Purification and Characterization of Phosphoenolpyruvate Carboxylase from Developing Seeds of Brassica

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified to apparent homogeneity with about 29% recovery from developing seeds of Brassica using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sepharose CL-6S. The purified enzyme with mol wt of about 400 kD exhibited maximum activity at pH 8.0. The enzyme had an absolute requirement for a divalent cation which was satisfied by Mg²⁺. The enzyme showed typical hyperbolic kinetics with PEP and HCO^?3 with Km of 0.125 and 0.104 mM, respectively. Glu-6-P could activate the enzyme, whereas other phosphate esters such as fru-1, 6-P₂, L-glycerophosphate and 3-PGA did not have any effect on the enzyme activity. Noneof the amino acids at 5 mM concentration had any significant effect on the enzyme activity. Nucleotide monophosphates and diphosphates did not inhibit the enzyme significantly, whereas ATP inhibited the enzyme activity. Oxaloacetate and malate inhibited the enzyme non-competitively with respect to PEP with Ki values of 0.127 and 1.25 mM, respectively. The enzyme activity in vivo seems to be regulated ’Tlainly by availability of its substrate and activation by glu-6-P, both of which are supplied through glycolysis.     

Changes in Sensitivity to Effectors of Maize Leaf Phosphoenolypyruvate Carboxylase during Light/Dark Transitions

Illumination of previously darkened maize (Zea mays L. cv Golden Cross Bantam T51) leaves had no effect on the concentration of phosphoenolpyruvate (PEP) carboxylase protein, but increased enzyme activity about 2-fold when assayed under suboptimal conditions (pH 7.0 and limiting PEP). In addition, sensitivity to effectors of PEP carboxylase activity was significantly altered; e.g. malate inhibition was reduced and glucose-6-phosphate activation was increased. Consequently, 10- to 20-fold differences in PEP carboxylase activity were observed during dark to light transitions when assayed in the presence of effectors. At pH 7.0 activity of purified PEP carboxylase was not proportional to enzyme concentrations. Below 0.7 microgram PEP carboxylase protein per milliliter, enzyme activity was disproportionately reduced. Including polyethylene glycol plus potassium chloride in the reaction mixture eliminated this discontinuity and substantially increased PEP carboxylase activity and reduced malate inhibition dramatically. Inclusion of polyethylene glycol in the assay mixture specifically increased the activity of PEP carboxylase extracted from dark leaves, and reduced malate inhibition of the enzyme from both light and dark leaves. Collectively, the results suggest that PEP carboxylase in maize leaves is subjected to some type of protein modification that affects both activity and effector sensitivity. We postulate that changes in quaternary structure (dissociation or altered subunit interactions) may be involved.     

Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development

In vivo CO₂ fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N₂ fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO₂ fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO₂ fixation in mature nodules was 7.9 nmol hour⁻¹ mg fresh weight⁻¹, similar to rates of N₂ fixation and reported values for amino acid translocation.     

The influence of temperature on malic Acid metabolism in grape berries: I. Enzyme responses

Phosphoenolpyruvate (PEP) carboxylase activity in immature `Carignane' grape berries (Vitis vinifera L.) had a temperature optimum of about 38 C, whereas malic enzyme activity rose with increasing temperature between 10 and 46 C. In vitro temperature inactivation rates for the PEP carboxylase were markedly greater than for the malic enzyme activity. From the simultaneous action of malic acid-producing enzymes (PEP carboxylase and malic dehydrogenase) and malic acid-degradating enzyme (malic enzyme) systems at different temperatures, the greatest tendency for malic acid accumulation in immature grape berries was at 20 to 25 C. Time-course measurements of enzymic activity from heated, intact berries revealed greater in vivo temperature stability for the malic enzyme activity than for the PEP carboxylase activity.     

Partial Characterization of Cell-bound Lipase of Candida paralipolytica

In accordance with the regulation by aspartate of phosphoenolpyrubate (PEP*) carboxylase, glutamate formation in Brevibacterium flavum, a glutamate-producing bacterium, was inhibited by the addition of aspartate. Furthermore, an increase in aspartate formation caused by a mutational decrease in citrate synthase specific activity was accompanied by a decrease in the total amount of glutamate and aspartate formed. However, a mutational decrease in glutamate dehydrogenase activity caused a decrease in the total amount without increasing the asparate formation but with accumulation of 2-oxoglutarate, suggesting that the feedback inhibition by the aspartate of PEP carboxylase was enhanced by 2-oxoglutarate. In fact, partially purified PEP carboxylase from this organism was found to be synergistically inhibited by aspartate and 2-oxoglutarate, citrate, cis-aconitase, or isocitrate. Among them, the effects of tricarboxylic acids were attributed to their non-specific chelating action with Mn²⁺, an activator of the enzyme. The synergistic action of 2-oxoglutarate was accompanied by a decrease in Hill coefficient for the aspartate of the enzyme.     

Phosphoenolpyruvate carboxylase in Hydrilla plants with varying CO2 compensation points

Incubation of the submersed aquatic macrophyte, Hydrilla verticillata Royle, for up to 4 weeks in growth chambers under winter-like or summer-like conditions produced high (130 to 150 μl CO₂/1) and low (6 to 8 μl CO₂/l) CO₂ compensation points (Γ), respectively. The activities of both ribulose bisphosphate (RuBP) and phosphoenolpyruvate (PEP) carboxylases increased upon incubation but the major increase was in the activity of PEP carboxylase under the summer-like conditions. This reduced the ratio of RuBP/PEP carboxylases from 2.6 in high Γ plants to 0.2 in low Γ plants. These ratios resemble the values in terrestrial C₃ and C₄ species, respectively. Kinetic measurements of the PEP carboxylase activity in high and low Γ plants indicated the Vmax was up to 3-fold greater in the low Γ plants. The Km (HCO₃ ^?) values were 0.33 and 0.22 mM for the high and low Γ plants, respectively. The Km (PEP) values for the high and low Γ plants were 0.23 and 0.40 mM, respectively; and PEP exhibited cooperative effects. Estimated Km (Mg²⁺) values were 0.10 and 0.22 mM for the high and low Γ plants, respectively. Malate inhibited both PEP carboxylase types similarly. The enzyme from low Γ plants was protected by malate from heat inactivation to a greater extent than the enzyme from high Γ plants. The results indicated that C₄ acid inhibition and protection were not reliable methods to distinguish C₃ and C₄ PEP carboxylases. The PEP carboxylase from low Γ plants was inhibited more by NaCl than that from hight Γ plants. These analyses indicated that Hydrilla PEP carboxylases had intermediate characteristics between those of terrestrial C₃ and C₄ species with the low Γ enzyme being different from the high Γ enzyme, and closer to a C₄ type.     

Phosphoenolpyruvate carboxylase in Hydrilla plants with varying CO2 compensation points

Incubation of the submersed aquatic macrophyte, Hydrilla vertieillata Royle, for up to 4 weeks in growth chambers under winter-like or summer-like conditions produced high (130 to 150 μl CO₂/l) and low (6 to 8 μl CO₂/l) CO₂ compensation points (Γ), respectively. The activities of both ribulose bisphosphate (RuBP) and phosphoenolpyruvate (PEP) carboxylases increased upon incubation but the major increase was in the activity of PEP carboxylase under the summer-like conditions. This reduced the ratio of RuBP/PEP carboxylases from 2.6 in high Γ plants to 0.2 in low Γ plants. These ratios resemble the values in terrestrial C₃ and C₄ species, respectively. Kinetic measurements of the PEP carboxylase activity in high and low Γ plants indicated the Vmax was up to 3-fold greater in the low Γ plants. The Km (HCO₃ ^-) values were 0.33 and 0.22 mM for the high and low Γ plants, respectively. The Km (PEP) values for the high and low Γ plants were 0.23 and 0.40 mM, respectively; and PEP exhibited cooperative effects. Estimated Km (Mg²⁺) values were 0.10 and 0.22 mM for the high and low Γ plants, respectively. Malate inhibited both PEP carboxylase types similarly. The enzyme from low Γ plants was protected by malate from heat inactivation to a greater extent than the enzyme from high Γ plants. The results indicated that C₄ acid inhibition and protection were not reliable methods to distinguish C₃ and C₄ PEP carboxylases. The PEP carboxylase from low Γ plants was inhibited more by NaCl than that from high Γ plants. These analyses indicated that Hydrilla PEP carboxylases had intermediate characteristics between those of terrestrial C₃ and C₄ species with the low Γ enzyme being different from the high Γ enzyme, and closer to a C₄ type.     

Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium.

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.     

Biochemical Specialization of Photosynthetic Cell Layers and Carbon Flow Paths in Suaeda monoica

Suaeda monoica Frossk. ex J. F. Gmel is a C₄ plant with three different photosynthesizing cell layers. The outer chlorenchymatous layer shows a high activity of phosphoenolpyruvate (PEP) carboxylase but none of ribulose bisphosphate (RuBP) carboxylase. The electrophoretic protein band of RuBP carboxylase was missing in this layer. The second chlorenchymatous cells layer shows a very high activity of RuBP carboxylase and NAD malic enzyme and only traces of activity of PEP carboxylase. The third photosynthesizing cell type is comprised of the water tissue. It has moderate activities of RuBP carboxylase and PEP carboxylase. A model for carbon flow in Suaeda monoica leaves is proposed.     

Kinetic evidence of the existence of a regulatory phosphoenolpyruvate binding site in maize leaf phosphoenolpyruvate carboxylase

Phenylphosphate, a structural analog of phosphoenolpyruvate (PEP), was found to be an activator of phosphoenolpyruvate carboxylase (PEP carboxylase) purified from maize leaves. This finding suggested the presence in the enzyme of a regulatory site, to which PEP could bind. We carried out kinetic studies on this enzyme using controlled concentrations of free PEP and of Mg-PEP complex and developed a theoretical kinetic model of the reaction. In summary, the main conclusions drawn from our results, and taken as assumptions of the model, were the following: (i) The affinity of the active site for the complex Mg-PEP is much higher than that for free PEP and Mg2+ ions, and therefore it can be considered that the preferential substrate of the PEP-catalyzed reaction is Mg-PEP. (ii) The enzyme has a regulatory site specific for free PEP, to which Mg2+ ions can not bind. (iii) The binding of free PEP, or an analog molecule, to this regulatory site yields a modified enzyme that has much lower apparent Km values and apparent Vmax values than the unmodified enzyme. So, free PEP behaves as an excellent activator of the reaction at subsaturating substrate concentrations, and as an inhibitor at saturating substrate concentrations. These findings may have important physiological implications on the regulation of the PEP carboxylase in vivo activity and, consequently, of the C4 pathway, since increased reaction rates would be obtained when the concentration of PEP rises, even at limiting Mg2+ concentrations.     

Maize leaf phosphoenolpyruvate carboxylase : oligomeric state and activity in the presence of glycerol

Maize (Zea mays L.) leaf phosphoenopyruvate (PEP) carboxylase activity at subsaturating levels of PEP was increased by the inclusion of glycerol (20%, v/v) in the assay medium. The extent of activation was dependent on H⁺ concentration, being more marked at pH 7 (with activities 100% higher than in aqueous medium) than at pH 8 (20% activation). The determination of the substrate concentration necessary to achieve half-maximal enzyme activity (S_0.5) (PEP) and maximal velocity (V) between pH 6.9 and 8.2 showed a uniform decrease in S_0.5 in the presence of glycerol over the entire pH range tested, and only a slight decrease in V at pH values near 8. Including NaCl (100 millimolar) in the glycerol containing assay medium resulted in additional activation, mainly due to an increase in V over the entire range of pH. Glucose-6-phosphate (5 millimolar) activated both the native and the glycerol-treated enzyme almost to the same extent, at pH 7 and 1 millimolar PEP. Inhibition by 5 millimolar malate at pH 7 and subsaturating PEP was considerably lower in the presence of glycerol than in an aqueous medium (8% against 25%, respectively). Size-exclusion high performance liquid chromatography in aqueous buffer revealed the existence of an equilibrium between the tetrameric and dimeric enzyme forms, which is displaced to the tetramer as the pH was increased from 7 to 8. In the presence of glycerol, only the 400 kilodalton tetrameric form was observed at pH 7 or 8. However, dissociation into dimers by NaCl could not be prevented by the polyol. We conclude that the control of the aggregation state by the metabolic status of the cell could be one regulatory mechanism of PEP carboxylase.     

Corn leaf phosphoenolpyruvate carboxylases: The effect of divalent cations on activity

Differences in the kinetic properties of corn leaf phosphoenolpyruvate (PEP) carboxylase isoenzymes were found, depending on whether Mg²⁺ or Mn²⁺ was used as the metal cofactor of the reaction. Also, differences in kinetic constants with respect to Mg²⁺ and Mn²⁺ were noticed between the two isoenzymes which further differentiates the two proteins. The catalytic activity of the enzyme in the Mg²⁺-activated system was dependent on a PEP-Mg²⁺ complex and not on the concentration of free Mg²⁺ or free PEP. Kinetics in the presence of total Mg²⁺ and those of PEP-Mg²⁺ suggest a negative cooperative effect with respect to ligand binding with concurrent progressive substrate activation. Magnesium ions, thus, have a special regulatory role in the corn leaf PEP carboxylase reaction.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate

Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5–30 °C) and weakly basic conditions (pH 8.5–10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.     

Junctions of catabolic and anabolic pathways in Zymomonas mobilis: phosphoenolpyruvate carboxylase and malic enzyme

Summary The synthesis of oxalacetate and malate in the ethanol-producing bacterium Zymomonas mobilis have been investigated. Cell-free extracts were examined for pyruvate carboxylase, phosphoenolpyruvate (PEP) carboxylase, PEP carboxytransphosphorylase, PEP carboxykinase, and malic enzyme, but only PEP carboxylase and nicotine adenine dinucleotide (NAD)-dependent malic enzyme activities could be detected. The PEP carboxylase, partially purified from extracts, was not affected by acetyl-coenzyme A. Intermediates of the tricarboxylic acid cycle and aspartate inhibited the enzyme competitively with PEP. Of these, citrate and -ketoglutarate were the strongest inhibitors. The physiological roles of PEP carboxylase and malic enzyme in Z. mobilis are discussed.Dedicated to Prof. Dr. A. Fiechter, ETH Zürich, on the occasion of his 65th birthday     

Effects of stabilizing solutes on salt activation of phosphoenolpyruvate carboxylase from various plant sources

Phosphoenolpyruvate carboxylase (PEP carboxylase EC 4.1.1.31) was extracted from various halophytic, semi-halophytic and glycophytic plant species. When the enzyme of those extracts was substrate protected, and in the presence of 1.6 m M PEP in the reaction mixture, the activity of PEP carboxylase was increased by 100 m M NaCl, and the activity range in the presence of NaCl was expanded. No correlation could be established between the response of the enzyme to ions and various plant characteristics, such as taxonomic status, salt tolerance or carbon fixation pathways. Salt activation of PEP carboxylase was substrate (PEP) dependent, but the minimal substrate concentration varied in different species.
Effects of the stabilizing solutes PEP, betaine, proline and glycerol on the kinetic properties of PEP carboxylase from Zea mays (L.) cv. Hazera were analyzed. In the absence of NaCl the slope of the Hill plot (n_It) tended to rise in the presence of these solutes. Stabilization of the enzyme with betaine or glycerol caused a decrease in K'. while K' and VTO increased in the presence of PEP. NaCl (100 mM) caused an increase in both K' and V^max in the protected as well as in the unprotected enzyme, except for PEP protection, where K' decreased somewhat. In the presence of the protectants, glycerol and PEP, the effect of NaCl on V^max, was 2–4 times higher than its effect on the non-protected enzyme.     

Interference by phosphatases in the spectrophotometric assay for phosphoenolpyruvate carboxylase

The aim of this work was to discover the extent of interference by phosphoenolpyruvate (PEP) phosphatase in spectrophotometric assays of PEP carboxylase (EC 4.1.1.31) in crude extracts of plant organs. The presence of PEP phosphatase and lactate dehydrogenase (EC 1.1.1.27) in extracts leads to PEP-dependent NADH oxidation that is independent of PEP carboxylase activity, and hence to overestimation of PEP carboxylase activity. In extracts of three organs of pea (Pisum sativum L.: leaves, developing embryos, and Rhizobium nodules), two organs of wheat (Triticum aestivum L.: developing grain and endosperm), and leaves of Moricandia arvensis (L.) D.C., lactate dehydrogenase activity was at most only 16% of that of PEP carboxylase at the pH optimum for PEP carboxylase activity. Endogenous PEP phosphatase and lactate dehydrogenase are thus unlikely to interfere seriously with the assay for PEP carboxylase at its optimum pH. Addition of lactate dehydrogenase to PEP carboxylase assays— a proposed means of correcting for nonenzymic decarboxylation of oxaloacetate to pyruvate—resulted in increases in PEP-dependent NADH oxidation from zero (Rhizobium nodules) to 131% (wheat grains). There was no obvious relationship between the magnitude of this increase and conditions in the assay that might promote oxaloacetate decarboxylation. However, the magnitude of the increase was highly positively correlated with the activity of PEP phosphatase in the extract. Addition of lactate dehydrogenase to PEP carboxylase assays can thus result in very large overestimations of PEP carboxylase activity, and should only be used as a means of correction for oxaloacetate decarboxylation for extracts with negligible PEP phosphatase activity.     

Environmentally controlled changes of phosphoenolpyruvate carboxylases in Mesembryanthemum

NaCl treated Mesembryanthemum crystallinum plants exhibit a Crassulacean acid metabolism. The activity of phosphoenolpyruvate (PEP) carboxylase, the enzyme responsible for CO₂ dark fixation, depends on leaf age showing maximum activity in mature leaves. Electrophoresis revealed that the young leaves possess only two protein bands with PEP carboxylase activity, while older leaves have 3 bands. The removal of NaCl from the soil resulted in the disappearance of the 3rd band obtained after electrophoresis and a decline in the total activity of the PEP carboxylase. The reintroduction of NaCl at the same concentration as before did not restore the activity of the PEP carboxylase nor did it restore the initial electrophoretic band pattern.     

Study on a prolyl endopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

A prolyl endopeptidase (PEP) was purified to homogeneity from the skeletal muscle of common carp using a procedure involving ammonium sulfate fractionation and column chromatography involving DEAE-Sephacel, Phenyl-Sepharose, DEAE-Sepharose Fast Flow, and hydroxyapatite. The molecular weight of the PEP was 82 kDa as determined by SDS-PAGE. Using Suc-Gly-Pro-MCA as a substrate, the optimal pH and temperature of the purified enzyme were pH 6.0 and 35 °C, respectively, and the K_m and k_cat were 8.33 μM and 1.71 S^?1, respectively. The activity of the PEP was inhibited by SUAM-14746, a specific inhibitor of prolyl endopeptidases, and was partially inhibited by the serine proteinase inhibitors PMSF and Pefabloc SC. According to peptide mass fingerprinting, 12 peptide fragments with a total of 134 amino acid residues were obtained, which were highly identical to prolyl endopeptidases from zebrafish (Danio rerio) and sponge (Amphimedon queenslandica), confirming the purified enzyme was a prolyl endopeptidase. Our present study for the first time reported the existence of a prolyl endopeptidase in fish muscle.     

Histochemical Approach to Properties of Vicia faba Guard Cell Phosphoenolpyruvate Carboxylase

Properties of phosphoenolpyruvate carboxylase in guard cells dissected from frozen-dried Vicia faba L. leaflets were studied using quantitative histochemical techniques. Control experiments with palisade cells and whole leaflet extract proved that the single cell approach was valid. Most characteristics of enzyme activity in guard cells were identical to those in the leaflet extract. The activities were highly dependent on temperature, with maximum activity at 25 to 35 C. Half-maximum activity (with 1 millimolar phosphoenolpyruvate [PEP]) was observed at 0.1 millimolar Mg²⁺. Two-hundred millimolar NaCl inhibited the reaction by 50%. With frozen-dried leaflet extract, the apparent K_m(PEP) was 0.15 millimolar at pH 7.7; with guard cells, the values were 1.49, 0.5 to 0.8, and 0.24 millimolar in three successive experiments. Additional experiments showed that apparent K_m(PEP) of guard cell activity from plants within a single growth lot was reproducible and did not change during stomatal opening. Mixed extract experiments proved that soluble compounds were not responsible for the difference observed between leaflet and guard cell activities. The differences in apparent K_m(PEP) of guard cell activity could not be unambiguously interpreted. The physiological implications of the properties of this enzyme in guard cells are discussed.