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

Abstract

The human NAD-dependent isocitrate dehydrogenase (IDH) is a heterotetrameric mitochondrial enzyme with 2␣:1␤:1␥ subunit ratio. The three subunits share 40 -52% identity in amino acid sequence and each includes a tyrosine in a comparable position: ␣Y126, ␤Y137, and ␥Y135. To study the role of the corresponding tyrosines of each of the subunits of human NAD-IDH, the tyrosines were mutated (one subunit at a time) to Ser, Phe, or Glu. Enzymes were expressed with one mutant and two wildtype subunits. The results of characterization of the mutant enzymes suggest that ␤Y137 is involved in NAD binding and allosteric activation by ADP. The ␣Y126 is required for catalytic activity and likely acts as a general acid in the reaction. The ␥Y135 is also required for catalytic activity and may be involved in proper folding of the enzyme. The corresponding tyrosines in the three dissimilar subunits of NAD-IDH thus have distinctive functions. Mammalian NAD-dependent isocitrate dehydrogenase (NAD-IDH)2 is a mitochondrial tricarboxylic acid cycle enzyme that catalyzes the oxidative decarboxylation of isocitrate to ␣-ketoglutarate, while reducing NAD to NADH. It is a heterotetramer with three subunits in the ratio 2␣:1␤:1␥ (1), with molecular masses of ϳ37,000, 39,000, and 39,000 Da, respectively (2, 3). The amino acid sequences of the ␤ and ␥ subunits of the human are 52.4% identical, whereas ␣ and ␤ subunits are only 40.4% and ␣ and ␥ subunits are only 41.6% identical. This enzyme is allosterically regulated by ADP, which decreases the K m for isocitrate by ϳ38-fold (4). NAD-IDH has two binding sites per tetramer for each ligand: isocitrate, NAD, Mn 2ϩ , and ADP (2, 5), raising the question of the function of each of the subunits.We have recently shown that homozygous mutations exclusively of the ␤ subunit of human NAD-IDH are a cause of Retinitis Pigmentosa, a hereditary degeneration of the retina that leads to blindness in patients (6). Characterization of these two types of mutant enzymes in lymphoblast cell extracts revealed a ϳ300-fold increase in K m,NAD and partial or complete loss of allosteric activation by ADP. The involvement of this enzyme in causing Retinitis Pigmentosa increases the importance of studying NAD-IDH in more detail.High-resolution crystal structures are available for the homodimeric NADP-dependent IDH of pig and Escherichia coli (7-11). The individual subunits of human NAD-IDH are only about 25-34% identical in amino acid sequence to the E. coli NADP-IDH (Fig. 1A) and about 12-18% identical to the pig NADP-IDH. Although there is a low % identity among these enzymes, the isocitrate-binding site is well conserved, including E. coli Arg-119, Arg-153, Tyr-160, and Lys-230. A previous study of pig NADP-IDH showed that Tyr-140 interacts with the ␤-carboxylate of isocitrate and acts as a general acid in the reaction (8). The Tyr-160 mutants of E. coli NADP-IDH showed a decreased k cat (12, 13) and the crystal structure revealed that the tyrosine is appropriately positioned to donate a proton to the carbanion fo...

Ligand binding and structural changes associated with allostery in yeast NAD+-specific isocitrate dehydrogenase

“…The sizes of the mammalian enzyme subunits are quite similar to those of the yeast enzyme, and respective catalytic and regulatory subunits share 40-50% sequence identity [51, 52]. The specific functions of the regulatory subunits are largely unknown, although there is evidence that the β subunit may contribute to allosteric activation by ADP [53]. Other potential functions to be determined for the different regulatory subunits include activation by Ca 2+ and inhibition by ATP.…”

A unique homodimeric NAD + ‐linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri

“…Eukaryotes usually possess a NAD + ‐dependent IDH (NAD‐IDH, EC 1.1.1.41) that is strictly mitochondrial and several types of NADP + ‐dependent IDH (NADP‐IDH, EC 1.1.1.42) isoforms that are distributed in the mitochondrial matrix, cell cytosol, and peroxisome (2–5). Mitochondrial NAD‐IDHs are believed to be heteromeric in solution, such as the hetero‐octameric yeast NAD‐IDH, composed of 4 heterodimers of regulatory IDH1 and catalytic IDH2 subunits (6), and the heterotetrameric human NAD‐IDH, with 3 types of subunits present in the ratio of 2α:1β:1γ (7–9), whereas most prokaryotic NAD‐IDHs are dimeric, such as NAD‐IDH from Acidithiobacillus thiooxidans (10) and Streptococcus mutans (11). In eukaryotes, mitochondrially localized NAD‐IDHs generate NADH to provide electrons for energy production (ATP), meanwhile NADP‐IDHs provide the reducing power (NADPH) and carbon skeleton (α‐ketoglutarate) for biosynthesis, cellular defense against oxidative damage and reactive oxygen species detoxification (12–15).…”

Enhanced Glutamate Uptake into Synaptic Vesicles Fueled by Vesicle-generated ATP from Phosphoenolpyruvate and ADP

“…It also affects bioenergetics by activating certain enzymes. It acts as an allosteric activator of the oxidative enzymes glutamate dehydrogenase [34–36] and isocitrate dehydrogenase [37–39] and the respiratory enzyme cytochrome c oxidase [40], all present in mitochondria. Moreover, mitochondrial oxidative phosphorylation is stimulated by cytosolic ADP concentration, with a second order kinetic function with respect to its concentration [41, 42].…”