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PubMed1975

Purification and properties of a soluble nicotinamide adenine dinucleotide-linked isocitrate dehydrogenase from Crithidia fasciculata.

A soluble NAD+-linked isocitrate dehydrogenase has been isolated from Crithidia fasciculata. The enzyme was purified 128-fold, almost to homogeneity, and was highly specific for NAD+ as the coenzyme. There is also a cytoplasmic NADP+-linked and a mitochondrial isocitrate dehydrogenase in the organism. Studies of the physical and kinetic properties of the soluble NAD+-isocitrate dehydrogenase from this organism showed that it resembled microbial NADP+-isocitrate dehydrogenases in general, all of which are cytoplasmic enzymes. The enzyme appeared not to be related to other NAD+-isocitrate dehydrogenases, which are found in the mitochondria of eukaryotic cells. The molecular weight of the soluble NAD+-isocitrate dehydrogenase was 105,000 which is within the range of the values for microbial NADP+-isocitrate dehydrogenases. Similar to the NADP+-isocitrate dehydrogenase in this organism, the enzyme was inhibited in a concerted manner by glyoxalate plus oxalacetate. Kinetic analysis revealed that Mn2+ was involved in the binding of isocitrate to the enzyme. Inhibition of the NAD+-linked isocitrate dehydrogenase by p-chloromercuribenzoate could be prevented by prior incubation of the enzyme with both Mn2+ and isocitrate; however, neither ion alone conferred protection. Free isocitrate, free Mn2+, and the Mn2+-isocitrate complex could all bind to the enzyme. Four different mechanisms with respect to the binding of isocitrate to the enzyme were tested. Of these, the formation of the active enzyme-Mn2+-isocitrate complex from (a) the random binding of Mn2+, isocitrate, and the Mn2+-isocitrate complex, or (b) the binding of Mn2+-isocitrate with free Mn2+ and isocitrate acting as dead-end competitors were both in agreement with these data.

Animals↗
PubMed1979

Activities of NAD-specific and NADP-specific isocitrate dehydrogenases in rat-liver mitochondria. Studies with D-threo-alpha-methylisocitrate.

The contributions of NAD-specific and NADP-specific isocitrate dehydrogenases to isocitrate oxidation in isolated intact rat liver mitochondria were examined using DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) to specifically inhibit flux through NADP-specific isocitrate dehydrogenase. Under a range of conditions tested with respiring mitochondria, the rate of isocitrate oxidation was decreased by about 20--40% by inhibition of NADP-isocitrate dehydrogenase, and matrix NADP became more oxidized. (a) For mitochondria incubated with externally added DL-isocitrate and citrate, the rate of isocitrate oxidation obtained by extrapolation to infinite alpha-methylisocitrate concentration was approximately 70% of the uninhibited rate in both state 3 and state 4. (b) With pyruvate plus malate added as substrates of citric acid cycle oxidation and isocitrate generated intramitochondrially, a concentration of alpha-methylisocitrate (400 microM) sufficient for 99.99% inhibition of NADP-isocitrate dehydrogenase inhibited isocitrate oxidation in states 4 and 3 by 21 +/- 6% and 19 +/- 11% (mean +/- SEM), respectively. (c) With externally added isocitrate and citrate, the addition of NH4Cl increased isocitrate oxidation by 3--4-fold, decreased NADPH levels by 30--40% and 2-oxoglutarate accumulation by about 40%. The further addition of 600 microM alpha-methylisocitrate decreased the NH4Cl-stimulated isocitrate oxidation by about 40% and decreased NADPH to about 30% of the level prevailing in the absence of NH4Cl; nevertheless, the rate of isocitrate oxidation was still twice as large in the presence of NH4Cl and alpha-methylisocitrate as in their absence. Experiments were also performed with intact mitochondria incubated with respiratory inhibitors to determine additional factors which might affect the flux through the two isocitrate dehydrogenases. (a) In the coupled reduction of acetoacetate by isocitrate, where the rate of reoxidation of reduced pyridine nucleotides is limited by NAD-specific 3-hydroxybutyrate dehydrogenase, 85--100% of the rate of 3-hydroxybutyrate formation was retained in the presence of 400--900 microM alpha-methylisocitrate. (b) In a system where the rate of isocitrate oxidation is limited by the rate of NADPH reoxidation by glutathione reductase, the rate of glutathione reduction extrapolated to infinite alpha-methylisocitrate concentration was from 20--40% of the uninhibited rate. (c) In the coupled synthesis of glutamate from isocitrate and NH4Cl, where the reoxidation of NADPH and NADH can occur via glutamate dehydrogenase, the rate of glutamate production extrapolated to infinite alpha-methylisocitrate concentration was about 60% of the uninhibited rate.

Ammonium Chloride↗
PubMed1969

Th control of isocitrate oxidation by rat liver mitochondria.

1. The factors capable of affecting the rate of isocitrate oxidation in intact mitochondria include the rate of isocitrate penetration, the activity of the NAD-specific and NADP-specific isocitrate dehydrogenases, the activity of the transhydrogenase acting from NADPH to NAD(+), the rate of NADPH oxidation by the reductive synthesis of glutamate and the activity of the respiratory chain. A quantitative assessment of these factors was made in intact mitochondria. 2. The kinetic properties of the NAD-specific and NADP-specific isocitrate dehydrogenases extracted from rat liver mitochondria were examined. 3. The rate of isocitrate oxidation through the respiratory chain in mitochondria with coupled phosphorylation is approximately equal to the maximal of the NAD-specific isocitrate dehydrogenase but at least ten times as great as the transhydrogenase activity from NADPH to NAD(+). 4. It is concluded that the energy-dependent inhibition of isocitrate oxidation by palmitoylcarnitine oxidation is due to an inhibition of the NAD-specific isocitrate dehydrogenase. 5. Kinetic studies of NAD-specific isocitrate dehydrogenase demonstrated that its activity could be inhibited by one or more of the following: an increased reduction of mitochondrial NAD, an increased phosphorylation of mitochondrial adenine nucleotides or a fall in the mitochondrial isocitrate concentration. 6. Uncoupling agents stimulate isocitrate oxidation by an extent equal to the associated stimulation of transhydrogenation from NADPH to NAD(+). 7. A technique is described for continuously measuring with a carbon dioxide electrode the synthesis of glutamate from isocitrate and ammonia.

Adenine Nucleotides↗
PubMed1997

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus.

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP+. Optimal temperature for activity was 90 degrees C or above, and a half-life of 22 min was found for the enzyme when incubated at 90 degrees C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups.

Amino Acid Sequence↗
PubMed1988

Isotope effect studies of the chemical mechanism of pig heart NADP isocitrate dehydrogenase.

The catalytic mechanism of porcine heart NADP isocitrate dehydrogenase has been investigated by use of the variation of deuterium and 13C kinetic isotope effects with pH. The observed 13C isotope effect on V/K for isocitrate increases from 1.0028 at neutral pH to a limiting value of 1.040 at low pH. The limiting 13C isotope effect with deuteriated isocitrate at low pH is 1.016. This decrease in 13(V/KIc) upon deuteriation indicates a stepwise mechanism for the oxidation and decarboxylation of isocitrate. This predicts a deuterium isotope effect on V/K of 2.9, but D(V/K) at low pH only increases to a maximum of 1.08. It is not known why 13(V/KIc) with deuteriated isocitrate decreases more than predicted. The pK seen in the 13(V/KIc) pH profile for isocitrate is 4.5. This pK is displaced 1.2 pH units from the true pK of the acid/base functionality of 5.7 seen in the pKi profile for oxalylglycine, a competitive inhibitor for isocitrate. From this displacement, catalysis is estimated to be 16 times faster than substrate dissociation. By use of the pH-dependent partitioning ratio of the reaction intermediate oxalosuccinate between decarboxylation to 2-ketoglutarate and reduction to isocitrate, the forward commitment to catalysis for decarboxylation was determined to be 7.3 at pH 5.4 and 3.2 at pH 5.0. This gives an intrinsic 13C isotope effect for decarboxylation of 1.050. 3-Fluoroisocitrate is a new substrate oxidatively decarboxylated by NADP isocitrate dehydrogenase. At neutral pH, D(V/K3-F-Ic) = 1.45 and 13(V/K3-F-Ic) = 1.0129. At pH 5.2, 13(V/K3-F-Ic) increases to 1.0186, indicating that a finite, but diminished, external commitment remains at neutral pH. The product of oxidative decarboxylation of 3-hydroxyisocitrate by NADP isocitrate dehydrogenase is 2-hydroxy-3-ketoglutarate. This results from enzymatic protonation of the cis-enediol intermediate at C2 rather than C3 (as seen with isocitrate and 3-fluoroisocitrate). 2-Hydroxy-3-ketoglutarate further decarboxylates in solution to 2-hydroxy-3-ketobutyrate, which further decarboxylates to acetol. This makes 3-hydroxyisocitrate unsuitable for 13C isotope effect studies.

Animals↗
PubMed2000

Identification by mutagenesis of arginines in the substrate binding site of the porcine NADP-dependent isocitrate dehydrogenase.

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The crystal structure of Escherichia coli isocitrate dehydrogenase and sequence alignment of porcine with E. coli isocitrate dehydrogenase suggests that the porcine Arg(101), Arg(110), Arg(120), and Arg(133) are candidates for roles in substrate binding. The four arginines were separately mutated to glutamine using a polymerase chain reaction method. Wild type and mutant enzymes were each expressed in E. coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase. The R120Q mutant has a specific activity, as well as K(m) values for isocitrate, Mn(2+), and NADP(+) similar to wild type enzyme, indicating that Arg(120) is not needed for function. The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 micromols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme. The R110Q and R133Q enzymes exhibit K(m) values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. The K(m) values for Mn(2+), but not for NADP(+), are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants. It is proposed that the positive charges of Arg(110) and Arg(133) normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction. The R101Q mutant shows smaller, but significant increases in the K(m) values for isocitrate and Mn(2+); however, the marked decrease in k(cat) suggests a role for Arg(101) in catalysis. The V(max) of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK(aes) is similar for the R101Q and R120Q enzymes. In contrast, the pK(aes) for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg(110) and Arg(133) normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of the E. coli enzyme.

Amino Acid Sequence↗