Identification of Mn2+-binding Aspartates from α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase

Abstract

The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2␣:1␤:1␥, 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 ␣, ␤, or ␥ 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 ␣ subunit and those corresponding to ␣-Asp-206: 217 of the ␤ subunit and 215 of the ␥ subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, ␣-D206N, ␣-D230C, ␣-D234C, ␤-D217N, and ␥-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 mol of NADH/min/mg, respectively, whereas ␣-D230N and ␣-D234N enzymes showed no activity. The K m,Mn 2؉ for ␣-D230C and ␥-D215N are increased 32-and 100-fold, respectively, along with elevations in K m,isocitrate . The K m,NAD of ␣-D230C is increased 16-fold, whereas that of ␤-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 ␣-Asp-230 and ␣-Asp-234 are critical for catalytic activity, but ␣-Asp-206 is not needed; ␣-Asp-230 and ␥-Asp-215 may interact directly with the Mn 2؉ ; and ␣-Asp-230 and ␤-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 ␣ and ␥ subunits and between ␣ and ␤ subunits.Mammalian NAD-dependent isocitrate dehydrogenase (threo-D s -isocitrate:NAD ϩ oxidoreductase (decarboxylating), EC 1.1.1.41) is a citric acid cycle enzyme that catalyzes the metaldependent oxidative decarboxylation of isocitrate to form ␣-ketoglutarate and CO 2 . This allosteric mitochondrial enzyme is regulated by ADP (1). The enzyme has a heterooligomeric structure with subunits, present in the ratio 2␣:1␤: 1␥, of molecular masses 37,000, 39,000, and 39,000 Da, respectively, and distinctive isoelectric points (2, 3).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, Mn 2ϩ , NAD, ADP, NADH, and NADPH (4, 5). These binding studies either indicate that these distinctive subunits have specialized functions for particular ligand sites or that the binding site for each ligand is shared between two subunits (4, 5). The three types of subunits of the pig heart enzyme can be separated by chromatofocusing in the presence of urea (6). Isolated ␣, ␤, and ␥ subunits are either inactive or exhibit very low activity, but recombination of isolated ␣ with either ␤ or ␥ results in formation of either ␣␤ or ␣␥, which have substantial catalytic activity (6). These observations suggest that dimers may be the minimal functional subunits. The active...

Each Conserved Active Site Tyr in the Three Subunits of Human Isocitrate Dehydrogenase Has a Different Function

“…Ser Ͻ Phe Ͻ Glu) suggests that the same properties determine the allosteric effect of ADP on the enzyme. Previous studies have shown that mutations at other positions in the ␤ subunit resulted in a higher K m for NAD and complete or partial loss of allosteric activation by ADP (6,15,16,19). None of these studies showed a major effect on the V max of the enzyme.…”

“…All the mutants of IDH, except the ␥Y135F and ␣Y126E, were purified using the established protocol described by Soundar et al (15,16). As assessed by SDS-PAGE (Fig.…”

“…The ␣Y126 could be acting as a general acid that protonates the enolate after decarboxylation to form ␣-ketoglutarate; this role has been proposed for Tyr-140 of the pig NADP-IDH (8). Previous studies have shown that replacement of different amino acids in the ␣ subunit of human NAD-IDH (Arg-88, Asp-230, and Asp-234) yield inactive enzyme (15,16). These reports, together with the results presented in this study, indicate that alpha is the catalytic subunit.…”

“…Purity was assessed by SDS-PAGE (17). If required, the enzyme was concentrated to ϳ1 ml in Amicon Ultra tubes (YM-10) and passed through the Ultrogel gel filtration column as described before (15,16). In all cases, fractions exhibiting NAD-IDH activity or two bands at ϳ40 kDa by SDS-PAGE were pooled and concentrated in Amicon Ultra (Ultracel 10K) tubes.…”

“…NAD-IDH was purified as described by Soundar et al (15,16). The frozen pellet from 10 liters of cell culture was thawed and lysed under conditions described previously.…”