NAD-dependent Isocitrate Dehydrogenase Research Articles

Cloning and activity analysis ofin vitro expression of plant NAD-IDH genes

The plant NAD-dependent isocitrate dehydro-genase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidop-sis thaliana ecotype Columbia gl1 and three lines (Shaan 2A, Shaan 2B and Ken C1) of Brassica napus by RT-PCR method. By searching the sequences in GenBank Database, it is shown that these sequences from B. napus were novel. Their encoding regions of a functional protein were then inserted into the multiple cloning sites of pMID1 vector for the expression of recombinant protein. All the recombinants were successfully expressed in the in vitro expression system. When the transcripts of subunits 0, 1 and 2 were added to-gether to the in vitro system with 36 靏/mL of protein disul-fide isomerase, the expressed products had NAD-IDH enzy-matic activity. Comparison of different kinds of gene and molecular ratio of the transcripts showed that the NAD-IDH enzyme is composed of three subunits designated subunit 0, subunit 1 and subunit 2. All the three subunits are essential to catalytic activity. Missing subunit 0 could abolish activity. Missing either subunit 1 or subunit 2 would cause severe impact on activity. Deletions, which would cause frameshift mutation or nonsense mutation, were also found in some transcripts of subunit 1 gene from Shaan 2A and Shaan 2B of B. napus. The mutated subunit 1 lost its proper function. This may explain why there is difference of the NAD-IDH activity among three lines of B. napus.

Inhibition of Krebs cycle and activation of glyoxylate cycle in the course of chronological aging of Saccharomyces cerevisiae. Compensatory role of succinate oxidation

We investigated oxidative processes in mitochondria of Saccharomyces cerevisiae grown on ethanol in the course of chronological aging. We elaborated a model of chronological aging that avoids the influence of exhaustion of medium, as well as the accumulation of toxic metabolites during aging. A decrease in total respiration of cells and, even more, of the contribution of respiration coupled with ATP-synthesis was observed during aging. Aging is also related with the decrease of the contribution of malonate-insensitive respiration. Activities of citrate-synthase (CS), α-ketoglutarate dehydrogenase (KGDH) and malate dehydrogenase (MDH) were threefold decreased. The activity of NADP-dependent isocitrate dehydrogenase (NADP-ICDH) decreased more significantly, while the activity of NAD-dependent isocitrate dehydrogenase (NAD-ICDH) fell even greater, being completely inactivated on the third week of aging. In contrast, succinate dehydrogenase (SDH), enzymes of glyoxylate cycle (GCL) (isocitrate lyase (ICL) and malate synthase (MLS)), and enzymes of ethanol oxidation (alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH)), were activated by 50% or more. The behavior of oxidative enzymes and metabolic pathways are apparently inherent to a more viable, long-lived cells in population, selected in the course of chronological aging. This selection allows cells to reveal the mechanism of their higher viability as caused by shunting of complete Krebs cycle by glyoxylate cycle, with a concomitant increased rate of the most efficient energy source, namely succinate formation and oxidation. Thiobarbituric-reactive species (TAR species) increased during aging. We supposed that to be the immediate cause of damage of a part of yeast population. These data show that a greater succinate contribution to respiration in more active cells is a general property of yeast and animal tissues.

Evaluation by Mutagenesis of the Importance of 3 Arginines in α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase

Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2alpha:1beta:1gamma. Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, alpha-Arg88, beta-Arg99, and gamma-Arg97 of human NAD-dependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation. A plasmid harboring cDNA that encodes alpha, beta, and gamma subunits of the human isocitrate dehydrogenase (Kim, Y. O., Koh, H. J., Kim, S. H., Jo, S. H., Huh, J. W., Jeong, K. S., Lee, I. J., Song, B. J., and Huh, T. L. (1999) J. Biol. Chem. 274, 36866-36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E. coli. Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with alpha-R88Q, beta-R99Q, or gamma-R97Q) were purified to homogeneity yielding enzymes with 2alpha:1beta:1gamma subunit composition and a native molecular mass of 315 kDa. Specific activities of 22, 14, and 2 micromol of NADH/min/mg were measured, respectively, for WT, beta-R99Q, and gamma-R97Q enzymes. In contrast, mutant enzymes with normal beta and gamma subunits and alpha-R88Q mutant subunit has no detectable activity, demonstrating that, although beta-Arg99 and gamma-Arg97 contribute to activity, alpha-Arg88 is essential for catalysis. For WT enzyme, the Km for isocitrate is 2.2 mm, decreasing to 0.3 mm with added ADP. In contrast, for beta-R99Q and gamma-R97Q enzymes, the Km for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP. These results suggest that beta-Arg99 and gamma-Arg97 are needed for normal ADP activation. In addition, the gamma-R97Q enzyme has a Km for NAD 10 times that of WT enzyme. This study indicates that a normal alpha subunit is required for catalytic activity and alpha-Arg88 likely participates in the isocitrate site, whereas the beta and gamma subunits have roles in the nucleotide functions of this allosteric enzyme.

Involvement of enzymes of amino acid metabolism and tricarboxylic acid cycle in bovine oocyte maturation in vitro

Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumulus cells as a result of IVM (P<0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P<0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P<0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.

Involvement of enzymes of amino acid metabolism and tricarboxylic acid cycle in bovine oocyte maturation in vitro

Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumulus cells as a result of IVM (P<0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P<0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P<0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.

Comparison of Isocitrate Dehydrogenase from Three Hyperthermophiles Reveals Differences in Thermostability, Cofactor Specificity, Oligomeric State, and Phylogenetic Affiliation

With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90 degrees C or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9 degrees C, respectively, compared with 98.3 and 98.5 degrees C for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (K(m)((NADP)) of 55.2 and 44.4 microm, respectively) whereas PfIDH was NAD-dependent (K(m)((NAD)) of 68.3 microm). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di- and multimeric IDHs are described and discussed.

Plasma membrane H +-ATPase, succinate and isocitrate dehydrogenases activities of vessel-associated cells in walnut trees

Summary In winter and spring, walnut trees exhibit variations of sugar content in the vascular sap. According to their location, the vessel-associated cells (VACs, also called contact cells) could control nutrient exchanges between the storage parenchyma and the xylem vessels. According to the literature, the recovery of sap (influx) occurs at the VAC/vessel interface via an H+/sugar symport that depends on the transmembrane pH gradient generated by the plasma membrane H+-ATPase. The aim of this study was to investigate the ATPase activity, using a perfusion technique that allows the use of several effectors: carbonyl cyanide m-chlorophenylhydrazone (CCCP) and fusicoccin (FC). During winter, the uncoupler CCCP revealed a low pH gradient between the xylem vessels and the vessel-associated cells. Under these conditions, FC, an activator of the H+-ATPase, had no effect on the pH of the perfusion solution, suggesting that the enzyme could be lightly active. In contrast, close to bud break, a high pH gradient was revealed by the use of CCCP, and an acidification of the perfusion solution was observed in the presence of FC. Moreover, cytochemical investigation showed high activity of two respiratory enzymes located in mitochondria: NAD-dependent isocitrate dehydrogenase and succinate dehydrogenase. The hypothesis is that in spring this high respiratory activity of VACs provides a consequent increase in available ATP that can be utilized by the plasma membrane H+-ATPase.

Affinity cleavage at the divalent metal site of porcine NAD-specific isocitrate dehydrogenase.

A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD-specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2alpha:1beta:1gamma. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa alpha subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS-PAGE. Amino-terminal sequencing reveals that these peptides arise by cleavage of the Val262-His263 bond of the alpha subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the alpha subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates alpha as the catalytic subunit of the multisubunit porcine NAD-dependent isocitrate dehydrogenase.

Simultaneous expression of NAD-dependent isocitrate dehydrogenase and other krebs cycle genes after nitrate resupply to short-term nitrogen-starved tobacco

Mitochondrial NAD-dependent (IDH) and cytosolic NADP-dependent isocitrate dehydrogenases have been considered as candidates for the production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The increase in IDH transcripts in leaf and root tissues, induced by nitrate or NH4+ resupply to short-term N-starved tobacco (Nicotiana tabacum) plants, suggested that this enzyme could play such a role. The leaf and root steady-state mRNA levels of citrate synthase, acotinase, IDH, and glutamine synthetase were found to respond similarly to nitrate, whereas those for cytosolic NADP-dependent isocitrate dehydrogenase and fumarase responded differently. This apparent coordination occurred only at the mRNA level, since activity and protein levels of certain corresponding enzymes were not altered. Roots and leaves were not affected to the same extent either by N starvation or nitrate addition, the roots showing smaller changes in N metabolite levels. After nitrate resupply, these organs showed different response kinetics with respect to mRNA and N metabolite levels, suggesting that under such conditions nitrate assimilation was preferentially carried out in the roots. The differential effects appeared to reflect the C/N status after N starvation, the response kinetics being associated with the nitrate assimilatory capacity of each organ, signaled either by nitrate status or by metabolite(s) associated with its metabolism.

Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA) function using a collection of mutants defective in all TCA cycle genes.

The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomyces cerevisiae. We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins. The 15 major TCA cycle genes can be sorted into five phenotypic categories on the basis of their growth on nonfermentable carbon sources. We have previously reported a novel phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH). Null and nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the TCA cycle citrate synthase and in other genes of oxidative metabolism. The TCA cycle mutant collection was utilized to search for other genes that can suppress idh2 mutants and to identify TCA cycle genes that display a similar suppressible growth phenotype on glycerol. Mutations in 7 TCA cycle genes were capable of functioning as suppressors for growth of idh2 mutants on glycerol. The only other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype was IDH1, which encodes the companion Idh1p subunit of NAD-IDH. These results provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function.

Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants.

NAD-dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling the activity of the citric acid cycle. Despite more than 30 years of work, the plant enzyme remains poorly characterized. In this paper, a molecular characterization of the plant IDH is presented. Starting from probes defined according to sequence comparisons, three full-length cDNAs named Ntidha, Ntidhb and Ntidhc encoding different IDH subunits have been isolated from a Nicotiana tabacum cell suspension library. Sequence comparisons of the tobacco IDH subunits with the E. coli NADP-dependent enzyme, and the yeast IDH1 and IDH2 subunits suggested that only IDHa had the capacity to be catalytic as IDHb and IDHc were lacking certain residues implied in catalysis. The ability of antibodies raised against the recombinant IDHa protein to preferentially cross-react with IDH2 indicated that IDHa was more closely related to IDH2 than to IDH1. Complementation of yeast single IDH mutants showed that IDHb and IDHc could replace the function of the yeast regulatory IDH1 subunit. Although IDHa was unable to complement the IDH2 mutant, its catalytic function was revealed by the ability of two heteromeric enzymes, composed of either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yeast double mutant lacking both subunits. Expression studies at the protein and mRNA levels show that each subunit is present in both root and leaf tissues and that the three IDH genes respond in the same way to nitrate addition. Taken together, such observations suggest that the physiologically active enzyme is composed of the three different subunits. These results show for the first time that the plant IDH is heteromeric and that IDH subunit composition appears to be conserved between plant and animal kingdoms.

Identification of the subunit and important target peptides of pig heart NAD-dependent isocitrate dehydrogenase modified by the affinity label adenosine 5'-O-[S-(4-bromo-2, 3-dioxobutyl)thiophosphate

Pig heart NAD-dependent isocitrate dehydrogenase is inactivated by adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPS-BDB) with incorporation of 1.78 mol of reagent/mol of average subunit. Complete protection against the inactivation is provided by 20 mM isocitrate + 1 mM Mn2+, and the incorporation is decreased to about 1.3 mol of reagent/mol of average subunit. The addition of NAD, NADH, or Mn2+ alone has little effect on the functional changes produced by AMPS-BDB, while ADP gives only partial protection against the inactivation. The ability of ADP to decrease the Km for isocitrate is not affected by the AMPS-BDB modification of the enzyme. These results indicate that the isocitrate substrate site is the target of AMPS-BDB. The enzyme has three types of subunits with a tetramer having the composition alpha2 beta gamma. Here, [2-3H]AMPS-BDB-modified subunits are separated by HPLC on a C4 reverse-phase column, after the treatment of the modified enzyme with 4 M urea. The predominant radioactivity is distributed in alpha and gamma subunits. However, evidence based on recombination of subunits from modified and unmodified enzymes indicates that only labeling of the alpha subunit is responsible for inactivation by AMPS-BDB. Subsequently, the separated modified subunits were chemically cleaved by CNBr and then purified by HPLC using a C18 column. The labeled peptides were further digested by pepsin, purified by HPLC, and sequenced. These results indicate that R88 and R98 from the alpha subunit are the major targets of AMPS-BDB which cause inactivation and that these are at or near the isocitrate site of the enzyme.

Effect of nitrogen and carbon sources on growth and activities of NAD and NADP dependent isocitrate dehydrogenases of Laccaria bicolor

The ectomycorrhizal Laccaria bicolor , cultivated axenically in modified Pachlewski's medium, can use a broad spectrum of nitrogen and carbon sources. The fungus exhibited greater growth in the presence of ammonium or nitrate than with ammonium nitrate. With the latter salt, the drop in pH of the culture medium indicated that ammonium was taken up preferentially to nitrate. L. bicolor grew more poorly on amino acids such as glycine, alanine, aspartate and glutamate, the latter three being very poor carbon sources. By contrast, glycine was used as nitrogen and carbon sources. Glucose and maltose in mixture, with a 20:5 ratio, were the most effective carbohydrates for promoting growth, followed by starch, dextrins, maltose, glucose and sorbitol. L. bicolor failed to grow in the presence of sucrose and galactose.

Identification of the Subunits and Target Peptides of Pig Heart NAD-Specific Isocitrate Dehydrogenase Modified by the Affinity Label 8-(4-Bromo-2,3-dioxobutylthio)NAD

Pig heart NAD-dependent isocitrate dehydrogenase reacts with 8-(4-bromo-2,3-dioxobutylthio)-NAD (8- BDB-TNAD) with incorporation of 1.21 mol of reagent/mol of average subunit when the enzyme reaches the limit of 25% residual activity (Kumar, A., and Colman, R. F.,Arch. Biochem. Biophys.308, 357–366, 1994). Inclusion of NADPH decreases both the extent of inactivation and the reagent incorporation to 0.55 mol/mol of average subunit. We have now isolated the peptides labeled by radioactive 8-(4-bromo-2,3-dioxobutylthio)-[2-3H]NAD and have located them within the sequence of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is composed of three types of subunits, present as α2βγ. We have separated the subunits from unmodified and 8-BDBT[2-3H]NAD-modified enzymes by HPLC on a C4reverse-phase column, after pretreatment of the enzymes with sodium dodecyl sulfate or urea, and compared the subunit sequences of the porcine enzyme with those of the corresponding subunits from other mammalian NAD-dependent isocitrate dehydrogenases. The predominant radioactivity of 8-BDBT[2-3H]NAD is observed in the α and γ peaks, and the NADPH-protected enzyme exhibits marked reduction in incorporation into these peaks. However, evidence based on recombination of subunits from modified and unmodified enzymes indicates that only labeling of the α-subunit is responsible for inactivation by 8-BDB-TNAD. Cyanogen bromide was used to cleave the modified enzyme, and we purified one labeled peptide from the α-subunit (amino acids 84–177) as well as one from the γ-subunit (amino acids 67–186). In the α-subunit, decreased modification by [7-14C]phenylglyoxal of Arg88and Arg98after prior labeling of the enzyme by 8-BDB-TNAD indicates that these residues are the critical target sites of the reactive nucleotide analogue. We conclude that α subunit's Arg88and Arg98are both at or near the allosteric NADPH sites of the pig heart isocitrate dehydrogenase.

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7.

The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.

Isocitrate dehydrogenases from liver ofSqualus acanthias (spiny dogfish) and citrate formation by isolated mitochondria

Isolated mitochondria from liver of Squalus acanthias (spiny dogfish) catalyze formation of citrate from α-ketoglutarate at a rate corresponding to >0.03 μmoles per min per g tissue. Malate alone does not support citrate formation, but stimulates citrated formation from α-ketoglutarate ≈︂ threefold. These reactions appear to proceed by reductive carboxylation catalyzed by the mitochondrial NADP-dependent isocitrate dehydrogenase, i.e., reversal of the Krebs cycle from α-ketoglutarate to citrate. The NADP-dependent isocitrate dehydrogenases present in the mitochondria and cytosol were isolated and shown to have different N-terminal amino acid sequences, indicating that they are encoded by different genes. The kinetic and structural properties of the mitochondrial enzyme were found to be similar to that of NADP-dependent isocitrate dehydrogenases from other species. NAD-dependent isocitrate dehydrogenase activity could not be detected in liver extracts or in mitochondrial or cytosolic subreactions of liver extracts. The level of activity of the reverse reaction (≈︂0.4 μmoles per min per g liver), i.e., reductive carboxylation, of the mitochondrial isocitrate dehydrogenase is sufficient to account for the observed rate of citrate formation by isolated mitochondria. Isolated mitochondria also catalyze citrate formation from pyruvate plus malate at a rate that is ≈︂70% of the rate observed with α-ketoglutarate plus malate. The levels of ATP citrate lyase (shown to be present in the cytosol) and cytosolic NADP-dependent isocitrate dehydrogenase activities (≈︂0.5 and 2.5 μmoles per min per g liver, respectively) are sufficient to utilize the observed optimal rate of mitochondrial citrate formation from α-ketoglutarate and pyruvate in the presence of malate (≈︂0.12 μmoles per min per g liver) for acetyl-CoA and NADPH formation required for lipid biosynthesis and-or for oxalacetate formation required for gluconeogensis. © 1996 Wiley-Liss, Inc.

Assembly and Function of a Cytosolic Form of NADH-specific Isocitrate Dehydrogenase in Yeast

Mitochondrial NAD-dependent isocitrate dehydrogenase catalyzes a rate-limiting step in the tricarboxylic acid cycle. Yeast isocitrate dehydrogenase is an octomer composed of two subunits (IDH1 and IDH2) encoded by different genes and possessing independent mitochondrial targeting presequences. Oligonucleotide-directed mutagenesis was used to remove the presequences from each gene and from both genes carried on centromere-based expression plasmids. Effects on cellular localization were examined in a yeast strain containing chromosomal disruptions of IDH1 and IDH2 loci. Each subunit was found to be dependent upon its presequence for mitochondrial localization, and the subunits are independently imported into mitochondria under most growth conditions. Furthermore, an active holoenzyme can be assembled in the cytosol and this ''cytosolic'' form of isocitrate dehydrogenase can reverse the acetate- growth phenotype characteristic of the DeltaIDH1/ DeltaIDH2 disruption strain, indicating functional replacement of the mitochondrial enzyme. However, transformants containing plasmids lacking either the IDH1 or IDH2 presequence coding regions were unexpectedly found to be capable of growth on acetate medium. Further investigation demonstrated that cellular localization of the IDH1 subunit can be biased by this stringent growth pressure.

Cadmium-113 and magnesium-25 NMR study of the divalent metal binding sites of isocitrate dehydrogenases from pig heart

The metal activator sites of NAD +-dependent and NADP +-dependent isocitrate dehydrogenases from pig heart have been probed using 113Cd- and 25Mg-NMR. In the presence of isocitrate and ADP, a broad resonance for cadmium bound to NAD-dependent isocitrate dehydrogenase was observed ( −8 ppm) arising from exchange with isocitrate (−20 ppm) and/or ADP (27 ppm) complexes. The Cd shift with ADP suggests interaction of the metal with the nucleotide ring nitrogen. Increasing shifts with excess ADP are indicative of macrochelate formation. 25Mg-NMR demonstrates that, unlike manganese, magnesium has a similar dissociation constant (1.8 mM) from NADP-dependent isocitrate dehydrogenase as from the enzyme-isocitrate complex (1.1 mM). The extrapolated line width of bound magnesium increases from 674 Hz in the binary complex to 10 200 Hz in the ternary complex. The quadrupole coupling constant, calculated from relaxation rates, is larger in the ternary complex. indicative of greater distortion in the magnesium coordination sphere. The line widths of magnesium complexed to NAD-dependent isocitrate dehydrogenase are broader, as expected for the larger octamer. 113Cd- and 25Mg-NMR both show that the metal sites have anisotropic octahedral symmetry. 25Mg relaxation rates yield correlation times corresponding to motions of a domain with motion independent of the enzyme multimers.

8-(4-Bromo-2,3-dioxobutylthio)NAD: A New Affinity Label for NAD-Specific Isocitrate Dehydrogenase

8-(4-Bromo-2,3-dioxobutylthio)nicotinamide adenine dinucleotide (8-BDB-TNAD), a new reactive NAD analog, was synthesized by coupling 8-thio-AMP with NMN, followed by condensation with 1,4-dibromobutanedione. Incubation of 160 μM 8-BDB-TNAD with the allosteric pig heart NAD-dependent isocitrate dehydrogenase causes time-dependent inactivation to a limit of 25% residual activity concomitant with incorporation of ∼ 1 mol reagent/mol average subunit. In addition to binding sites for NAD and NADH, this enzyme has been shown to have regulatory sites for NADPH and for ADP (R. S. Ehrlich and R. F. Colman 1982, J. Biol. Chem. 257, 4769-4774). Marked protection against enzyme inactivation by 8-BDB-TNAD and incorporation is provided by the regulatory nucleotides NADPH or ADP, while NAD and NADH are less effective. The rate constant for inactivation shows a nonlinear dependence on 8-BDB-TNAD concentration which can be ascribed to reversible formation of an enzyme-reagent complex (KI = 83 μM) prior to an irreversible reaction (kmax = 0.0625 min−1). Analysis of the kinetic properties and binding characteristics of modified enzyme indicates that this enzyme retains the ability to bind ADP, but does not bind NADPH. Thus, 8-BDB-TNAD reacts at or near the allosteric NADPH site of pig heart NAD-dependent isocitrate dehydrogenase.

Unfolding and refolding of the NAD-dependent isocitrate dehydrogenase from yeast

The unfolding of the NAD +-dependent isocitrate dehydrogenase from yeast in guanidinium chloride (GdnHCI) has been monitored by changes in c.d. and fluorescence. Major structural changes occur over the range of GdnHCI concentrations from 0.5 to 1.5 M, although loss of catalytic activity is complete at 0.3 M. After incubation in GdnHCI, activity can be regained on dilution; however, the extent of this regain is dependent on the initial concentration of GdnHCI and is very small at a concentration of 2 M or above. Under these conditions there is only limited regain of the secondary and tertiary structure of the enzyme. Considerably more structure and activity can be regained if the concentration of GdnHCI is lowered by dialysis. The implications of these results for the folding and assembly of the enzyme are discussed.