TPN-dependent Isocitrate Dehydrogenase Research Articles

Modification of TPN-dependent isocitrate dehydrogenase by the 2‘, 3‘-dialdehyde derivatives of TPNH and TPN.

Catalytic reaction of the 2', 3'-dialdehyde analog of TPN (oTPN) with pig heart TPN-dependent isocitrate dehydrogenase in the presence of the substrate manganous isocitrate results in the formation of the dialdehyde derivative of TPNH (oTPNH). In the absence of the substrate, modification by oTPN leads to a progressive inactivation of the enzyme. The dependence of the pseudo-first order rate constants on the reagent concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification (kmax = 5.5 X 10(-2) min-1; K1 = 290 microM). Reaction of [14C]oTPN with the enzyme results in the incorporation of 2 mol of oTPN/mol of peptide chain. No appreciable protection against either inactivation or incorporation by the natural ligands TPN and TPNH was obtained, suggesting different modes of binding of the analog in the presence and absence of the substrate isocitrate. Enzymatically synthesized oTPNH has been isolated and demonstrated to act as an affinity label for a TPNH-binding site of isocitrate dehydrogenase. The inactivation process exhibits saturation kinetics (kmax = 2.67 X 10(-3) min-1; K1 = 33 microM). Protection against activity loss, as well as a decrease in incorporation from 2 to 1 eq of [14C]oTPNH bound/peptide chain was observed in the presence of 1 mM TPNH. From the TPNH concentration dependence of the inactivation rate by oTPNH, a dissociation constant of 3.4 microM is calculated for TPNH, indicating binding of the analog to a specific TPNH-binding site on the enzyme. Although dialdehyde derivatives are frequently assumed to form Schiff bases with proteins, the evidence presented suggests the formation of morpholino derivatives as the products of the covalent reaction of isocitrate dehydrogenase with the dialdehyde derivatives of TPN and TPNH. The new reagent, oTPNH, may serve as an affinity label for other dehydrogenases.

The reaction of 4-iodoacetamidosalicylic acid with TPN-dependent isocitrate dehydrogenase from pig heart.

The reaction of 4-iodoacetamidosalicylic acid with TPN-dependent isocitrate dehydrogenase from pig heart.

Cysteine in the manganous-isocitrate binding site of pig heart TPN-specific isocitrate dehydrogenase: I. Kinetics of chemical modification and properties of thiocyano enzyme

Pig heart TPN-dependent isocitrate dehydrogenase is inactivated by reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). The dependence of the rate constant for inactivation on the reagent concentration is nonlinear, and can be analyzed in terms of the existence of two mechanisms for reaction with the enzyme, one involving reversible binding prior to inactivation and the other a bimolecular reaction. Cyanide reacts with the inactive modified enzyme to yield thiocyano-isocitrate dehydrogenase without increasing the catalytic activity; this result suggests that inactivation by DTNB is not due to steric hindrance by the bulky thionitrobenzoate group bound to the enzyme. The inactive thiocyano enzyme binds manganous ion normally. In contrast to its effect on native enzyme, however, isocitrate does not strengthen the binding of Mn 2+ to the thiocyano enzyme; the tightened binding of manganous-isocitrate may be critical for the catalytic activity of the enzyme. Protection against inactivation by DTNB is provided by isocitrate plus the activator, manganous ion, or the competitive inhibitor, calcium ion. The concerted inhibitors oxalacetate and glyoxylate, when present together with Mn 2+ and TPN, also protect against loss of activity. A marked decrease in the inactivation rate constant to a finite limiting value is caused by saturating concentrations of TPNH and Mn 2+, indicating that these ligands do not bind directly at the sites attacked by DTNB. The number of cysteine residues which react with DTNB concomitant with inactivation depends on the ligands present in the reaction mixture. In all cases, the equivalent of one -SH reacts without affecting activity. In the presence of Mn 2+ and α-ketoglutarate, which do not appreciably affect the inactivation rate, loss of activity is proportional to reaction with two -SH groups. These results suggest that the integrity of a maximum of two cysteine residues is essential for the function of the pig heart isocitrate dehydrogenase, and that at least one cysteine residue may be located within the manganous-isocitrate binding site.

Alkylation of methionine in human heart TPN-dependent isocitrate dehydrogenase.

Alkylation of methionine in human heart TPN-dependent isocitrate dehydrogenase.

Structure-function relationships in TPN-dependent isocitrate dehydrogenase. I. Electron paramagnetic resonance studies of the interaction of enzyme-bound Mn(II) with substrates, cofactors, and substrate analogues.

Electron paramagnetic resonance (EPR) spectra were obtained for various isocitrate dehydrogenase-Mn(II) complexes. The qualitative effects of the binding of substrates, nucleotides, and substrate analogues on the isotropic character of the electronic environment of enzyme-bound Mn(II) were subsequently investigated. The addition of isocitrate produces a markedly anisotropic spectrum whereas alpha-ketoglutarate does not alter the spectrum of enzyme-Mn(II) substantially. This suggests direct coordination of isocitrate to the Mn(II) but perphaps a different mode of binding for alpha-ketoglutarate. Other studies demonstrated mutually exclusive binding relationships between TPN and TPNH, between Mn-isocitrate and TPNH, and between HCO3-(CO2) and formate or thiocyanate. Indirect evidence supporting CO2 rather than HCO3-as the actual reactive species which binds to the enzyme in the reductive carboxylation reaction is presented on the basis of the results of the formate and thiocyanate studies. From the EPR results recorded for ternary, quaternary, and quinary enzyme-substrate complexes, correlations between the appearance of fine structure signals and the binding of individual substrates and/or nucleotides are found, and tentative assignments of such signals are made on this basis. Additional studies were conducted to determine binding constants for Mg(II) Co(II), and Co-isocitrate, and a comparison was made with kinetically determined binding constants.

Structure-function relationships in TPN-dependent isocitrate dehydrogenase. II. Determination of the paramagnetic relaxation rates of water protons in complexes of enzyme, Mn(II), substrates, cofactors, and inhibitors.

Structure-function relationships in TPN-dependent isocitrate dehydrogenase. II. Determination of the paramagnetic relaxation rates of water protons in complexes of enzyme, Mn(II), substrates, cofactors, and inhibitors.

Mechanisms for the oxidative decarboxylation of isocitrate: Implications for control

Mammalian tissues contain two isocitrate dehydrogenases: the DPN-dependent enzyme which is an oligomeric protein subject to allosteric activation by ADP, and the TPN-specific enzyme which is a monomeric protein not generally considered to be subject to regulation. Yet any controls exerted on the oxidative decarboxylation of isocitrate must necessarily encompass both enzymes since the reactions involve common substrates and metal ions. The two enzymes have been isolated in homogeneous state from porcine cardiac muscle. Similarity in their catalytic mechanisms is indicated by kinetic studies and evidence from chemical modification of enzymic functional groups. No appreciable deuterium isotope effect is observed when isocitrate-2- 2H is substituted for isocitrate-2- 1H as substrate for either the DPN- or TPN-dependent isocitrate dehydrogenase. The rates of both reactions are decreased about 5-fold in D 2O as compared with H 2O as solvent, implying that a proton transfer (perhaps from the hydroxyl group of isocitrate) may be involved in the rate-determining step. The pH dependence of Vmax measured at different temperatures suggests that an enzyme carboxylate ion is essential for the activity of both enzymes, although its pK is approximately 5.7 for the TPN enzyme and 6.6 for the DPN enzyme. This conclusion is strengthened by the inactivation of both enzymes by carbodiimide in the presence of nucleophiles. Similarly, chemical modification studies have implicated cysteinyl residues in the function of both enzymes. A single methionyl residue has been shown to be critical for catalytic activity of the TPN enzyme but a comparable residue has not yet been identified for the DPN enzyme. A lysyl residue has been shown to be directly involved only in the DPN enzyme. Mechanisms are proposed to account for catalysis of the oxidative decarboxylation of isocitrate by the two enzymes. Of the four stereoisomers, threo-D s-isocitrate is used as substrate by both isocitrate dehydrogenases. However, the pH dependence of K m for isocitrate suggests that dibasic isocitrate is the actual substrate for the DPN enzyme, whereas tribasic isocitrate is utilized by the TPN enzyme. Both enzymes require a divalent metal ion for activity. Kinetic and binding studies indicate that the TPN enzyme interacts with the preformed metal-tribasic isocitrate complex, whereas the DPN enzyme (depending on the available metal concentration) can either bind sequentially free metal ion followed by free dibasic isocitrate or can bind the preformed metal-dibasic isocitrate complex. Manganous ion produces the highest maximum velocity of each enzyme, but marked differences in the relative effectiveness of other metal ions, such as zinc and magnesium, are apparent. ADP, by binding to the allosteric site of the DPN enzyme, uniquely lowers the K m for dibasic isocitrate and its metal complex. However, other metabolic chelators which affect the distribution of free and metal-bound isocitrate can also indirectly alter the affinity of the substrate for both enzymes. The DPN- and TPN-specific isocitrate dehydrogenases are complementary. Several factors, including changes in the concentrations of different metal ions, subtle variations in pH or fluctuations in the levels of physiological chelators, influence the relative flux through the two pathways of oxidative decarboxylation of isocitrate. These changes may contribute to an alteration in the ratio of DPNH to TPNH in the intracellular environment.

Substrate Independence of Molecular Weight of Triphosphopyridine Nucleotide-specific Isocitrate Dehydrogenase

The techniques of gel filtration and light scattering were used to evaluate a report that the molecular weight of the TPN-specific isocitrate dehydrogenase of pig heart can be influenced by its substrates. The results here presented indicate that the elution position of the enzyme from a column of Sephadex G-150 is not appreciably altered by the addition of isocitrate, manganous ion, TPN, α-ketoglutarate, or TPNH. The Stokes radius is estimated as 39.3 A. Similarly the weight average molecular weight of the enzyme, as measured by light scattering, does not vary significantly from 58,000 when isocitrate, α-ketoglutarate, metal ion, and the coenzymes are added. It is concluded that the pig heart TPN-dependent isocitrate dehydrogenase exists as a single molecular weight species independent of the presence of substrates.

Role of Metal Ions in Reactions Catalyzed by Pig Heart Triphosphopyridine Nucleotide-dependent Isocitrate Dehydrogenase: I. MAGNETIC RESONANCE AND BINDING STUDIES OF THE COMPLEXES OF ENZYME, MANGANOUS ION, AND SUBSTRATES

Abstract Native TPN-dependent isocitrate dehydrogenase binds 1 mole of Mn(II) per mole of enzyme with a dissociation constant of 45 µm which remains unchanged in the presence of coenzymes, α-ketoglutarate, or bicarbonate, but is lowered to 2.2 µm in the presence of isocitrate. A specific influence of isocitrate on the affinity of enzyme for manganese is implied. Inactive N-ethylmaleimide-modified enzyme binds 1 mole of manganous ion with a dissociation constant of 48 µm in the presence or absence of isocitrate, suggesting that modification of critical sulfhydryl groups disrupts interaction between the manganous ion and isocitrate sites. In contrast, carboxymethylmethionyl enzyme exhibits a dissociation constant of 8.0 µm in the presence and 18 µm in the absence of isocitrate. Isocitrate dehydrogenase enhances the effect of Mn(II) on the proton relaxation rate of water at 22° and 24.3 MHz giving an enhancement, eb, due to bound manganous ion of 11.0 ± 0.5. Isocitrate and α-ketoglutarate (but not TPN or TPNH) lower this enhancement by approximately twothirds. Dissociation constants of isocitrate and α-ketoglutarate from their respective ternary complexes were calculated to be 2 ± 1 µm and 290 ± 80 µm, and are essentially unchanged in the presence of TPN or TPNH. These results are consistent with the formation of ternary metal bridge complexes involving enzyme, Mn(II), and substrates.

The Role of Sulfhydryl Groups in the Catalytic Function of Isocitrate Dehydrogenase: II. EFFECT OF N-ETHYLMALEIMIDE ON KINETIC PROPERTIES

Abstract Dehydrogenase and decarboxylase activity are both lost as a result of incubation of TPN-dependent isocitrate dehydrogenase with N-ethylmaleimide (NEM). The rate of inactivation is decreased 450-fold by isocitrate in the presence of MnSO4, indicating a specific reaction at the active site of the enzyme. No other substrates or coenzymes significantly decrease the reaction rate, although TPNH plus MnSO4 have been shown to protect against the reaction of essential sulfhydryl groups of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid). The larger derivative of NEM, N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, also inactivates the enzyme, but protection in that case is provided by TPNH plus MnSO4, suggesting that steric factors may account for the different results with the three reagents. Inactive NEM-enzyme retains the ability to bind 1 mole each of radioactive isocitrate and α-ketoglutarate. Inactivation must therefore be attributed to disruption of a catalytic step subsequent to the adsorption of these substrates. Partially inactive NEM-enzymes exhibit a 3- to 4-fold increase in the Michaelis constants for isocitrate and oxalosuccinate, whereas the Km values for other substrates are normal. In addition, an increase of 0.3 logarithmic unit is noted in the pK of an essential ionizable group in the enzyme-substrate complex. Enzyme incubated with NEM in the presence of isocitrate and manganous ions retains full activity but still exhibits abnormal Km and pK values. Reaction of 1 functional group (A) may thus be responsible for inactivation, while modification of a second group (B) may cause the changes in the kinetic parameters.

The Role of Sulfhydryl Groups in the Catalytic Function of Isocitrate Dehydrogenase: III. EFFECT OF N-ETHYLMALEIMIDE ON CHEMICAL AND PHYSICAL PROPERTIES

Abstract Incubation of the TPN-dependent isocitrate dehydrogenase from pig heart with 1-14C-N-ethylmaleimide (NEM) leads to inactivation and altered Michaelis constants for isocitrate and oxalosuccinate concomitant with incorporation of 2 moles of reagent. Reaction of enzyme with radioactive NEM in the presence of isocitrate and Mn++ yields a catalytically functional enzyme, with the same altered Michaelis constants, which also contains 2 moles of reagent. Inactive enzyme with a single carboxymethyl methionyl residue exhibits the same ability to bind radioactive NEM as does native enzyme; however, prior inactivation by reaction of 5 sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) prevents binding of NEM. Cysteine is the sole type of amino acid modified in the inactive NEM-enzyme, as determined by paper chromatographic identification of radioactive cysteinosuccinic acid and S-(N-ethylsuccinimido)cysteine in acid and proteolytic digests of 14C-labeled modified enzyme. Thirteen moles of cysteic acid are found after performic acid oxidation of native enzyme, the same as the number of free —SH groups observed by reaction with DTNB after denaturation; therefore, no disulfide bonds are present in isocitrate dehydrogenase. A decrease in the number of measurable —SH groups after reaction with NEM is consistent with modification of cysteinyl residues. By contrast, the recovery of NH2-terminal alanine after the Edman reaction was approximately the same for native and modified enzyme. Inactive enzymes with a single carboxymethyl methionyl residue, with 2 or 5 altered sulfhydryl groups, give a reaction of antigenic identity to that of native enzyme as measured against rabbit antibody to isocitrate dehydrogenase. The molecular size of the inactive NEM-enzyme is the same as that of native enzyme, as determined by gel filtration on Sephadex G-150, and the rates of proteolytic digestion by Pronase are not significantly different. These data argue against a generalized structural change in the modified enzyme. However, a small conformational change is indicated by a decrease in the reaction rate of DTNB with the residual —SH groups of the inactive NEM-enzyme. The magnitude of conformational change is not directly related to loss in enzymatic activity, since an observed decrease in the amplitude of the optical rotatory dispersion curves is greater for active NEM-enzyme ([α]234 -5610°) than it is for inactive NEM-enzyme ([α]234 -7430°), as compared to native isocitrate dehydrogenase ([α]234 -8050°). These results, in conjunction with those of the preceding paper, suggest that, in the absence of isocitrate and MnSO4, modification of Sulfhydryl Group A produces inactivation, whereas modification of Sulfhydryl Group B leads to a small conformational change which is reflected in altered Michaelis constants. In the presence of substrates, NEM reacts with 2 sulfhydryl groups, including Group B but not Group A, which produces a further change in the conformation of the enzyme but no concomitant change in the kinetic properties.

Deuterium solvent isotope effects in reactions catalyzed by isocitrate dehydrogenase

The result of substituting deuterium oxide for water on the rates of the overall and partial reactions catalyzed by the pig heart TPN-dependent isocitrate dehydrogenase has been studied. Although the overall oxidative decarboxylation of isocitrate proceeds 4.9 times as fast in H 2O as in D 2O, the rate of the separately measured oxalosuccinate decarboxylation reaction is only 1.4–1.7 times greater in water. In contrast, the oxalosuccinate reduction reaction proceeds 5.0 times faster in H 2O. These results suggest that dehydrogenation is the slow reaction in the oxidative decarboxylation of isocitrate and that a proton transfer is involved in this rate determining step.

Effects of various anions and cations on the release of four dehydrogenases from brain mitochondria by a non-ionic detergent.

Summary It has been found that a number of agents greatly affects the release by Triton-X-ioo of certain dehydrogenases from rabbit-brain mitochondria. The enzymes studied were DPN-dependent isocitrate dehydrogenase ( L s-isocitrate:DPN+ oxidoreductase (decarboxylating), EC 1.1.1.41) and TPN-dependent isocitrate dehydrogenase ( L s-isocitrate:TPN+ oxidoreductase (decarboxylating), EC 1.1.1.42), gluta-mate dehydrogenase ( L -glutamate:DPN+ (TPN+) oxidoreductase (deaminating), EC 1.4.1.3) and lipoamide dehydrogenase (DPNH:lipoamide oxidoreductase, EC 1.6.4.3). The effects of Triton X-100 on the morphology of the mitochondria were checked with the electron microscope. 1. The detergent was shown to disrupt the architecture of the mitochondria, leaving single and double membranes and amorphous material. 2. ATP, ADP, pyrophosphate, citrate and isocitrate enhanced release of the enzymes. DPN was less effective, and α-ketoglutarate, adenylic acid and adenosine were ineffective, as was EDTA. 3. K+ and Na+ (80 mM) enhanced release of both isocitrate dehydrogenases, but partially prevented release of glutamate dehydrogenase. Ca2+ and Mg2+ at 4–5 mM enhanced release of TPN-isocitrate dehydrogenase but inhibited that of DPN-isocitrate dehydrogenase and glutamate dehydrogenase. Mg2+ had little effect on the release of lipoamide dehydrogenase. 4. It is suggested that metal ions play a role in the binding of these four dehydrogenases to the mitochondria, but that the details of the attachments may be different in each case.

The Comparative Activity of Some Enzymes in Sheep, Cattle and Rats—Normal Serum and Tissue Levels and Changes During Experimental Liver Necrosis

SUMMARY A survey has been carried out of the distribution of some enzymes in the tissues, whole blood and serum of normal sheep, cattle and rats. The enzymes studied were glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), glutamate dehydrogenase (GD), L-lactate dehydrogenase (LD) and TPN-dependent isocitrate dehydrogenase (ICD). Carbon tetrachloride was given orally to 4 sheep, 2 calves, 1 cow and 26 rats. Centrilobular liver necrosis was observed histologically in liver samples obtained post-mortem from the rats, and by biopsy from the cow, 3 of the sheep and both calves. The activities of the enzymes were assayed in serum collected at intervals from these animals. These activities, except of GPT in the cow, sheep and calves, increased during the period of necrosis of liver cells up to 48 hours. There was a tendency for enzymes most active in liver to increase most. There also appeared to be some relationship between the amount of the increase and the extent of hepatic necrosis. The pattern of serum enzyme elevations was similar in the cow, sheep and calves and differed from that in rats. The serum enzyme most affected by liver damage was GPT in the rat and GD in sheep and calves.