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:4.1.1.32 (phosphoenolpyruvate carboxykinase)
4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To analyze the role of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) during seed development, two cDNA clones encoding two isoforms of PEPCase were isolated from a seed-specific library of Vicia faba. The two sequences (VfPEPCase1 and VfPEPCase2) have a sequence identity of 82 and 89% on the nucleotide and amino acid levels. The VfPEPCase1 mRNA was found to be predominantly expressed in roots and developing cotyledons whereas the VfPEPCase2 mRNA was more abundant in green and maternal tissues. In the cotyledons, PEPCase mRNAs accumulated from early to mid cotyledon stage and decreased thereafter. The PEPCase activity increased continuously during cotyledon development. The enzyme was strongly activated by glucose-6-phosphate, but not by glucose, fructose or sucrose. Asparagine was weakly activating whereas malate, aspartate and glutamate were inhibitory. The inhibitors became less effective with increasing pH. Aspartate was a much stronger inhibitor of cotyledonary PEPCase than glutamate at both pH 7.0 and 7.5. The sensitivity of PEPCase to malate inhibition decreased from early to mid cotyledon stage at a time when storage proteins are synthesized. This indicates activation on the protein level, possibly by protein phosphorylation. Nitrogen starvation in the presence of hexoses but not sucrose decreased mRNA levels of VfPEPCase1 and enzyme activity, indicating control on the mRNA level by both carbon and nitrogen. It is concluded that in developing cotyledons PEPCase is probably important for the synthesis of organic acids to provide carbon skeletons for amino acid synthesis.
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PMID:Phosphoenolpyruvate carboxylase in developing seeds of Vicia faba L.: gene expression and metabolic regulation. 1021 2

In Escherichia coli, aspartate aminotransferase (encoded by aspC) and aromatic amino acid aminotransferase (encoded by tyrB) share overlapping substrate specificity in the syntheses of aromatic amino acids. Through the transamination reactions catalyzed by AspC or TyrB, L-phenylalanine (L-Phe) can be produced from phenylpyruvate with aspartic acid as the amino donor. To modulate and enhance the production levels of proteins, both aspC and tyrB were subcloned into a runaway-replication vector. As a result, the specific activities of AspC and TyrB obtained showed 65-fold and 50-fold increases, respectively, compared with the wild-type level. Employing resting cells of AspC- and TyrB-overproducing E. coli K-12 strains for L-Phe productions resulted in molar conversion yields of 70% and 55%, respectively. With an additional introduction of phosphoenolpyruvate carboxykinase (encoded by pck) into the transamination reactions, the conversion yields were improved to 93% from 70% and to 75% from 55% in a relatively short time. These results account for more than an 8-fold increase in productivity, as compared to the previous report (Calton et al., 1985). In addition, a four-run reuse of the recombinant cells for L-Phe production gave a total yield of 91 g/L with a 93% conversion.
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PMID:Enhanced conversion rate of L-phenylalanine by coupling reactions of aminotransferases and phosphoenolpyruvate carboxykinase in Escherichia coli K-12. 1035 62

We have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A resolution, and specified the location of enzyme-bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the alpha/beta barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP.
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PMID:Plausible phosphoenolpyruvate binding site revealed by 2.6 A structure of Mn2+-bound phosphoenolpyruvate carboxylase from Escherichia coli. 1048 Oct 43

Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201-fold to electrophoretic homogeneity and a final specific activity of 51 micromol phosphoenolpyruvate utilized per min per mg protein. SDS/PAGE and gel filtration analyses of the final preparation indicated that this PKc is a 220-kDa homotetramer composed of 56-kDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of pH 6.8. PKc activity was absolutely dependent upon the simultaneous presence of a bivalent and univalent cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed for phosphoenolpyruvate, ADP, Mg2+ and K+ (apparent Km values = 0.12, 0.075, 0.21 and 0.48 mM, respectively). Although the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate, oxalate, and the flavonoids rutin and quercetin were the most effective inhibitors (I50 values = 4, 0.3, 0.07, and 0.10 mM, respectively). L-Aspartate functioned as an activator (Ka = 0.31 mM) by causing a 40% increase in Vmax while completely reversing the inhibition of PKc by L-glutamate. Reciprocal control by L-aspartate and L-glutamate is specific for these amino acids and provides a rationale for the in vivo activation of PKc that occurs during periods of enhanced NH +4-assimilation. Allosteric features of B. napus PKc are compared with those of B. napus phosphoenolpyruvate carboxylase. A model is presented that highlights the pivotal role of L-aspartate and L-glutamate in the coordinate regulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.
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PMID:Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures: implications for the integration of glycolysis with nitrogen assimilation. 1088 Sep 71

In most Gram-positive bacteria, catabolite repression is mediated by a bifunctional enzyme, the histidine-containing protein kinase/phosphatase (HprK/P). Based either on its primary sequence or on its recently solved three-dimensional structure, no straightforward homology with other known proteins was found. However, we showed here that HprK/P exhibits a restricted homology with an unrelated phosphotransferase, the phosphoenolpyruvate carboxykinase. This includes notably two consecutive Asp residues from the phosphoenolpyruvate carboxykinase active site, whose equivalent residues were mutated in Bacillus subtilis HprK/P. Characterization of the corresponding mutants emphasizes the crucial role of these Asp residues in the HprK/P functioning. Furthermore, superimposition of HprK/P and phosphoenolpyruvate carboxykinase active sites supports the view that both enzymes bear significant resemblance in their overall mechanism of functioning showing that these two enzymes constitute a new family of phosphotransferases.
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PMID:A new family of phosphotransferases with a P-loop motif. 1179 14

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe. The S. vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa. SvPEPC was expressed in E. coli BL21 Codonplus (DE3), using pET32a as a vector. The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE. The enzyme forms homotetramer as judged by gel filtration. SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)). SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln. Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.
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PMID:Molecular characterization of a phosphoenolpyruvate carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties. 1186 95

Our objective was to understand the influence of dietary gluconeogenic amino acids on hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss). We analyzed the effects of partial substitution of dietary protein by a single gluconeogenic dispensable amino acid (DAA: alanine, aspartic acid or glutamic acid), on the regulation of hepatic glycolytic and gluconeogenic enzymes. We fed juvenile rainbow trout with isonitrogenous and isoenergetic diets in which part of nitrogen from fishmeal was replaced by nitrogen from one of the three DAA. Fish were fed over 9 weeks and samples withdrawn 6 h after feeding or 5 days after food deprivation. Our data did not show a clear effect of an excess of DAA on activities of glycolytic enzymes (glucokinase and pyruvate kinase) compared to the control diet. In contrast, feeding caused a significant repression of gluconeogenic enzyme activities (glucose-6-phosphatase, fructose-1,6-bisphosphatase and mitochondrial phosphoenolpyruvate carboxykinase) only in fish fed the three DAA substituted diets. However, these differences were insufficient to affect postprandial glycemia significantly. In conclusion, an excess of dietary DAA tested does not seem to modify glycemia or to have a negative impact on dietary carbohydrate utilization in rainbow trout.
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PMID:Effect of partial substitution of dietary protein by a single gluconeogenic dispensable amino acid on hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss). 1254 63

Transgenic Medicago truncatula plants were produced harboring chimeric gene constructs of the glutamine synthetase (GS) cDNA clones (MtGS1a or MtGS1b) fused in sense or antisense orientation to the nodule-specific leghemoglobin promoter Mtlb1. A series of transgenic plants were obtained showing a 2- to 4-fold alteration in nodule GS activity when compared with control plants. Western and northern analyses revealed that the increased or decreased levels of GS activity correlate with the amount of cytosolic GS polypeptides and transcripts present in the nodule extracts. An analysis of the isoenzyme composition showed that the increased or decreased levels of GS activity were attributable to major changes in the homo-octameric isoenzyme GS1a. Nodules of plants transformed with antisense GS constructs showed an increase in the levels of both asparagine synthetase (AS) polypeptides and transcripts when compared with untransformed control plants, whereas the sense GS transformants showed decreased AS transcript levels but polypeptide levels similar to control plants. The polypeptide abundance of other nitrogen metabolic enzymes NADH-glutamic acid synthase and aspartic acid amino-transferase as well as those of major carbon metabolic enzymes phosphoenolpyruvate carboxylase, carbonic anhydrase, and sucrose synthase were not affected by the GS-gene manipulations. Increased levels of AS polypeptides and transcripts were also transiently observed in nodules by inhibiting GS activity with phosphinothricin. Taken together, the results presented here suggest that GS activity negatively regulates the level of AS in root nodules of M. truncatula. The potential role of AS in assimilating ammonium when GS becomes limiting is discussed.
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PMID:Nodule-specific modulation of glutamine synthetase in transgenic Medicago truncatula leads to inverse alterations in asparagine synthetase expression. 1297 Apr 90

An investigation was made of the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant Ananas comosus (pineapple) in malate metabolism during CAM phase III. Pineapple mitochondria showed very high malate dehydrogenase (MDH), and low malic enzyme (ME) and glutamate-oxaloacetate transaminase (GOT) activities. The mitochondria readily oxidized succinate and NADH with high rates and coupling, while they only oxidized NADPH in the presence of Ca(2+). Pineapple mitochondria oxidized malate with low rates under most assay conditions, despite increasing malate concentrations, optimizing pH, providing cofactors such as coenzyme A, thiamine pyrophosphate, and NAD(+), and supplying individually external glutamate or GOT. However, providing glutamate and GOT simultaneously strongly increased the rates of malate oxidation. The OAA easily permeated the mitochondrial membranes to import into or export out of pineapple mitochondria during malate oxidation, but the mitochondria did not consume external Asp or alpha-KG. These results suggest that OAA played a significant role in the mitochondrial malate metabolism of pineapple, in which malate was mainly oxidized by active mMDH to produce OAA which could be exported outside the mitochondria via a malate-OAA shuttle. Cytosolic GOT then consumed OAA by transamination in the presence of glutamate, leading to a large increase in respiration rates. The malate-OAA shuttle might operate as a supporting system for decarboxylation in phase III of PCK-CAM pineapple. This shuttle system may be important in pineapple to provide a source of energy and substrate OAA for cytosolic PCK activity during the day when cytosolic OAA and ATP was limited for the overall decarboxylation process.
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PMID:Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne. 1536 38

Actinobacillus succinogenes can produce, via fermentation, high concentrations of succinate, an important industrial commodity. A key enzyme in this pathway is phosphoenolpyruvate carboxykinase (PCK), which catalyzes the production of oxaloacetate from phosphoenolpyruvate and carbon dioxide, with the concomitant conversion of adenosine 5'-diphosphate to adenosine 5'-triphosphate. 1.85 and 1.70 A resolution structures of the native and a pyruvate/Mn(2+)/phosphate complex have been solved, respectively. The structure of the complex contains sulfhydryl reducing agents covalently bound to three cysteine residues via disulfide bonds. One of these cysteine residues (Cys285) is located in the active-site cleft and may be analogous to the putative reactive cysteine of PCK from Trypanosoma cruzi. Cys285 is also part of a previously unreported conserved motif comprising residues 280-287 and containing the pattern NXEXGXY(/F)A(/G); this new motif appears to have a structural role in stabilizing and positioning side chains that bind substrates and metal ions. The first few residues of this motif connect the two domains of the enzyme and a fulcrum point appears to be located near Asn280. In addition, an active-site Asp residue forms two coordinate bonds with the Mn(2+) ion present in the structure of the complex in a symmetrical bidentate manner, unlike in other PCK structures that contain a manganese ion.
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PMID:Structure of PEP carboxykinase from the succinate-producing Actinobacillus succinogenes: a new conserved active-site motif. 1598 13


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