Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.1.49 (phosphoenolpyruvate carboxykinase)
4,654 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have cloned and sequenced the pckA gene of Rhizobium sp. NGR234, a broad host-range strain. The gene encodes phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of gluconeogenesis. The locus was isolated and subcloned from a genomic library of NGR234 employing hybridization with an R. meliloti pck gene probe and complementation of a Tn5 mutant in this species. The DNA sequence of pckA (NGR234) was determined and encoded a PEPCK protein of 535 amino acids with a molecular weight of 58.4 kDa. The deduced polypeptide sequence was compared to those of three known ATP-dependent PEPCKs. Slightly higher homology was observed with yeast and trypanosome polypeptides than with that of Escherichia coli. We have identified several regions that are conserved in all four PEPCK proteins. A mutant constructed in the pck gene by site-directed mutagenesis with interposon omega failed to grow on succinate, malate and arabinose but grew on glucose and glycerol as sole carbon sources. These data show that NGR234 requires PEPCK-driven gluconeogenesis to grow on TCA cycle intermediates. A host-dependent effect of the pckA mutation was observed on nodule development and nitrogen fixation. Nodules formed by the site-directed mutant on Leucaena leucocephala and Macroptilium atropurpureum were FixRed, but on Vigna unguiculata were Fix-. The expression of the gene was positively regulated in free-living cells of NGR234 by either succinate or host-plant exudates, and was subject to catabolite repression by glucose.
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PMID:Site-directed mutagenesis and DNA sequence of pckA of Rhizobium NGR234, encoding phosphoenolpyruvate carboxykinase: gluconeogenesis and host-dependent symbiotic phenotype. 172 Aug 62

Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.
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PMID:Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitata. 177 15

The results from the experiments performed with a mutant deficient in citrate synthase activity can be summarized as follows. (1) Totally blocking entry into the TCA cycle did not appreciably alter the cellular ATP yield. The unchanged yield suggests that for growth on abundant glucose, the sensitivity of ATP yield to TCA cycle flux is low. ATP production in the mutant is altered, in part, by modulating the relative amounts of formate and acetate produced. (2) The in vivo operation of pyruvate-formate lyase and malic enzyme corresponds to proposals developed from in vitro studies. Namely, pyruvate activates the former, and acetyl CoA inhibits the latter. Overall, the diversion of pyruvate to formate under aerobic conditions constitutes an adaptation of the mutant to the enzymatic lesion. The low alpha-ketoglutarate dehydrogenase flux estimated for the mutant indicates that the enzyme is highly repressed in cells growing rapidly on glucose, which is in accord with prior induction-repression studies. Moreover, the lack of a change in uptake flux during the bulk of batch growth is consistent with prior induction-repression studies. (3) The mutant exhibits a heightened sensitivity to CO2 as compared to wild-type counterparts. Growth rate is increased, and the production of formate, malate, glycerate, and pyruvate is reduced. This sensitivity illustrates that citrate synthase is more than an expendable component in an amphibolic pathway. Its presence in wild-type cells "immunizes" against the effect of CO2 fluctuations. (4) The effects of CO2 can be tentatively explained by assuming that the PEP carboxylase-catalyzed reaction is stimulated.
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PMID:Flux adaptations of citrate synthase-deficient Escherichia coli. 783 22

Activities of enzymes in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and glutaminolysis have been determined in the mouse myeloma SP2/0.Ag14. Cells were grown on IMDM medium with 5% serum in steady-state chemostat culture at a fixed dilution rate of 0.03 h-1. Three culture conditions, which differed in supply of glucose and oxygen, were chosen so as to change catabolic fluxes in the central metabolism, while keeping anabolic fluxes constant. In the three steady-state situations, the ratio between specific rates of glucose and glutamine consumption differed by more than twentyfold. The specific rates of glucose consumption and lactate production were highest at low oxygen supply, whereas the specific rate of glutamine consumption was highest in the culture fed with low amounts of glucose. Under low oxygen conditions, the specific production of ammonia increased and the consumption pattern of amino acids showed large changes compared with the other two cultures. For the three steady states, activities of key enzymes in glycolysis, the pentose phosphate pathway, glutaminolysis, and the TCA cycle were measured. The differences in the in vivo fluxes were only partially reflected in changes in enzyme levels. The largest differences were observed in the levels of glycolytic enzymes, which were elevated under conditions of low oxygen supply. High activities of phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) in all cultures suggest an important role for this enzyme as a link between glutaminolysis and glycolysis. For all enzymes, in vitro activities were found that could accommodate the estimated maximum in vivo fluxes. These results show that the regulation of fluxes in central metabolism of mammalian cells occurs mainly through modulation of enzyme activity and, to a much lesser extent, by enzyme synthesis.
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PMID:Fluxes and enzyme activities in central metabolism of myeloma cells grown in chemostat culture. 1009 11

Quantifying the contribution of the various skeletal muscle fiber types toward lactate disposal has proven elusive. In part, this can be attributed to the lack of adequate preparations for the study of all potential metabolic pathways involved. Toward this end our laboratory developed several perfused muscle preparations that are homogeneous for specific fiber types. This paper briefly reviews our findings regarding the influence of fiber type on lactate disposal in resting skeletal muscle and the metabolic pathways involved. Perfusing over a range of lactate concentrations, 1-12 mM, all fiber types were shown to switch from net production at low lactate concentrations to net consumption at higher concentrations. This transition occurred at lower lactate concentrations for Type I and IIa fibers, when compared with IIb fibers. For Type I and IIa fibers oxidation was observed to be the primary route of disposal accounting for approximately 50% of the lactate removed. For all fiber types, transamination was a significant pathway for the disposal of lactate carbon, whereas glyconeogenesis was the primary pathway for disposal in Type IIb fibers. The glyconeogenic capacity was quantitatively similar for Type IIa and IIb fibers but was negligible for Type I fibers. The pathway for glyconeogenesis in skeletal muscle was shown to be substantially different from that employed in hepatic glyconeogenesis. Results indicated that neither the TCA cycle nor phosphoenolpyruvate carboxykinase is involved in skeletal muscle glyconeogenesis. Our findings suggested that PEP formation in skeletal muscle glyconeogenesis occurs by "reversal" of the pyruvate kinase reaction.
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PMID:Quantitative assessment of pathways for lactate disposal in skeletal muscle fiber types. 1077 96

The activities of carbon metabolism enzymes were determined in cellular extracts of the moderately thermophilic, chemolithotrophic, acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41, grown either at an atmospheric content of CO2 in the gas phase (autotrophically, heterotrophically, or mixotrophically) or autotrophically at a CO2 content increased to 5-10%. Regardless of the growth conditions, all TCA cycle enzymes (except for 2-oxoglutarate dehydrogenase), one glyoxylate cycle enzyme (malate synthase), and some carboxylases (ribulose bisphosphate carboxylase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase) were detected in the cellular extracts of strain 41. During autotrophic cultivation of strains 41 and 1269, the increase in the CO2 content of the supplied air to 5-10% resulted in the activation of growth and iron oxidation, a 20-30% increase in the cellular content of protein, enhanced activity of the key TCA enzymes (citrate synthase and aconitase), and, in strain 41, a decrease in the activity of carboxylases.
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PMID:[Carbon metabolism in Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41]. 1092 Aug 1

Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h(-1)) and high (0.44 h(-1)) dilution rates. Using a mixture of 10% [U-(13)C] and 90% glucose labeled at natural abundance, (13)C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [(13)C, (1)H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a chi(2) test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.
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PMID:Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. 1150 84

A stoichiometric model of central metabolism was developed based on new information regarding metabolism in this bacterium to evaluate the steady-state growth capabilities of the serine cycle facultative methylotroph Methylobacterium extorquens AM1 during growth on methanol, succinate, and pyruvate. The model incorporates 20 reversible and 47 irreversible reactions, 65 intracellular metabolites, and experimentally-determined biomass composition. The flux space for this underdetermined system of equations was defined by finding the elementary modes, and constraints based on experimental observations were applied to determine which of these elementary modes give a reasonable description of the flux distribution for each growth substrate. The predicted biomass yield, on a carbon atom basis, is 49.8%, which agrees well with the range of published experimental yield measurements (37-50%). The model predicts the cell to be limited by reduced pyridine nucleotide availability during methylotrophic growth, but energy-limited when growing on multicarbon substrates. Mutation and phenotypic analysis was used to explore a previously unknown region of the metabolic map and to confirm the stoichiometry of the pathways in this region used in the metabolic model. Based on genome sequence data and simulation results, three enzymes involved in C(3)-C(4) interconversion pathways were predicted to be mutually redundant: malic enzyme, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate synthase. Insertion mutations in the genes predicted to encode these enzymes were made and these mutants were capable of growing on all substrates tested, confirming the redundancy of these pathways. Likewise, pathway analysis suggests that the TCA cycle enzymes citrate synthase and succinate dehydrogenase are essential for all growth substrates. In keeping with these predictions, null mutants could not be obtained in these genes. Finally, a similar model was developed for the ribulose monophosphate pathway obligate methylotroph Methylobacillus flagellatum KT to compare the efficiency of carbon utilization in the two types of methylotrophic carbon utilization pathways. The predicted yield for this organism on methanol is 65.9%.
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PMID:Stoichiometric model for evaluating the metabolic capabilities of the facultative methylotroph Methylobacterium extorquens AM1, with application to reconstruction of C(3) and C(4) metabolism. 1192 Apr 46

Exposure of Leishmania donovani culture promastigotes to ethyleneglycol-bis-((-aminoethyl ether) N,N,N(1),N(1),-tetraacetic acid (EGTA) concentrations of between 0.2 to 1.6 mg/ml significantly inhibited their growth, though the different concentrations did not significantly differ between themselves on their effect on promastigotes in cell free media. EGTA concentrations of between 0.05 and 0.1 mg/ml were non-toxic to mouse peritoneal macrophages in vitro. Treatment of L. donovani-infected macrophages with EGTA at concentrations of 0.05, 0.1 and 0.2 mg/ml contributed significantly to a decline in amastigote parasite-loads in the macrophages. The higher the chelator concentration within the acceptable toxic levels for macrophages, the greater was the rate at which parasites were cleared from the macrophages. It is speculated that EGTA chelates manganese from phosphoenol pyruvate (PEP) carboxykinase, a TCA-rate limiting enzyme in the metabolism of Leishmania parasites. A manganese-complex is also probably used by these parasites as a defense mechanism against oxygen-derived radicals. Chelation of manganese would destabilise PEP carboxykinase, and therefore severely interfere with the parasite metabolism. All these factors would render the Leishmania parasite susceptible to digestion in the lysosomal vacuoles of the macrophage, hence the observed significant reduction in parasite-loads of L. donovani-infected EGTA-treated macrophages in vitro. These results suggest an exciting future potential use of chelators in the experimental chemotherapy of visceral leishmaniasis.
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PMID:Ethyleneglycol-Bis-((-aminoethyl ether) N,N,N(1),N(1), -Tetraacetic acid (EGTA) inhibits Leishmania donovani in vitro. 1215 41

The metabolic mechanism of hepatic glucose overproduction was investigated in 3,3'-5-triiodo-l-thyronine (T3)-treated rats and Zucker diabetic fatty (ZDF) rats (fa/fa) after a 24-h fast. 2H2O and [U-13C3]propionate were administered intraperitoneally, and [3,4-13C2]glucose was administered as a primed infusion for 90 min under ketamine-xylazine anesthesia. 13C NMR analysis of monoacetone glucose derived from plasma glucose indicated that hepatic glucose production was twofold higher in both T3-treated rats and ZDF rats compared with controls, yet the sources of glucose overproduction differed significantly in the two models by 2H NMR analysis. In T3-treated rats, the hepatic glycogen content and hence the contribution of glycogenolysis to glucose production was essentially zero; in this case, excess glucose production was due to a dramatic increase in gluconeogenesis from TCA cycle intermediates. 13C NMR analysis also revealed increased phosphoenolpyruvate carboxykinase flux (4x), increased pyruvate cycling flux (4x), and increased TCA flux (5x) in T3-treated animals. ZDF rats had substantial glycogen stores after a 24-h fast, and consequently nearly 50% of plasma glucose originated from glycogenolysis; other fluxes related to the TCA cycle were not different from controls. The differing mechanisms of excess glucose production in these models were easily distinguished by integrated 2H and 13C NMR analysis of plasma glucose.
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PMID:Differing mechanisms of hepatic glucose overproduction in triiodothyronine-treated rats vs. Zucker diabetic fatty rats by NMR analysis of plasma glucose. 1556 53


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