reduce the inhibitory effect of atranscript from the mitochondrial COX2 mRNA. In contrast, residue changes altering binding of allostericregulators do not prevent inhibition by the COX2 RNA transcript but do prevent alleviation of inhibitionby AMP. Results obtained using surface plasmon resonance methods suggest that the mRNA transcriptmay bind at the active site of IDH. Also, the presence of AMP has little effect on overall affinity butrenders the binding of mRNA ineffective in catalytic inhibition of IDH. Finally, by expressing mutantforms of IDH in vivo, we determined that detrimental effects on levels of mitochondrial translation productscorrelate with a substantial reduction in catalytic activity. However, concomitant loss of IDH and ofcitrate synthase eliminates these effects, suggesting that any role of IDH in mitochondrial translation isindirect.
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