Coenzyme binding by native and chemically modified pig heart triphosphopyridine nucleotide dependent isocitrate dehydrogenase.

The binding of TPNH to native and chemically modified pig heart TPN-dependent isocitrate dehydrogenase was studied by the techniques of ultrafiltration and fluorescence enhancement. A single site (per peptide chain) was found for TPNH with a dissociation constant (KD = 1.45 muM) that is quantitatively comparable to the Michaelis constant. The oxidized coenzyme, TPN+, weakens the binding of TPNH. The substrate manganous isocitrate also inhibits the binding of TPNH and, reciprocally, TPNH inhibits the binding of manganous isocitrate, suggesting that binding to the reduced coenzyme and substrate sites is mutually exclusive. Ultrafiltration experiments with carbonyl [14C]TPN+ revealed the existence of two sites with a dissociation constant (49 muM) more than ten times higher than the Michaelis constant. This observation excludes a random mechanism for isocitrate dehydrogenase or a sequential mechanism in which TPN+ binds first. Four chemically modified isocitrate dehydrogenases have been prepared: enzyme inactivated by reaction of a single methionyl residue with iodoacetate, by modification of a glutamyl residue by glycinamide (in the presence of a water soluble carbodiimide), by reaction of four cysteines successively with 5,5'-dithiobis(2-nitrobenzoic acid) and potassium cyanide, or by addition of two cysteine residues to N-ethylmaleimide. These enzymes were tested for their ability to bind TPN+, TPNH, and manganous isocitrate. In the cases of the cysteinyl and glutamyl-modified enzymes, inactivation appears to be due primarily to loss of the ability to bind the substrate manganous isocitrate. In constrast, the methionyl residue may participate in the coenzyme binding site or, more likely, may be involved in a step in catalysis subsequent to binding.     

Isotope effect studies of the role of metal ions in isocitrate dehydrogenase.

Pig heart NADP+-dependent isocitrate dehydrogenase requires a metal ion for activity. Under optimum conditions (pH 7.5, Mg2+ present), the carbon isotope effect is k12/k13 = 0.9989 +/- 0.0004 for the carboxyl carbon undergoing decarboxylation and hydrogen isotope effects are VmaxH/VmaxD = 1.09 +/- 0.04 and (Vmax/Km)H/(Vmax/Km)D = 0.76 +/- 0.12 with threo-D,L-[2-2H]isocitric acid. Deuterium isotope effects measured by the equilibrium perturbation technique under the same conditions are VH/VD = 1.20 for the forward reaction and 1.02 for the reverse reaction. Under these conditions the rate-determining step in the enzymatic reaction must be product release. Dissociation of isocitrate from the enzyme-isocitrate complex and the enzyme-NADP+ complex must be two or more orders of magnitude slower than the chemical steps. The catalytic activity of the enzyme is about tenfold lower in the presence of Ni2+ than in the presence of Mg2+. The carbon isotope effect in the presence of Ni2+ at pH 7.5 is k12/k13 = 1.0051 +/- 0.0012 and the hydrogen isotope effects are VmaxH/VmaxD = 0.98 +/- 0.07 and (Vmax/Km)H/(Vmax/Km)D = 1.11 +/- 0.14. Thus, the rate decrease caused by substitution of Ni2+ for Mg2+ must result from the effects of metal on substrate and product binding and dissociation, rather than effects of metal on catalysis. However, a more detailed analysis of the carbon isotope effects reveals that there is also a large metal effect on the rate of the decarboxylation step, consistent with the view that the carbonyl oxygen of the oxalosuccinate intermediate is coordinated to the metal during decarboxylation.     

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.     

Spectroscopic studies of the interactions of coenzymes and coenzyme fragments with pig heart, oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase

Spectroscopic, ultrafiltration, and kinetic studies have been used to characterize interactions of reduced and oxidized triphosphopyridine nucleotides (TPNH and TPN), 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P), and adenosine 2',5'-bisphosphate [Ado(2',5')P2] with with TPN-specific isocitrate dehydrogenase. Close similarity of the UV difference spectra and of the protein fluorescence changes accompanying the formation of the binary complexes provides evidence for the binding of these nucleotides to the same site on the enzyme. From the pH dependence of the dissociation constants for TPNH binding to TPN-specific isocitrate dehydrogenase in the absence and in the presence of Mn2+, over the pH range 5.8-7.6, it has been demonstrated that the nucleotide binds to the enzyme in its unprotonated, metal-free form. The involvement of positively charged residues, protonated over the pH range studied, has been postulated. One TPNH binding site per enzyme subunit has been measured by fluorescence and difference absorption titrations. A dramatic effect of ionic strength on binding has been demonstrated: about a 1000-fold decrease in the dissociation constant for TPNH has been observed at pH 7.6 upon decreasing ionic strength from 0.336 (Kd = 1.2 +/- 0.2 microM) to 0.036 M (Kd = 0.4 +/- 0.1 nM) in the presence and in the absence of 100 mM Na2SO4, respectively. Weak competition of sulfate ions for the nucleotide binding site has been observed (KI = 57 +/- 3 mM). The binding of TPN in the presence of 100 mM Na2SO4 at pH 7.6 is about 100-fold weaker (Kd = 110 +/- 22 microM) than the binding of the reduced coenzyme and is similarly affected by ionic strength. These results demonstrate the importance of electrostatic interactions in the binding of the coenzyme to TPN-specific isocitrate dehydrogenase. The large enhancement of protein fluorescence caused by binding of TPN and Rib-P2-Ado-P (delta Fmax = 50%) and of Ado(2',5')P2 (delta Fmax = 41%) has been ascribed to a local conformational change of the enzyme. An apparent stoichiometry of 0.5 nucleotide binding site per peptide chain was determined for TPN, Rib-P2-Ado-P, and Ado(2',5')P2 from fluorescence titrations, in contrast to one binding site per enzyme subunit determined from UV difference spectral titration and ultrafiltration experiments. Thus, the binding of one molecule of the nucleotide per dimeric enzyme molecule is responsible for the total increase in protein fluorescence, while binding to the second subunit does not cause further change.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides with dihydrofolate reductase from amethopterin-resistant L1210 cells.

The 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides (TPN and TPNH) epsilon-TPN and epsilon-TPNH) have been synthesized and used as fluorescent probes to examine the pyridine nucleotide binding site of L1210 dihydrofolate reductase. Epsilon-TPNH (Km = 16.7 muM) was able to replace TPNH (Km = 3.8 muM) in the enzyme-catalyzed reduction of dihyrdofolate, and both epsilon-TPN and epsilon-TPNH formed binary complexes with the enzyme that were stable to polyacrylamide gel electrophoresis. The fluorescence of epsilon-TPN was enhanced and the emission maximum shifted from 415 to 405 nm when the nucleotide was bound to the enzyme. The ethenoadenine moiety in epsilon-TPNH behaved similarily, but the fluorescence changes were complicated by concurrent effects of binding upon the dihydronicotinamide fluorophore. Fluorescence enhancement titrations yielded values of 1.8 and 0.59 muM, respectively, for the dissociation constants of the enzyme-epsilon-TPN and enzyme-epsilon-TPNH complexes. Titration experiments based upon quenching of enzyme fluorescence gave similar values, viz., 2.1 and 0.53 muM for the dissociation constants of these complexes. Fluorimetric titration of the enzyme-TPNH complex with epsilon-TPN (or of the enzyme-TPN complex with epsilon-TPNH) failed to reveal the presence of a second pyridine nucleotide binding site. The fluorescence enhancement of enzyme-bound epsilon-TPN or dihydrofolate was quenched when amethopterin or epsilon-TPN, respectively, was added to form a ternary complex. These results provide information concerning the nature of the pyridine nucleotide binding site and its spatial relationship to the dihydrofolate/amethopterin binding site.     

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 Mn²⁺ 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 Mn²⁺ 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 Mn²⁺, 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 Mn²⁺ 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.     

pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme.

The pH variation of the kinetic parameters for the oxidative decarboxylation of L-malate and decarboxylation of oxalacetate catalyzed by malic enzyme has been used to gain information on the catalytic mechanism of this enzyme. With Mn2+ as the activator, an active-site residue with a pK of 5.4 must be protonated for oxalacetate decarboxylation and ionized for the oxidative decarboxylation of L-malate. With Mg2+ as the metal, this pK is 6, and, at high pH, V/K for L-malate decreases when groups with pKs of 7.8 and 9 are deprotonated. The group at 7.8 is a neutral acid (thought to be water coordinated to Mg2+), while the group at 9 is a cationic acid such as lysine. The V profile for reaction of malate shows these pKs displaced outward by 1.4 pH units, since the rate-limiting step is normally TPNH release, and the chemical reaction, which is pH sensitive, is 25 times faster. TPN binding is decreased by ionization of a group with pK 9.3 or protonation of a group with pK 5.3. The pH variation of the Km for Mg shows that protonation of a group with pK 8.7 (possibly SH) decreases metal binding in the presence of malate by a factor of 1400, and in the absence of malate by a factor of 20. A catalytic mechanism is proposed in which hydride transfer is accompanied by transfer of a proton to the group with pK 5.4-6, and enolpyruvate is protonated by water coordinated to the Mg2+ (pK 7.8) after decarboxylation and release of CO2.     

Determination of the rate-limiting steps for malic enzyme by the use of isotope effects and other kinetic studies.

Isotope effects have been measured with Mg2+ as the activator and L-malate labeled with deuterium or tritium at carbon 2 as the substrate over the pH range 4-10. Comparison of the nearly pH-independent deuterium-isotope effect on V/Kmalate of 1.5 with the tritium effect of 2.0 by the method of Northrop (Northrop, D.B. (1975), Biochemistry 14, 2644) gives limits on the true effect of deuterium substitution on the bond-breaking step of 5-8 in the forward reaction and 4-6.5 in the reverse direction. Comparison of the deuterium effect on V/K with the 13C-isotope effect of 1.031 reported by Schimerlik et al. (Schimerlik, M.I., Rife, J.E., and Cleland, W.W. (1975), Biochemistry 14, 5347) allows the deduction that at pH 8 reverse hydride transfer is six to eight times faster than decarboxylation, which is thus largely rate limiting for the catalytic reaction. The absence of a deuterium-isotope effect on V at pH 7-8 and comparison of the Ki of pyruvate as an uncompetitive inhibitor of the forward reaction and a substrate for the reverse reaction indicate that at neutral pH the release of TPNH from enzyme-reduced triphosphopyridine nucleotide (E-TPNH) is the rate-limiting step in the forward direction. The observation of a deuterium effect on V that approaches 3 at pH 4 and 10 shows, however, that, at very low and very high pH, hydride transfer may become partly rate limiting. In the reverse reaction, the probable rate-limiting step at pH 7 is the isomerization of E-TPNH, while at pH 8.5 and above V becomes too large to measure and appears infinite. Substitution of Co2+, Ni2+, or low levels of Mn2+ for Mg2+ gives similar deuterium-isotope effects, although the values of V and Kmalate vary considerably with metal. The kinetics of Mn2+ show pronounced negative cooperativity, with Km values of 7 muM and 5 mM for concentration ranges from 4 to 100 muM and over 1 mM.     

Histidine in the nucleotide-binding site of NADP-linked isocitrate dehydrogenase from pig heart.

Incubation of pig heart NADP-dependent isocitrate dehydrogenase with ethoxyformic anhydride (diethylpyrocarbonate) at pH 6.2 results in a 9-fold greater rate of loss of dehydrogenase than of oxalosuccinate decarboxylase activity. The rate constants for loss of dehydrogenase and decarboxylase activities depend on the basic form of ionizable groups with pK values of 5.67 and 7.05, respectively, suggesting that inactivation of the two catalytic functions results from reaction with different amino acid residues. The rate of loss of dehydrogenase activity is decreased only slightly in the presence of manganous isocitrate, but is reduced up to 10-fold by addition of the coenzymes or coenzyme analogues, such as 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P). Enzyme modified at pH 5.8 fails to bind NADPH, but exhibits manganese-enhanced isocitrate binding typical of native enzyme, indicating that reaction takes place in the region of the nucleotide binding site. Dissociation constants for enzyme . coenzyme-analogue complexes have been calculated from the decrease in the rate of inactivation as a function of analogue concentration. In the presence of isocitrate, activating metals (Mn2+, Mg2+, Zn2+) decrease the Kd value for enzyme . Rib-P2-Ado-P, while the inhibitor Ca2+ increases Kd. The strengthened binding of nucleotide produced by activating metal-isocitrate complexes may be essential for the catalytic reaction, reflecting an optimal orientation of NADP+ to facilitate hydride transfer. Measurements of ethoxyformyl-histidine formation at 240 nm and of incorporation of [14C]ethoxy groups in the presence and absence of Rib-P2-Ado-P indicate that loss of activity may be related to modification of approximately one histidine. The critical histidine appears to be located in the nucleotide binding site in a region distal from the substrate binding site.     

Properties of the nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from Blastocladiella emersonii.

The nicotinamide adenine dinucleotide phosphate (NADP)-specific isocitrate dehydrogenase from Blastocladiella emersonii was purified. The enzyme was very unstable. Satisfactory stability was obtained in the presence of 0.2% ovalbumin. The enzyme had a molecular weight of about 100,000. It did not exhibit homotropic cooperativity for any of it substrates and was not affected by the allosteric modifiers citrate and adenosine monophosphate, diphosphate, and tri-phosphate. The substrate saturation studies showed both intercept and slope effects in Lineweaver-Burk plots. The Km values for isocitrate and NADP were found to be 20 and 10 muM, respectively. The product inhibition pattern was compatible with a random sequential reaction mechanism. The enzyme catalyzed the oxidative decarboxylation of isocitrate about six times better than the reductive carboxylation of alpha-ketoglutarate. The enzyme was inhibited by glyoxylate plus oxalacetate. Assays conducted in the presence of low Mg2+ concentrations exhibited a lag. This lag could be abolished by the addition of reduced NADP to the assay mixture.     

Alpha-methylisocitrate. A selective inhibitor of TPN-linked isocitrate dehydrogenase from bovine heart and rat liver.

Alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a potent inhibitor, competitive with isocitrate (1-hydroxy-1,2,3-propanetricarboxylate), of the TPN-linked isocitrate dehydrogenase from bovine heart and rat liver; it does not inhibit the DPN-specific enzyme from these tissues. In the presence of magnesium ion, values of Kis for DL-alpha-methylisocitrate for purified bovine heart enzyme, rat liver cytosol, and rat liver mitochondrial extract were in the range of 0.1 muM to 0.3 muM. This compared to values of apparent Km for DL-isocitrate for the same tissue preparations of 14 muM to 20 muM. One of the DL isomer pairs of alpha-methylisocitrate was inactive; the observations suggest that it is threo-alpha-methylisocitrate which inhibits TPN-linked isocitrate dehydrogenase. A method of synthesis of DL-threo-alpha-methylisocitric lactone (2-methyl-5-oxo-2,3-furandicarboxylic acid) from dimethyl trans-epoxymethylsuccinate and dimethylmalonate is described.     

Purification, properties, and kinetic observations on the isoenzymes of NADP isocitrate dehydrogenase of maize.

A successful method for the purification of NADP isocitrate dehydrogenase from a plant source, Zea mays, is reported. Two mitochondrial isoenzymes were found and purified to homogeneity by a course of acetone fractionation, bulk exchange on DEAE-cellulose, cellulose hydroxylapatite column chromatography, and continuous elution electrophoresis. The mitochondrial isoenzymes are very similar with respect to kinetic properties, response to solvent perturbation, and temperature dependence of the pH/V relationship of isocitrate dehydrogenation. The Michaelis constant for isocitrate is identical for both isoenzymes. The enzymes have a molecular weight of 81,000 as estimated by permeation chromatography and an isoelectric point of 5.5 as extrapolated from gel-electrophoretic mobilities. Detectable differences are confined to differences in electrophoretic mobilities and heat denaturation. In D₂O the rate of the overall reaction from isocitrate to α-ketoglutarate and CO₂ was about 3.6 times slower than the same reaction in H₂O. Both the forward and reverse reactions, in which isocitrate is dehydrogenated or generated from oxalosuccinate, were observed to decrease by this amount in D₂O. The decarboxylation of oxalosuccinate was found to decrease by only about 25% in D₂O relative to the velocity of the reaction in H₂O. Thus the slow step in the overall reaction must be the initial dehydrogenation step rather than the decarboxylation of oxalosuccinate. The pK of the overall reaction did not change in D₂O as compared to H₂O.     

Kinetic properties of NADP-specific isocitrate dehydrogenase from bovine adrenals

At low concentrations of Mg2+ or Mn2+ the reaction catalyzed by isocitrate dehydrogenase from bovine adrenal cortex proceeds with a lag period which disappears as a result of the enzyme saturation with Mn2+ or Mg2+. The nu o versus D,L-isocitrate concentration curve is non-hyperbolic, which may be interpreted either by the presence of two active sites with different affinity for the substrate (K'mapp = 2.3 and 63 microM) within the enzyme molecule or by the "negative" cooperativity of these sites. The apparent Km value for NADP lies within the range of 3.6-9 microM. High concentrations of NADP inhibit isocitrate dehydrogenase (Ki = 1.3 mM). NADP.H inhibits the enzyme in a mixed manner with respect to NADP (Ki = 0.32 mM). In the presence of NADP.H the curve nu o dependence on NADP concentration shows a "negative" cooperativity between NADP binding sites. The reverse enzyme-catalyzed reaction of reductive carboxylation of 2-oxoglutarate does not exhibit any significant deviations from the Michaelis-Menten kinetics. The Km value for 2-oxoglutarate is 120 microM, while that for NADP.H is 10 microM.     

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.     

Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases: Oxalosuccinate Reductase as an Ancestral Form of Isocitrate Dehydrogenase

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and NAD⁺) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and NAD⁺ were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.     

A novel oxalosuccinate-forming enzyme involved in the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6

We have previously demonstrated that the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6 is not simply a reversal of the oxidative decarboxylation catalysed by isocitrate dehydrogenase (ICDH). The reaction involves a novel biotin protein (carboxylating factor for ICDH-CFI) and ATP. In this study, we have analysed the ICDH/CFI system responsible for the carboxylation reaction. Sequence analysis revealed a close relationship between CFI and pyruvate carboxylase. Rather unexpectedly, the rate of ATP hydrolysis was greater than that of isocitrate formation or NADH oxidation. Furthermore, ATP hydrolysis catalysed by CFI was dependent on 2-oxoglutarate but not on ICDH, suggesting that a carboxylated product is formed in the absence of ICDH. The product, which was detectable only at low temperatures, was identified as oxalosuccinate. Thus, CFI was confirmed to be a novel enzyme that catalyses the carboxylation of 2-oxoglutarate to form oxalosuccinate, which corresponds to the first step of the reductive carboxylation from 2-oxoglutarate to isocitrate. The CFI-ICDH system may also be present in mammals, where it could play a significant role in modulating central metabolism.     

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.     

Inactivation of pig heart NADP-specific isocitrate dehydrogenase by two affinity reagents is due to reaction with a cysteine not essential for function.

Pig heart NADP-dependent isocitrate dehydrogenase is 65% inactivated by 3-bromo-2-ketoglutarate (Ehrlich, R.S., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,614-12,619) and 90% inactivated by 2-(4-bromo-2,3-dioxobutylthio)-1,N6- ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) (Bailey, J.M., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,620-12,626). Both inactivation reactions result in enzyme with an incorporation of 1.0 mol reagent/mol enzyme dimer and both modified enzymes bind only 1.0 mol manganous isocitrate or NADPH/mol enzyme dimer as compared to 2.0 mol manganous isocitrate or NADPH/mol enzyme dimer for unmodified enzyme. The inactivation reactions, which occur at or near the nucleotide binding site, are mutually exclusive. Reaction with either affinity reagent led to the isolation of the same modified triskaidekapeptide, DLAGXIHGLSNVK. We have isolated from isocitrate dehydrogenase a peptide, DLAGCIHGLSNVK, that had been modified by N-ethylmaleimide (NEM) with no loss of enzymatic activity. We now show that enzyme modified by NEM in the presence of isocitrate plus Mn2+ retains full catalytic activity but is not inactivated by either of the affinity reagents; thus, all three reagents appear to react at the same site. The analysis of HPLC tryptic maps of isocitrate dehydrogenase treated under denaturing conditions with iodo[3H]acetic acid or [3H]NEM demonstrates that both bromoketoglutarate and 2-BDB-T epsilon A-2',5'-DP react with the cysteine residue of DLAGCIHGLSNVK. We conclude that the cysteine of this triskaidekapeptide is close to the coenzyme binding site but is not essential for catalytic function.     

The Histochemical Localization of Triphosphopyridine Nucleotide Diaphorase

A histochemical method is described for the localization of triphosphopyridine nucleotide diaphorase using a recently synthesized tetrazolium salt (Nitro-BT). By virtue of the favorable histochemical properties of this reagent, it has been possible to demonstrate that whereas DPN diaphorase is usually restricted to the mitochondria, the TPN diaphorase activity of corresponding cells was distributed throughout the cytoplasm in granules too fine to be considered mitochondria. Furthermore, although the diaphorase alone is responsible for the passage of electrons from TPNH to the tetrazole, it has been found that sites of activity of different TPN-linked dehydrogenases can be visualized in tissue sections, and characteristic loci for each enzyme may be observed. For example, whereas TPN diaphorase and isocitric dehydrogenase have an extensive distribution in the kidney cortex, 6-phosphogluconic dehydrogenase is limited to the cells of the macula densa.     

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.