Effect of AMP on mRNA binding by yeast NAD+-specific isocitrate dehydrogenase.
Yeast mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) has previously been shown to bind specifically to 5'-untranslated regions of yeast mitochondrial mRNAs, and transcripts containing these regions have been found to allosterically inhibit activity of the enzyme. This inhibition is relieved by AMP, an allosteric activator of this regulatory enzyme of the tricarboxylic acid cycle. We further investigated these enzyme/ligand interactions to determine if binding of RNA and AMP by IDH is competitive or independent. Gel mobility shift experiments indicated no effect of AMP on formation of an IDH/RNA complex. Similarly, sedimentation velocity ultracentrifugation experiments used to analyze interactions in solution indicated that AMP alone had little effect on the formation or stability of an RNA/IDH complex. However, when these sedimentation experiments were conducted in the presence of isocitrate, which has been shown to be essential for binding of AMP by IDH, the proportion of RNA sedimenting in a complex with IDH was significantly reduced by AMP. These results suggest that AMP can affect the binding of RNA by IDH but that this effect is apparent only in the presence of substrate. They also suggest that the catalytic activity of IDH in vivo may be subject to complex allosteric control determined by relative mitochondrial concentrations of mRNA, isocitrate, and AMP. We also found evidence for binding of 5'-untranslated regions of mitochondrial mRNAs by yeast mitochondrial NADP+-specific isocitrate dehydrogenase (IDP1) but not by the corresponding cytosolic isozyme (IDP2). However, this appears to be a nonspecific interaction since no evidence was obtained for any effect on the catalytic activity of IDP1.
Anderson
Sondra L
SL
Department of Biochemistry and Center for Analytical Ultracentrifugation of Macromolecular Assemblies, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Schirf
Virgil
V
McAlister-Henn
L
L
D020121:5' Untranslated Regions / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D001667:Binding, Competitive / Q000502:physiology; D003600:Cytosol / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004789:Enzyme Activation; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism*; D046911:Macromolecular Substances; D008928:Mitochondria / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D014461:Ultracentrifugation / Q000379:methods; D015003:Yeasts / Q000201:enzymology
D020121:5' Untranslated Regions; D007523:Isocitrates; D046911:Macromolecular Substances; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000249:Adenosine Monophosphate; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)