Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.1.32 (phosphoenolpyruvate carboxykinase)
4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of reversible transfer of the gamma-phosphate group of ATP by Escherichia coli phosphoenolpyruvate carboxykinase (PCK) on to its substrate is of great interest. It is known that metallofluorides are accurate analogs of the transition state in the context of kinase mechanisms. Therefore, two complexes of PCK, one with AlF(3), Mg(2+) and ADP (complex I), the other with AlF(3), Mg(2+), ADP and pyruvate (complex II) were crystallized. The X-ray crystal structures of these two complexes were determined at 2.0 A resolution. The Al atom has trigonal bipyramidal geometry that mimics the transition state of phosphoryl transfer. The Al atom is at a distance of 2.8 A and 2.9 A from an oxygen atom of the beta-phosphoryl group of ADP in complex I and II, respectively. A water molecule in complex I and an oxygen atom of the pyruvate in complex II are located along the axis of the trigonal bipyramid on the side opposite to the beta-phosphoryl oxygen with respect to the equatorial plane, suggesting that the complexes are close mimics of the transition state. Along with the presence of positively charged species around the AlF(3) moiety, these results indicate that phosphoryl transfer occurs via a direct displacement mechanism with associative qualities.
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PMID:The phosphoryl-transfer mechanism of Escherichia coli phosphoenolpyruvate carboxykinase from the use of AlF(3). 1172 34

The effect of Mn2+/Mg2+ concentration on the activity of intact, homogeneous phosphoenolpyruvate carboxykinase (PEPCK) from leaves of the C4 grass, Guinea grass (Panicum maximum), have been investigated. Assay conditions were optimized so that PEPCK activity could be measured at concentrations of Mn2+/Mg2+ similar to those found in the cytosol (low micromolar Mn2+ and millimolar Mg2+). PEPCK activity was totally dependent on Mn2+ and was activated at low micromolar concentrations of Mn2+ by millimolar concentrations of Mg2+. Therefore, at physiological concentrations of Mn2+, PEPCK has a requirement for Mg2+. Assay at physiological concentrations of Mn2+/Mg2+ led to a marked decrease in its affinity for ATP and a 13-fold increase in its affinity for CO2. The Km (CO2) was further decreased by assay at physiological ATP to ADP ratios, reaching values as low as 20 microM CO2, comparable with the Km (CO2) of ribulose 1,5-bisphosphate carboxylase-oxygenase. This means that PEPCK will catalyze a reversible reaction and that it could operate as a carboxylase in vivo, a feature that could be particularly important in algal CO2-concentrating systems.
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PMID:Phosphoenolpyruvate carboxykinase assayed at physiological concentrations of metal ions has a high affinity for CO2. 1178 61

In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves. To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves. The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms. In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate. The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations. Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass. Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves. There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation.
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PMID:Effects of phosphorylation on phosphoenolpyruvate carboxykinase from the C4 plant Guinea grass. 1178 62

Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional (13)C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.
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PMID:Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures. 1191 94

The effect of gene knockout on metabolism in the pflA-, pflB-, pflC-, and pflD- mutants of Escherichia coli was investigated. Batch cultivations of the pfl- mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA- and pflB- mutants, but not pflC- and pflD- mutants, produced large amounts of D-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD+ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA- and pflB- mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD+ ratio in pfl- mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.
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PMID:The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli. 1467 46

Antibodies against Brassica napus cytosolic pyruvate kinase (PKc) (EC 2.7.1.40) were employed to examine PKc subunit composition and developmental profiles in castor and soybean seeds. A 56-kDa immunoreactive polypeptide was uniformly detected on immunoblots of clarified extracts from developing castor endosperm or soybean embryos. Maximal PKc activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (micromol of pyruvate produced/min) g(-1) FW, respectively) and were up to 25-fold greater than those of fully mature seeds. Time-course studies revealed a close correlation between extractable PKc activity and the relative amount of the immunoreactive 56-kDa PKc polypeptide. PKc from developing COS was purified 1,874-fold to homogeneity and a final specific activity of 73.1 (micromol of pyruvate produced/min) mg(-1) protein. Gel filtration and SDS-PAGE indicated that this PKc exists as a 230-kDa homotetramer composed of 56-kDa subunits. The mass fingerprint of tryptic peptides of the 56-kDa COS PKc subunit best matched three putative PK(c)s from Arabidopsis thaliana. The purified enzyme was relatively heat-stable and displayed a broad pH optimum of 6.4. However, more efficient substrate utilization (in terms of Vmax /Km for phosphoenolpyruvate or ADP) was observed at pH 7.4. Glutamate was the most effective inhibitor, whereas aspartate functioned as an activator by partially relieving glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PKc and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PKc exists as tissue-specific isozymes that exhibit substantial differences in their respective physical and regulatory properties.
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PMID:Cytosolic pyruvate kinase: subunit composition, activity, and amount in developing castor and soybean seeds, and biochemical characterization of the purified castor seed enzyme. 1604 77

Here, nodulated lupins (Lupinus angustifolius (cv Wonga)) were hydroponically grown at low phosphate (LP) or adequate phosphate (HP). Routes of pyruvate synthesis were assessed in phosphorus (P)-starved roots and nodules, because P-starvation can enhance metabolism of phosphoenolpyruvate (PEP) via the nonadenylate-requiring PEP carboxylase (PEPc) route. Since nodules and roots may not experience the same degree of P stress, it was postulated that decreases in metabolic inorganic phosphorus (Pi) of either organ, should favour more pyruvate being synthesized from PEPc-derived malate. Compared with HP roots, the LP roots had a 50% decline in Pi concentrations and 55% higher ADP : ATP ratios. However, LP nodules maintained constant Pi levels and unchanged ADP : ATP ratios, relative to HP nodules. The LP roots had greater PEP metabolism via PEPc and synthesized more pyruvate from PEPc-derived malate. In nodules, P supply did not influence PEPc activities or levels of malate-derived pyruvate. These results indicate that nodules were more efficient than roots in maintaining optimal metabolic Pi and adenylate levels during LP supply. This caused an increase in PEPc-derived pyruvate synthesis in LP roots, but not in LP nodules.
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PMID:Routes of pyruvate synthesis in phosphorus-deficient lupin roots and nodules. 1641 42

A detailed comparison of green leaf phosphoenolpyruvate carboxylases from the C(4)-species Atriplex spongiosa and the C(3)-species Atriplex hastata revealed significant physical and kinetic differences. The two alloenzymes can be separated by anion exchange chromatography but have comparable molecular weights (350,000). Maximal velocity estimates were 38.0 and 1.48 micromoles per minute per milligram of chlorophyll for the carboxylases of A. spongiosa and A. hastata, respectively. Km phosphoenolpyruvate estimates were 0.49 and 0.08 mm for the C(4)A. spongiosa and C(3)A. hastata and the Km Mg estimates were 0.33 mm for the C(4) species and 0.017 mm for the C(3) species. The activity of the phosphoenolpyruvate carboxylase of A. spongiosa is more sensitive to chloride and phosphate than the phosphoenolpyruvate carboxylase of A. hastata, but both are equally sensitive to Mg chelating substances such as ATP, ADP, and citrate if assayed at their respective Km Mg values. A survey of the phosphoenolpyruvate carboxylases from 18 C(4) and C(3) species resulted in mean maximal velocity estimates of 29.0 +/- 13.2 and 1.50 +/- 0.57 micromoles per minute per milligram of chlorophyll for the C(4) species and C(3) species, respectively. Km phosphoenolpyruvate estimates were 0.59 +/- 0.35 mm and 0.14 +/- 0.07 mm for the C(4) and C(3), and Km Mg estimates were 0.50 +/- 0.30 and 0.097 +/- 0.057 mm for C(4) and C(3). All differences between means were significant at the 0.01 confidence level, supporting our hypothesis that the phosphoenolpyruvate carboxylase alloenzymes of C(4) and C(3) plants are functionally different and are associated with different photosynthetic roles. Both function in the photosynthetic production of C(4) acids, the phosphoenolpyruvate carboxylase of C(4) species largely producing malate or aspartate (or both) as a photosynthetic intermediate and the phosphoenolpyruvate carboxylase of C(3) species producing malate or aspartate (or both) as a photosynthetic product.
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PMID:Photosynthetic phosphoenolpyruvate carboxylases: characteristics of alloenzymes from leaves of c(3) and c(1) plants. 1665 48

Phosphoenolpyruvate carboxykinase, EC 4.1.1.32 (PEPCK), was purified 43-fold from the grass Panicum maximum. Michaelis constants (Km) were determined for the exchange reaction, the carboxylation reaction, and the decarboxylation reaction. The Km values for oxaloacetate and ATP in the decarboxylation reaction were found to be lower than the Km values for the substrates used in the exchange reaction and in the carboxylation reaction. Phosphoenolpyruvate carboxylase was not detectable in the purified PEPCK preparation.Studies on the nucleotide specificity of the oxaloacetate decarboxylation reaction indicate that ATP serves as the best nucleotide for this reaction and that ADP is about 60% as effective as ATP. The pH optimum for decarboxylase activity is near 6.8. The decarboxylation reaction has a divalent cation requirement with both Mn(2+) and Mg(2+) needed for full activity.Temperature curves of the three PEPCK reactions indicate optimum activities between 38 and 45 C. There is a pronounced drop in the decarboxylation and carboxylation activities as the temperature is decreased from these optima. Below 30 C the energy of activation was 8.2 kcal/mol for the decarboxylation reaction.These studies are consistent with the proposal that under physiological conditions PEPCK catalyzes the decarboxylation of oxaloacetate in the bundle sheath cells of Panicum maximum leaves during C(4) dicarboxylic acid photosynthesis.
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PMID:Characterization of Phosphoenolpyruvate Carboxykinase from Panicum maximum. 1665 27

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.
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PMID:Characterization of Phosphoenolpyruvate Carboxykinase from Pineapple Leaves Ananas comosus (L.) Merr. 1665 5


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