The characterization and regulatory properties of pyruvate kinase from cotton seeds

Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was partially purified from cotton seeds. The enzyme shows normal kinetics toward phosphoenol-pyruvate, ADP, and magnesium or manganese. Of nearly 50 metabolites tested, the enzyme is inhibited only by ATP, UTP, citrate, and malate, and activated by AMP, GMP, and fumarate. The inhibition by citrate and ATP is not due to metal chelation; both compounds appear to directly affect the enzyme. The kinetics of the activations by AMP and by fumarate suggest the existence of separate activator sites for the two compounds.It is suggested that cotton seed pyruvate kinase is a regulatory enzyme, although it differs markedly from the regulatory pyruvate kinases which have been described in animals and in microorganisms. This is the first instance in which regulatory properties have been reported for a pyruvate kinase from a higher plant.     

Purification and characterization of ATP:citrate lyase from Hydrogenobacter thermophilus TK-6.

ATP:citrate lyase [ATP citrate (pro-3S)-lyase; EC 4.1.3.8] was purified and characterized from the cells of Hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle. The enzyme was quite stable, even in the absence of sulfhydryl reagents. Optimum pH for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees C. The molecular weight of native enzyme was estimated to be 260,000 by gel filtration analysis, and that of a subunit was estimated to be 43,000 by sodium dodecyl sulfate-polyacrylamide gel analysis. Km values for reaction components were as follows: citrate, 6.25 mM; ATP, 650 microM; coenzyme A, 40.8 microM; and Mg2+, 8 mM. The enzyme showed citrate synthase activity in the presence of Mg2+, but the reaction rate was very low (less than 1/200 of the lyase activity).     

Identification and characterization of a re-citrate synthase in Dehalococcoides strain CBDB1

The genome annotations of all sequenced Dehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that a Re face-specific citrate synthase is synthesized by Dehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession number CAI83711), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified. The enzyme catalyzed the condensation of oxaloacetate and acetyl coenzyme A (acetyl-CoA) to citrate. The protein did not have homocitrate synthase activity and was inhibited by citrate, and Mn2+ was needed for full activity. The stereospecificity of the heterologously expressed citrate synthase was determined by electrospray ionization liquid chromatography-mass spectrometry (ESI LC/MS). Citrate was synthesized from [2-(13)C]acetyl-CoA and oxaloacetate by the Dehalococcoides recombinant citrate synthase and then converted to acetate and malate by commercial citrate lyase plus malate dehydrogenase. The formation of unlabeled acetate and 13C-labeled malate proved the Re face-specific activity of the enzyme. Shotgun proteome analyses of cell extracts of strain CBDB1 demonstrated that cbdbA1708 is expressed in strain CBDB1.     

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.     

Regulation of citrate synthase from blue-green bacteria by succinyl coenzyme A

Citrate synthase (EC 4.1.3.7) was prepared from nine species of blue-green bacteria. In every case the citrate synthase was of the large type otherwise found only in Gram-negative bacteria.In addition to inhibition by -oxoglutarate, the enzymes were all sensitive to inhibition by succinyl coenzyme A, acting competitively with respect to acetyl coenzyme A. Desensitization by potassium chloride and a sigmoidal dependence of inhibition on succinyl coenzyme A concentration suggested the possibility of an allosteric mechanism. Multiple-inhibition analysis using pairs of the competitive inhibitors succinyl coenzyme A, bromoacetyl coenzyme A and ATP confirmed the existence of a distinct site for succinyl coenzyme A.It is suggested that the specific sensitivity of bluegreen bacterial citrate synthases to succinyl coenzyme A, as well as to -oxoglutarate, is related to the particular metabolic role of the enzyme in these organisms. The absence of a complete energy-yielding citric acid cycle, resulting from the lack of -oxoglutarate dehydrogenase, confers a strictly biosynthetic role on citrate synthase, which initiates a branched pathway leading to the two end-products -oxoglutarate and succinyl coenzyme A. Inhibition of the enzyme by these compounds constitutes a plausible regulatory mechanism.     

The interaction of yeast citrate synthase with yeast mitochondrial inner membranes

The specific interaction of yeast citrate synthase with yeast mitochondrial inner membranes was characterized with respect to saturability of binding, pH optimum, effect of ionic strength, temperature response, and inhibition by oxalacetate. The binding ability of the inner membranes is inhibited by proteolysis and heat treatment, which implies that the membrane component(s) responsible for binding is a protein. A protein fraction from inner membranes when added to liposomes will bind citrate synthase. In addition, the binding of yeast fumarase, mitochondrial malate dehydrogenase, and cytosolic malate dehydrogenase to yeast inner membranes was examined. For these studies the yeast mitochondrial matrix enzymes, citrate synthase (from two types of yeast), malate dehydrogenase, and fumarase, as well as cytosolic malate dehydrogenase, were purified using rapid new techniques.     

Quantitation of the interaction between citrate synthase and malate dehydrogenase

Formation of a bienzyme complex of pig heart mitochondrial malate dehydrogenase and citrate synthase in a buffered system is demonstrated by means of a covalently attached fluorescent probe to citrate synthase. Assuming 1:1 stoichiometry of the enzymes in the complex, an apparent dissociation constant of 10(-6) M was calculated from fluorescence anisotropy measurements. The effect of various metabolites on the interaction was tested. NAD+, oxalacetate, citrate, ATP, and L(-)- or D(+)-malate had no effect on the association of the two enzymes, whereas alpha-ketoglutarate increased and NADH decreased it. The interaction of mitochondrial citrate synthase with cytosolic malate dehydrogenase was found to be much weaker, whereas interaction of citrate synthase with another cytosolic enzyme, aldolase, could not be detected. In kinetic experiments, the activation of malate dehydrogenase by citrate synthase was observed. The effect of pyridine nucleotides and alpha-ketoglutarate is discussed in relation to the direction of the metabolic flow of oxalacetate.     

Induction of citric acid cycle enzymes during initiation of sporulation by guanine nucleotide deprivation

In Bacillus subtilis, conditions causing partial deprivation of guanine nucleotides initiated sporulation and caused the synthesis of citrate synthase, aconitase, and alpha-ketoglutarate dehydrogenase. Alpha-ketoglutarate dehydrogenase could also be induced by acetate, and the specific activity of this enzyme was elevated in mutants that had high intracellular acetyl coenzyme A concentrations because they lacked citrate synthase activity. After deprivation of guanine nucleotides, the intracellular concentration of acetyl coenzyme A also increased, which explained the induction of alpha-ketoglutarate dehydrogenase. Furthermore, the decreases in alpha-ketoglutarate and L-malate concentrations observed during this deprivation accounted for the observed increases in citrate synthase activity (which was repressed by alpha-ketoglutarate and malate) and aconitase activity (which was repressed by alpha-ketoglutarate).     

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.     

Regulation of biosynthesis of secondary metabolites

Partially purified citrate synthase (EC 4.1.3.7) was isolated from submerged mycelium ofStreptomyces aureofaciens RIA 57, a strain producing chlortetracycline. Enzymatic activity was determined spectrophotometrically by means of 5,5′-dithio-bis-(2-nitrobenzoic acid). Citrate synthase was inhibited by ATP, inhibition being competitive with respect to acetyl coenzyme A. In 5mm concentration, ATP caused 55% inhibition of the enzyme. ADP, in the same concentration, caused 30% inhibition, while AMP caused none. Other natural nucleoside di- and triphosphates, in 1–5mm concentration, did not significantly affect citrate synthase inStreptomyces aureofaciens. Mg²⁺ inhibited the activity of the enzyme, but also reduced the negative effect of ATP. The role of ATP in regulation of the metabolic paths of acetyl CoA is discussed.     

Crystal structure of Escherichia coli malate dehydrogenase. A complex of the apoenzyme and citrate at 1.87 A resolution.

The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A. Molecular replacement, using the partially refined structure of porcine mitochondrial malate dehydrogenase as a probe, provided initial phases. The structure of this prokaryotic enzyme is closely homologous with the mitochondrial enzyme but somewhat less similar to cytosolic malate dehydrogenase from eukaryotes. However, all three enzymes are dimeric and form the subunit-subunit interface through similar surface regions. A citrate ion, found in the active site, helps define the residues involved in substrate binding and catalysis. Two arginine residues, R81 and R153, interacting with the citrate are believed to confer substrate specificity. The hydroxyl of the citrate is hydrogen-bonded to a histidine, H177, and similar interactions could be assigned to a bound malate or oxaloacetate. Histidine 177 is also hydrogen-bonded to an aspartate, D150, to form a classic His.Asp pair. Studies of the active site cavity indicate that the bound citrate would occupy part of the site needed for the coenzyme. In a model building study, the cofactor, NAD, was placed into the coenzyme site which exists when the citrate was converted to malate and crystallographic water molecules removed. This hypothetical model of a ternary complex was energy minimized for comparison with the structure of the binary complex of porcine cytosolic malate dehydrogenase. Many residues involved in cofactor binding in the minimized E. coli malate dehydrogenase structure are homologous to coenzyme binding residues in cytosolic malate dehydrogenase. In the energy minimized structure of the ternary complex, the C-4 atom of NAD is in van der Waals' contact with the C-3 atom of the malate. A catalytic cycle involves hydride transfer between these two atoms.     

Malate Metabolism in Leaf Mitochondria from the Crassulacean Acid Metabolism Plant Kalanchoë blossfeldiana Poelln

The mechanisms and the controlling factors of malate oxidation by mitochondria from leaves of Kalanchoë blossfeldiana Poelln. plants performing Crassulacean acid metabolism were investigated using Percollpurified mitochondria. The effects of pH and of various cofactors (ATP, NAD⁺, coenzyme A) on malate dehydrogenase (EC 1.1.1.37) and malic enzyme (EC 1.1.1.39) solubilized from these mitochondria were examined. The crucial role of cofactor concentrations in the mitochondrial matrix on the pathways of malate oxidation is shown. The distribution of the electrons originating from malate between the different electron transport pathways and its consequence on the phosphorylation yield was studied. It was found that, depending on the electron transport pathway used, malate oxidation could yield from 3 to 0 ATP. Assayed under conditions of high reducing power and high energy charge, the ability of malic enzyme to feed electrons to the cyanide-resistant nonphosphorylating alternative pathway was found to be higher than that of other dehydrogenases linked to the functioning of the Krebs cycle (pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase). The physiological significance of such a functional relationship between malic enzyme activity and the nonphosphorylating alternative pathway is discussed in relation to Crassulacean acid metabolism.     

Apparent Inhibition of ATP Citrate Lyase by l-Glutamate In Vitro Is Due to the Presence of Glutamine Synthetase

Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.     

Oxidized NADP as a potential active-site-directed reagent of pigeon liver malic enzyme.

Pigeon liver malic enzyme (malate dehydrogenase (decarboxylating), EC 1.1.1.40) was reversibly inactivated by periodate-oxidized NADP in a biphasic manner. The reversibility could be made irreversible by treating the modified enzyme with sodium borohydride. The inactivation showed saturation kinetics and could be prevented by nucleotide (NADP or NADPH). Fully protection was afforded by the combination of NADP, Mn²⁺ and L-malate. Oxidized NADP was also found to be a coenzyme and noncompetitive inhibitor of L-malate in the oxidative decarboxylase reaction catalyzed by malic enzyme.     

Amino Acid Catabolism and Malic Enzyme in Differentiating Dictyostelium discoideum

Amino acids produced from protein degradation are the major energy source for differentiation and aging in Dictyostelium discoideum. Considering the reactions involved in the conversion of amino acids from an average protein into tricarboxylic acid cycle intermediates, a route from a cycle intermediate (probably malate) to acetyl coenzyme A is required for the complete utilization of amino acids. Citrate was isolated from cells pulse-labeled with (14)C-labeled amino acids and was cleaved with citrate lyase. When cells were pulse-labeled with [U-(14)C]-glutamate the specific radioactivity of the acetate and oxaloacetate portions of citrate were consistent with the conclusion that one-third of the carbon flowing through the tricarboxylic acid cycle is removed for the synthesis of acetyl coenzyme A. The data were also consistent with the patterns of carbon flux required to maintain steady-state levels of cycle intermediates in cells catabolizing amino acids. It is suggested that the malic enzyme (EC 1.1.1.40) catalyzes the synthesis of acetyl coenzyme A from malate and is responsible for the observed citrate labeling pattern. In cell extracts the activity of this enzyme increased markedly with the onset of differentiation. The properties of partially purified (40-fold) malic enzyme isolated at culmination indicated that the enzyme was allosteric and was positively affected by aspartate and glutamate. Thus, amino acid production from protein degradation would stimulate a reaction essential for the efficient utilization of these amino acids for energy.     

Inhibition by alloxan of mitochondrial aconitase and other enzymes associated with the citric acid cycle

Considerable variations were found in the in vitro effect of alloxan on mouse liver enzymes associated with the citric acid cycle. The following approximative alloxan concentrations induced 50% inhibition of enzyme activity: 10(-6)M for aconitase, 10(-4)M for NAD-linked isocitrate dehydrogenase, glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase and fumarase, and 10(-3)M for citrate synthase and NADP-linked isocitrate dehydrogenase. Pyruvate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were not inhibited by 10(-3)M alloxan. The inhibition of aconitase was competitive both when using mouse liver and purified porcine heart enzyme. The Ki values for the purified enzyme in the presence of 5 microM alloxan were 0.22 microM with citrate, 4.0 microM with cis-aconitate and 0.62 microM with isocitrate as substrate. The high sensitivity of aconitase for inhibition by alloxan probably plays a prominent role for the toxic effects of alloxan.     

Characterization of a malate dehydrogenase in the cyanobacterium Coccochloris peniocystis

A malate dehydrogenase (MDH) was characterized from the cyanobacterium Coccochloris peniocystis. The enzyme was purified approximately 180-fold and had a molecular weight of about 90000. The enzyme had a pH optimum of pH 6.7 to 7.5; a K_m (malate) of 5.6 mM and K_ms for NAD and NADP of 24 M and 178 M, respectively, although similar V_max were obtained with either pyridine nucleotide. Enzyme activity was inhibited by ATP, citrate, oxalacetate, acetyl CoA and CoA. Enzyme assays with uniformly ¹⁴C-labelled malate caused no ¹⁴CO₂ release, indicating this MDH is not a malic enzyme. Electrophoresis and S-200 gel filtration of the partially purified enzyme indicated a single MDH was present in this preparation. A second, less abundant, MDH was present in crude extracts. The presence of MDH in this organism is consistent with the operation of a glyoxylate cycle which, in the absence of a TCA cycle, would provide organic acids required in secondary carbon metabolism. ATP inhibition of MDH may allow for light regulation of MDH activity since, in the light, oxaloacetic acid is generated by phosphoenolpyruvate carboxylase activity.Abbreviations MDH malate dehydrogenase - PEPcase phosphoenolpyruvate carboxylase - MOPS 3-[N-Morpholino] propane sulfonic acid - TRIS Tris(hydroxymethyl)-aminomethane - EDTA Disodium Ethylenadiamine Tetraacetate - MES 2[N-Morpholino]-ethane Sulfonic Acid - EPPS N-2-Hydroxyethylpiperazine Propane - MW Molecular weight - OAA Oxaloacetic acid     

Complexes between mitochondrial enzymes and either citrate synthase or glutamate dehydrogenase

Experiments performed in polyethylene glycol and with a divalent crosslinker indicate that both mitochondrial malate dehydrogenase and aspartate aminotransferase can form hetero enzyme—enzyme complexes with either glutamate dehydrogenase or citrate synthase. In general, these as previous results indicate that complexes with the aminotransferase are favored over those with malate dehydrogenase and complexes with glutamate dehydrogenase are favored over those with citrate synthase. When the levels of enzymes are low, the only detectable complex is between the aminotransferase and glutamate dehydrogenase. Under these conditions, palmitoyl-CoA is required for complexes between the other three enzyme pairs, however, palmitoyl-CoA also enhances interactions between glutamate dehydrogenase and the aminotransferase. DPNH disrupts complexes with malate dehydrogenase and has little effect on those with the aminotransferase, while oxalacetate disrupts complexes with citrate synthase but has little effect on those with glutamate dehydrogenase. The citrate synthase-aminotransferase complex was favored in the presence of DPNH plus malate, which disrupt the other three enzyme-enzyme complexes. Glutamate dehydrogenase has a higher affinity and capacity than citrate synthase for palmitoyl-CoA. Consequently, lower levels of palmitoyl-CoA are required to enhance interactions with glutamate dehydrogenase. Furthermore, glutamate dehydrogenase can compete with citrate synthase for palmitoyl-CoA and thus can prevent palmitoyl-CoA from enhancing interactions between citrate synthase and either malate dehydrogenase or the aminotransferase.     

Lipid metabolism by rat lung in vitro. Utilization of citrate by normal and starved rats

1. The utilization of [1,5-(14)C(2)]citrate by lung slices and cell cytosol preparations, and the activities of liver and lung cytosol citrate-cleavage enzyme (EC 4.1.3.8), l-malate-NAD oxidoreductase (malate dehydrogenase, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (EC 4.1.1.32) were examined in normal and starved rats. 2. Lipogenesis from citrate was decreased by approx. 70% in both the phospholipid and neutral lipid fractions of lung slices from starved rats as compared with fed controls. 3. Incorporation of citrate by lung cytosol preparations into fatty acids was decreased by approx. 35% in the starved rats. The apparent inhibition by avidin of fatty acid synthesis was overcome partially by preincubation of lung cytosol preparations with biotin. These results are consistent with the presence in lung tissue of the malonyl-CoA pathway for fatty acid synthesis. 4. Lung citrate-cleavage enzyme activity decreased in rats that had been starved for 72h whereas malate dehydrogenase and phosphoenolpyruvate carboxylase activities remained unchanged. The results suggest that the pattern of utilization of lipid precursors by rat lung may be altered during various nutritional states.     

The kinetic properties of citrate synthase from rat liver mitochondria

1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16mum for acetyl-CoA and 2mum for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2.5. 5. The pH optimum of the enzyme is 8.7, and is not significantly affected by ATP. 6. Mg(2+) inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6.3s, equivalent to molecular weight 95000.