Abstract
The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2alpha:1beta:1gamma, requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn(2+), we mutated aspartates on the alpha, beta, or gamma subunits. Mutagenesis target sites were based on crystal structures of metal-isocitrate complexes of Escherichia coli and pig mitochondrial NADP-IDH and sequence alignments. Aspartates replaced by asparagine or cysteine were 206, 230, and 234 of the alpha subunit and those corresponding to alpha-Asp-206: 217 of the beta subunit and 215 of the gamma subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, alpha-D206N, alpha-D230C, alpha-D234C, beta-D217N, and gamma-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 micromol of NADH/min/mg, respectively, whereas alpha-D230N and alpha-D234N enzymes showed no activity. The K(m,Mn(2+)) for alpha-D230C and gamma-D215N are increased 32- and 100-fold, respectively, along with elevations in K(m,isocitrate). The K(m,NAD) of alpha-D230C is increased 16-fold, whereas that of beta-D217N is elevated 10-fold. For all the mutants K(m,isocitrate) is decreased by ADP, indicating that these aspartates are not needed for normal ADP activation. This study demonstrates that alpha-Asp-230 and alpha-Asp-234 are critical for catalytic activity, but alpha-Asp-206 is not needed; alpha-Asp-230 and gamma-Asp-215 may interact directly with the Mn(2+); and alpha-Asp-230 and beta-Asp-217 contribute to the affinity of the enzyme for NAD. These results suggest that the active sites of the human NAD-IDH are shared between alpha and gamma subunits and between alpha and beta subunits.
Highlights
Our previous work on pig heart NAD-dependent isocitrate dehydrogenase showed that this enzyme has two binding sites per tetramer for each of its ligands: isocitrate, Mn2ϩ, NAD, ADP, NADH, and NADPH [4, 5]
Certain amino acids in the E. coli and pig mitochondrial NADP-isocitrate dehydrogenases that are known to interact with Mn2ϩ and isocitrate are conserved among the three subunits of the human NAD-IDH
The recombinant human NAD-IDHs were purified to homogeneity as described under “Experimental Procedures,” giving a yield of enzyme higher than that obtained from the IDH- deficient E. coli EB 106 cells that we used previously [11]
Summary
Materials—Ammonium sulfate, citric acid, rotenone, adenosine-5Ј-diphosphate, -NAD, DL-isocitrate (trisodium salt), triethanolamine chloride, Coomassie Brilliant Blue-R, DL-dithiothreitol (DTT), ampicillin (monosodium salt), MES, and PIPES were obtained from Sigma. These were pooled and concentrated to a 1–1.5-ml volume After dialyzing this pool against 4 liters of 50 mM citrate-Tris, pH 7.2, buffer containing 20% glycerol, 0.2 mM MnSO4, and 0.1 mM DTT (Buffer C) for 6 h, the enzyme was separated from the other proteins by elution through an Ultragel ACA 34 (molecular mass cut-off 350 kDa) gel filtration column (1.0 ϫ 124 cm). To minimize interference from NADH oxidase while assaying for IDH activity in crude preparations or ammonium sulfate fractions containing lower amounts of expressed mutant enzymes, the NADH oxidase inhibitor, 10 l of rotenone (dissolved in 100% ethanol), was added to the 1 ml of standard assay solution to give a final concentration of 2.5 M rotenone. As described under “Experimental Procedures,” the specific activities for wild-type and mutant purified human NAD-IDHs were generally determined using standard assay solutions
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