Phosphoenolpyruvate carboxykinase from Fasciola hepatica

Behm C. A. and Bryant C. 1982. Phosphoenolpyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology12: 271–278. The kinetic properties of a partially purified preparation of phosphoenolpyruvate carboxykinase (PEPCK) from F. hepatica were examined. The pH optimum for the carboxylation reaction is 5.8–6.2. The enzyme is more active with Mn²⁺ than Mg²⁺ and the Mn²⁺ saturation curve was sigmoid. Apparent K_m values for the substrates GDP, IDP, PEP and HCO₃^? were determined and found to be in the same range as those reported for other helminths except that the enzyme is less sensitive to low PEP concentrations. GTP and ATP at 0.5 and 1.0 mM inhibit the enzyme; the GTP inhibition was greater in the presence of Mg²⁺ than Mn²⁺ and was competitive with GDP. It was concluded that the activity of PEPCK from F. hepatica is controlled by the concentration of reactants and the ambient pH, that the accumulation of GTP is a sensitive mechanism for inhibiting the carboxylation reaction and that PEPCK activity in the cytosol is likely to be favoured over that of pyruvate kinase except when pH is high and PEP concentration low.     

Kinetic and structural analysis of Escherichia coli phosphoenolpyruvate carboxykinase mutants

BackgroundPhosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO₂. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn²⁺ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis.MethodsKey residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography.ResultsMutation of residues D269 and H232, which comprise part of the coordination sphere of Mn²⁺, reduced k_cat by 14-fold, and significantly increased the K_m values for Mn²⁺ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the K_m value for MgATP and reduced k_cat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the K_m value for OAA and reduced k_cat respectively.ConclusionsOur results show that mutation of residues involved in coordinating OAA, MgATP and Mn²⁺ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK.General significanceIn higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

PROPERTIES OF PHOSPHOENOLPYRUVATE CARBOXYKINASE FROM ASCOPHYLLUM NODOSUM (PHAEOPHYCEAE)1

Phosphoenolpyruvate carboxykinase was purified to electrophoretic homogeneity from the brown alga. Ascophyllum nodosum (L.) le Jol. Its molecular mass was 60 kDa as determined by gel filtration. The pH optimum of the carboxylating reaction was 7.9 and the apparent K_m for PEP⁴, ADP and HCO₃^- were 0.036, 0.0116 and 50 mol · m^-3, respectively. Rates of light and dark carbon fixation are also reported for A. nodosum apices and it is shown that rates of in vitro PEPCK activity would account for the observed rate of dark fixation. The physiological role of PEPCK is discussed in relation to light-independent carbon fixation.     

EXTRACTION,PARTIAL PURIFICATION AND CHARACTERIZATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE FROM ASCOPHYLLIUM NODOSUM (PHAEOPHYCEAE)1

Phosphoenolpyruvate carboxykinase (PEPCK) from Ascophyllum nodosum (L.) Le Jolis was partially purified and characterized to investigate its role in inorganic carbon assimilation in macroalgae. Inorganic carbon isotopic disequilibrium studies showed that the carboxylation of phosphoenolpyruvate utilized CO₂ rather than HCO₃^?as its source of inorganic carbon. This is consistent with the enzyme being a phosphoenolpyruvate carboxykinase rather than a phosphoenolpyruvate carboxylase. Pre-incubation with Mn²⁺alone activated PEPCK more effectively than when combinations of Mn²⁺, ADP and HCO₃^?were used as activators. Activation of PEPCK during catalysis was found not to occur. Although the activation of PEPCK reduced the K_m for CO₂ by a factor of 2.25, the value reported here of 1.084 mM CO₂ for the activated enzyme at pH 7.0 is at the top of the range of previously reported values for brown algal PEPCK. The specific activity of PEPCK was increased from 0.268 μmol·min^?1·mg^?1in the crude extract to 33.03 μmol·min^?1·mg^?1in the partially purified preparations. Whether PEPCK can act as an initial carboxylating enzyme is discussed. Triton X-100 at 0.57% (v/v) was found to be the optimum detergent and concentration for the extraction of enzymes from A. nodosum. When high concentrations of detergents -were used, a low (NH₄)₂SO₄ cut was required to remove the free detergent from solution, which was extracted by centrifugation. Q Sepharose was used to partially purify PEPCK and separate it from pyruvate kinase. Good protein separations were consistently obtained.     

Characterization of Phosphoenolpyruvate Carboxykinase from Pineapple Leaves Ananas comosus (L.) Merr

Phosphoenolpyruvate carboxykinase has been partially purified from pineapple (Ananas comosus [L.]) leaves. Specific activities obtained show it to be a major activity in this tissue. Above 15 C, the respective activation energies for decarboxylation and carboxylation are 13 and 12 kcal/mol. Below 15 C, there are discontinuities in Arrhenius plots with an associated large increase in activation energy. The adenine nucleotides are preferred to other nucleotides as substrates. The apparent Km values in the carboxylation direction are: ADP 0.13 mm, HCO(3) (-) 3.4 mm, and phosphoenolpyruvate 5 mm. In the decarboxylation direction, the apparent Km values are: ATP 0.02 mm, ADP 0.05 mm, and oxaloacetate 0.4 mm. The decarboxylation activity had an almost equal velocity with either ADP or ATP. The pH optima are between 6.8 and 7. Inhibition of the carboxylation reaction by ATP, pyruvate, and carbonic anhydrase was demonstrated. Decarboxylase specific activities are over twice carboxylation activities. The data support a model in which phosphoenolpyruvate carboxykinase is of physiological significance only during the light period and then only as a decarboxylase.     

Purification and characterization of phosphoenolpyruvate carboxykinase from the anaerobic ruminal bacterium Ruminococcus flavefaciens

Phosphoenolpyruvate (PEP) carboxykinase was purified 42-fold with a 25% yield from cell extracts of Ruminococcus flavefaciens by ammonium sulfate precipitation, preparative isoelectric focusing, and removal of carrier ampholytes by chromatography. The enzyme had a subunit molecular mass of ∼66.3 kDa (determined by mass spectrometry), but was retained by a filter having a 100-kDa nominal molecular mass cutoff. Optimal activity required activation of the enzyme by Mn²⁺ and stabilization of the nucleotide substrate by Mg²⁺. GDP was a more effective phosphoryl acceptor than ADP, while IDP was not utilized. Under optimal conditions the measured activity in the direction of PEP carboxylation was 17.2 μmol min^–1 (mg enzyme)^–1. The apparent K _m values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent K _m values for the substrates of the back reaction (oxaloacetate and GTP, respectively). The data are consistent with the involvement of PEP carboxykinase as the primary carboxylation enzyme in the fermentation of cellulose to succinate by this bacterium. Received: 20 August 1996 / Accepted: 28 December 1996     

Purification and characterization of cytosol phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver.

Cytosol PEP carboxykinase has been purified to electrophoretic homogeneity from bullfrog liver homogenate. The enzyme is a single polypeptide chain with a molecular weight of approximately 72,000-75,000. The purified enzyme catalyzed oxaloacetate decarboxylation (nucleoside triphosphate-supported), phosphoenolpyruvate carboxylation, and an exchange reaction between oxaloacetate and [14C]HCO3-in the presence of ITP or CTP. Manganese is absolutely required for the enzyme-catalyzed phosphoenolpyruvate carboxylation, whereas it can be replaced by Mg2+ for the oxaloacetate decarboxylation and the exchange reaction. The optimal pH of each reaction is dependent on the divalent metal ion used. The dependence of the enzyme activity on Mn2+ is markedly different in the phosphoenolpyuvate carboxylation and the oxaloacetate decarboxylation reactions.     

Regulation of mitochondrial glycine decarboxylase from pea mitochondria

Reactions of glycine cleavage were assayed in mitochondria isolated from cotyledons of germinating pea seeds. These reactions, which included the exchange of bicarbonate with C-1 of glycine and an NAD-stimulated decarboxylation of glycine, were maximal under aerobic conditions at pH 7·8. The apparent Michaelis-Menten constants for glycine and bicarbonate in the exchange reaction were 1·8 and 12·5 mM respectively. The K_m for NAD in the decarboxylation reaction was 47 μM. Maximal enzyme activity was observed when mitochon-drial integrity was maintained. Up to 40% inhibition of the decarboxylation reaction was observed when NADH, NADPH or l-methionine were added to the reaction system. When glycine-[2-¹⁴C] was incubated with the isolated mitochondria, labelled CO₂ was evolved in nanomolar quantities. It is concluded that glycine decarboxylase may be of importance in supplying C-1 units for the de novo synthesis of methionine in pea mitochondria.     

Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240

We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K _m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K _m value was 6.80 mM. In the presence of 3 M KHCO₃ and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K _m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.     

Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K_m values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N₂-fixing alfalfa nodules.     

Malate inhibition of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate²⁻ concentration. Of the reaction components only HCO₃⁻ increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in K_m, but no effect on V_max. Enzyme preincubated with malate shows the same effect of malate on K_m, but in addition V_max is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows V-type inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate.     

Properties of oxaloacetate decarboxylase from Veillonella parvula.

Oxaloacetate decarboxylase was purified to 136-fold from the oral anaerobe Veillonella parvula. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity of the enzyme was exhibited at pH 7.0 for both carboxylation and decarboxylation. At this pH, the Km values for oxaloacetate and Mg2+ were at 0.06 and 0.17 mM, respectively, whereas the Km values for pyruvate, CO2, and Mg2+ were 3.3, 1.74, and 1.85 mM, respectively. Hyperbolic kinetics were observed with all of the aforementioned compounds. The Keq' was 2.13 X 10(-3) mM-1 favoring the decarboxylation of oxaloacetate. In the carboxylation step, avidin, acetyl coenzyme A, biotin, and coenzyme A were not required. ADP and NADH had no effect on either the carboxylation or decarboxylation step, but ATP inhibited the carboxylation step competitively and the decarboxylation step noncompetitively. These types of inhibition fitted well with the overall lactate metabolism of the non-carbohydrate-fermenting anaerobe.     

Purification and properties of myo-inositol-1-phosphate dehydrogenase from germinating mung bean seeds

A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of M_r 32,000 each was discernible. K_m values for NAD⁺ and myo-inositol 1-phosphate have been recorded as 2.8 × 10^?4 and 5.0 × 10^?4m, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD⁺ are reduced.     

The synergistic decarboxylation of glyoxylate and 2-oxoglutarate by an enzyme system from pig-liver mitochondria

1. An enzyme system that catalyses a synergistic decarboxylation of glyoxylate and 2-oxoglutarate has been purified from pig-liver mitochondria. 2. The purified system is specific for glyoxylate and 2-oxoglutarate as substrates, although in earlier stages of purification glycine and l-glutamate are also active. 3. The reaction is inhibited strongly by EDTA and N-ethylmaleimide. Substrate analogues, present at concentrations equimolar with respect to the substrates, are not effective as inhibitors. 4. The reaction proceeds in the absence of added cofactors. Magnesium chloride, mercaptoethanol and sucrose stimulate the reaction, and stabilize the activity of the enzyme. 5. The pH optimum of the reaction is 7·0. The K_m values of glyoxylate and 2-oxoglutarate, at saturating concentration of the corresponding co-substrate, are 16mm and 3·6mm respectively. 6. Isotopic work with specifically labelled [¹⁴C]glyoxylate and 2-oxo[¹⁴C]-glutarate suggests that the enzyme system catalyses an initial condensation of glyoxylate and 2-oxoglutarate that results in, or leads to, release of C-1 of both substrates as carbon dioxide. C-2 of glyoxylate and C-5 of 2-oxoglutarate do not appear as carbon dioxide. 7. The stoicheiometry of the reaction is complex. During the initial stages of the reaction, more carbon dioxide is recovered from 2-oxoglutarate than from glyoxylate. Subsequently, there is a disproportionate increase with time of carbon dioxide evolution from the carboxyl group of glyoxylate. The excess of decarboxylation of glyoxylate over 2-oxogluturate is further increased by treatment of reaction products with acid.     

Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity

Phosphoenolpyruvate carboxykinase (PEPCK)—the major decarboxylase in PEPCK-type C₄ plants—is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C₄ plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539–546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301–311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.     

Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.     

Does phosphoenolpyruvate carboxykinase have a role in both amino acid and carbohydrate metabolism?

Summary. Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO₂. The role of the enzyme in gluconeogenesis and anaplerotic reactions in a range of organisms is discussed, along with the important function in C₄ and CAM photosynthesis in higher plants. In addition, new data are presented indicating that PEPCK may play a key role in amino acid metabolism. It is proposed that PEPCK is involved in the conversion of the carbon skeleton of asparagine/aspartate (oxaloacetate) to that of glutamate/glutamine (2-oxoglutarate). This metabolism is particularly important in the transport system, seeds and fruits of higher plants. Received January 27, 2000 Accepted March 7, 2000