Kinetic and Physiological Effects of Alterations in Homologous Isocitrate-Binding Sites of Yeast NAD⁺-Specific Isocitrate Dehydrogenase

Abstract

Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four each of two homologous but nonidentical subunits designated IDH1 and IDH2. Models based on the crystallographic structure of Escherichia coli isocitrate dehydrogenase suggest that both yeast subunits contain isocitrate-binding sites. Identities in nine residue positions are predicted for the IDH2 site whereas four of the nine positions differ between the IDH1 and bacterial enzyme sites. Thus, we speculate that the IDH2 site is catalytic and that the IDH1 site may bind but not catalytically alter isocitrate. This was examined by kinetic analyses of enzymes with independent and concerted replacement of residues in each yeast IDH subunit site with the residues that differ in the other subunit site. Mutant enzymes were expressed in a yeast strain containing disrupted IDH1 and IDH2 loci and affinity-purified for kinetic analyses. The primary effects of various residue replacements in IDH2 were reductions of 30->300-fold in V(max) values, consistent with the catalytic function of this subunit. In contrast, replacement of all four residues in IDH1 produced a 17-fold reduction in V(max) under the same assay conditions, suggesting that the IDH1 site is not the primary catalytic site. However, single or multiple residue replacements in IDH1 uniformly increased half-saturation concentrations for isocitrate, implying that isocitrate can be bound at this site. Both subunits appear to contribute to cooperativity with respect to isocitrate, but AMP activation is lost only with residue replacements in IDH1. Overall, results are consistent with isocitrate binding by IDH2 for catalysis and with isocitrate binding by IDH1 being a prerequisite for allosteric activation by AMP. The effects of residue substitutions on enzyme function in vivo were assessed by analysis of various growth phenotypes. Results indicate a positive correlation between the level of IDH catalytic activity and the ability of cells to grow with acetate or glycerol as carbon sources. In addition, lower levels of activity are associated with increased production of respiratory-deficient (petite) segregants.

Global Transcription Analysis of Krebs Tricarboxylic Acid Cycle Mutants Reveals an Alternating Pattern of Gene Expression and Effects on Hypoxic and Oxidative Genes

“…As described above, second site nuclear mutations arise that enhance growth of cells lacking isocitrate dehydrogenase on glycerol. Second, isocitrate dehydrogenase dysfunction results in mtDNA instability, and strains lacking this enzyme have a high frequency of petite [ Ϫ ] mutations with large deletions in mtDNA (Elzinga et al, 1993;Lin et al, 2001). mtDNA instability is a phenotype associated with other TCA cycle defects and with a number of genes encoding proteins in mitochondrial oxidative phosphorylation and biogenesis (Contamine and Picard, 2000).…”

“…Isocitrate dehydrogenase is a bifunctional protein since it binds to mitochondrially encoded mRNA and appears to regulate translation of these transcripts (Elzinga et al, 1993;de Jong et al, 2000). However, it is not clear whether the mtDNA instability associated with isocitrate dehydrogenase dysfunction results from the loss of catalytic activity or from the aberrant expression and turnover of mitochondrial respiratory complexes (de Jong et al, 2000;Lin et al, 2001). These functions may not be distinct, because mRNA binding by isocitrate dehydrogenase inhibits its catalytic activity (Anderson and McAlister-Henn, 2000).…”

Suppression of metabolic defects of yeast isocitrate dehydrogenase and aconitase mutants by loss of citrate synthase

“…For derivatization, 10 µl samples of clarified supernatants or of standard metabolite mixtures (including TCA cycle intermediates plus aspartate and glutamate at concentrations ranging from 0-2.0 mM) were diluted with 10 µl of water containing labeled internal standards (1.0 mM [1,[5][6][7][8][9][10][11][12][13] Laboratories, respectively) and dried in a microfuge for 3 h using a Speed-Vac. The dry residues were dissolved in 50 µl of 20 mg/ml O-ethylhydroxylamine hydrochloride (Sigma-Aldrich) in pyridine, and reacted for 90 min at 30°C.…”

“…Similar suppression effects have also been observed for other IDH mutant phenotypes. For example, yeast strains lacking IDH or expressing catalytically defective forms of IDH exhibit elevated frequencies of generation of respiratory-deficient petite colonies [8]. Concomitant loss of CIT1 reduces these frequencies to levels observed for the parental strain [9].…”

Physiological Consequences of Loss of Allosteric Activation of Yeast NAD+-specific Isocitrate Dehydrogenase

“…The functions of each subunit have been analyzed by mutagenesis of analogous residues based on the well defined three-dimensional structure of the homodimeric Escherichia coli isocitrate dehydrogenase (ICD,7,8), which shares ϳ32% sequence identity with both IDH subunits. Results suggest that the basic functional unit of the yeast enzyme is a heterodimer of a catalytic IDH2 subunit and a regulatory IDH1 subunit (9,10). IDH2 contributes most of the residues in the catalytic substrate (isocitrate/Mg 2ϩ ) and cofactor (NAD ϩ ) binding sites, whereas the analogous sites in IDH1 have apparently evolved for cooperative binding of isocitrate and for allosteric binding of AMP (9 -12).…”