Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase.
Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated IDH1 and IDH2. IDH2 was previously shown to contain the catalytic site, whereas IDH1 contributes regulatory properties including cooperativity with respect to isocitrate and allosteric activation by AMP. In this study, interactions between IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions between identical subunit polypeptides were not detected with this or other methods. A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme. A corollary of this model, based on the three-dimensional structure of the homologous enzyme from Escherichia coli, is that some interactions between subunits occur at isocitrate binding sites. Based on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit were predicted to be important in the catalytic site of IDH2. We found that individually replacing these residues with alanine results in mutant enzymes that exhibit a drastic reduction in catalysis both in vitro and in vivo. Also based on this model, the two analogous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predicted to contribute to the regulatory site of IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate. A D222A substitution in IDH2 produces similar regulatory defects and a substantial reduction in V(max) in the absence of AMP. Collectively, these results suggest that the basic structural/functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the isocitrate binding site of the other.
Panisko
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EA
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
McAlister-Henn
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D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D017930:Genes, Reporter; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D021122:Protein Subunits; D011994:Recombinant Proteins; D007521:Isocitrate Dehydrogenase; D008274:Magnesium