Allosteric inhibition of NAD(+)-specific isocitrate dehydrogenase by a mitochondrial mRNA

NAD(+)-specific isocitrate dehydrogenase (IDH) has been reported to bind se quences in 5'-untranslated regions of yeast mitochondrial mRNAs. In the cur rent study, an RNA transcript containing the 5'-untranslated region of the mRNA from the yeast mitochondrial COX2 gene is shown to be an allosteric in hibitor of the affinity-purified yeast enzyme. At 0.1 mu M concentrations o f the transcript, velocity of the IDH reaction is reduced to 20% of the val ue obtained in the absence of the RNA transcript. This inhibition is due to a 2.5-fold increase in the S-0.5 value for isocitrate. Significant inhibit ion of IDH activity is also obtained with a transcript containing a portion of the 5'-untranslated region of the yeast mitochondrial ATP9 gene and wit h an antisense form of the COX2 transcript, both of which contain potential stem-loop secondary structures implicated in binding of IDH. In contrast, much higher concentrations of yeast tRNA or poly(A)mRNA, respectively, 33- and 60-fold greater than that required for the COX2 transcript, are require d to produce a 50% decrease in velocity. These results suggest that inhibit ion of activity is relatively specific for the 5'-untranslated regions of m itochondrial mRNAs. All measurable inhibition of IDH activity by RNA is eli minated by addition of 100 mu M concentrations of the allosteric activator AMP. At equivalent concentrations, dAMP is less efficient than AMP as an al losteric activator of IDH and is proportionally less effective in protectin g against inhibition of activity by the COX2 transcript. Other nucleotides that are not allosteric activators fail to protect IDH activity from inhibi tory effects of RNA. Thus, alleviation of catalytic inhibition of IDH by mi tochondrial mRNA correlates with the property of allosteric activation.