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)

The crystal structure of ATP-dependent phosphoenolpyruvate carboxykinase (ATP-oxaloacetate carboxy-lyase, (transphosphorylating), E.C. 4.1.1.49; PCK) from Escherichia coli strain K12 has been determined using a combination of multiple isomorphous replacement, density modification, and partial model phase combination, and refined to a conventional R-index of 0.204 (Rfree = 0.244) at 1.9 A resolution. Each PCK molecule consists of a 275 residue N-terminal domain and 265 residue C-terminal or mononucleotide-binding domain, with the active site postulated to be within a cleft between the two domains. PCK is an open-faced, mixed alpha/beta protein, with each domain having an alpha/beta folding topology as found in several other mononucleoside-binding enzymes. The putative phosphate-binding site of ATP adopts the P-loop motif common to many ATP and GTP-binding proteins, and is similar in structure to that found within adenylate kinase. However, the beta-sheet topology within the mononucleotide-binding fold of PCK differs from all other families within the P-loop containing nucleoside triphosphate hydrolase superfamily, therefore suggesting it represents the first member in a new family of such proteins. The mononucleotide-binding domain is also different in structure compared to the classical mononucleotide-binding fold (CMBF) common to adenylate kinase, p21ras, and elongation factor-Tu. Several amino acid residues, including R65, K212, K213, H232, K254, D269, K288 and R333 appear to make up the active site of the enzyme, and are found to be absolutely conserved among known members of the ATP-dependent PCK family. A cysteine residue is located near the active-site, as has been suggested for other PCKs, although in the E. coli enzyme C233 is buried and so is most likely not involved in substrate binding or catalysis. Two binding sites of the calcium-analog TB3+ have been determined, one within the active site coordinating to the side-chain of D269, and the other within the C-terminal domain coordinating to the side-chains of E508 and E511.
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PMID:Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold. 860 5

The reactivities of Cys365 and Cys458 of ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase against a range of sulfhydryl reagents have been investigated. The effect of pH on the second order reaction constants of N-(1-pyrenyl)maleimide with mutant C458S and C365S PEP carboxykinases allowed the determination of pKa values of 9.4 and 9.1 for Cys365 and Cys458, respectively. The analysis of the inactivation rates of C458S and C365S mutant enzymes by several sulfhydryl reagents of different hydrophobicity showed that the microenvironment of these residues is rather polar. Anisotrophy measurements and acrylamide quenching experiments carried out with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled mutant enzymes indicated a higher rotational freedom and solvent exposure for the probe linked to Cys458 than to Cys365. These findings point to differences in the protein microenvironments around Cys365 and Cys458 in S. cerevisiae PEP carboxykinase. A comparison of the results obtained with published data for GTP-dependent PEP carboxykinases, suggest significant differences for the protein region around the reactive cysteinyl residues in these enzymes.
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PMID:Reaction of wild-type C365S, and C458S saccharomyces cerevisiae phosphoenolpyruvate carboxykinases with fluorescent iodoacetamide derivatives. 861 82

Rabbit, pigeon and rat liver mitochondria convert exogenous phosphoenolpyruvate and acetylcarnitine to citrate at rates of 14, 74 and 8 nmol/15 min/mg protein. Citrate formation is dependent on exogenous HCO3-, is increased consistently by exogenous nucleotides (GDP, IDP, GTP, ADP, ATP) and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricarboxylate. Citrate is not made from pyruvate alone or combined with acetylcarnitine. Pigeon and rat liver mitochondria make large amounts of citrate from exogenous succinate, suggesting the presence of an endogenous source of acetyl units or means of converting oxalacetate to acetyl units. Citrate synthesis from succinate by pigeon and rabbit mitochondria is increased significantly by exogenous acetylcarnitine. Pigeon and rat liver contain 80 and 15 times, respectively, more ATP:citrate lyase activity than does rabbit liver. Data suggest that mitochondrial phosphoenolpyruvate carboxykinase in vivo could convert glycolysis-derived phosphoenolpyruvate to oxalacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA carboxylase, a carbon source via ATP:citrate lyase and NADPH via NADP:malate dehydrogenase or NADP:isocitrate dehydrogenase.
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PMID:Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit, pigeon and rat liver: implications for lipogenesis. 884 May 17

Recombinant phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) of an extreme thermophile, Thermus sp., which was expressed in Escherichia coli cells, was purified and its enzymological properties were investigated and compared with native Thermus PEPC. The enzyme activity was strongly dependent on acetyl-CoA, an allosteric activator, and inhibited by malate or aspartate. Contrary to the other known PEPCs, Thermus PEPC was not activated but rather inhibited by phosphorylated compounds such as fructose 1,6-bisphosphate and GTP. The specific activity in the presence of 0.3 mM acetyl-CoA and 2 mM phosphoenolpyruvate was highest at 70 degrees C. The half-saturation concentrations for both substrates at 70 degrees C were about twice those at 30 degrees C. Half-lives of the enzyme at 85, 90, and 95 degrees C were 220, 110, and 50 min, respectively. Thermus PEPC was highly tolerant also to guanidine hydrochloride (Gdn-HCl): the concentrations required for complete inactivation of Thermus and E. coli PEPCs after incubation at 30 degrees C for 20 h were 3.5 and 0.6 M, respectively. The properties of recombinant and native enzyme were similar to each other except for the catalytic activity after incubation with 1-2 M Gdn-HCl.
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PMID:Purification and characterization of recombinant phosphoenolpyruvate carboxylase of Thermus sp. 890 15

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 approximately 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 micromol min-1 (mg enzyme)-1. The apparent Km values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent Km 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.
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PMID:Purification and characterization of phosphoenolpyruvate carboxykinase from the anaerobic ruminal bacterium Ruminococcus flavefaciens. 909 26

Avian mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) was incubated with Co2+ and H2O2 to form a stable Co3+-PEPCK complex. PEPCK, similarly incubated with H2O2 and either Mg2+ or Mn2+, resulted in no significant loss in activity over 30 min. PEPCK, incubated with Co2+ and H2O2 at pH 7.4, showed rapid inhibition as observed by a 40% decrease in activity after 5 min. The loss of activity is linear with the incorporation of cobalt into PEPCK, resulting in 15-25% activity for the stoichiometric Co3+-PEPCK complex. The incorporation of and inhibition by Co3+ is protected by PEP and GTP (ITP). Treatment of the Co3+-PEPCK complex with beta-mercaptoethanol results in a loss of cobalt and full recovery of activity. The reduction and reactivation are protected by PEP and GTP (ITP). EPR, PRR, circular dichroism, and fluorescence studies all indicate that Co3+ has been selectively incorporated into the cation site of PEPCK, resulting in a catalytically active enzyme-cation species. The substrates form Michaelis complexes with Co3+-PEPCK, and the catalytic reaction occurs as a second sphere complex as previously suggested [Lee & Nowak (1984) Biochemistry 23, 6506); Duffy & Nowak (1985) Biochemistry 24, 1152]. Proteolytic digestion of the Co3+-PEPCK complex and isolation of the cobalt-containing peptide by reverse phase HPLC were performed to identify the location of the cation binding site. From mass, amino acid composition, and sequence analyses of the isolated cobalt-peptide, the region Thr276-Lys301 is responsible for metal chelation. This very homologous region, located in the central portion of PEPCK, contains two highly conserved aspartic acids, Asp295 and Asp296, that are the only feasible metal binding ligands.
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PMID:Formation and characterization of an active phosphoenolpyruvate carboxykinase-cobalt(III) complex. 911 19

The PCK1 gene encoding PEP carboxykinase (Pck1) of the fungal pathogen Candida albicans was isolated and sequenced. The deduced Pck1 protein has high homology to ATP-dependent Pck1 proteins in other species, especially to Pck1 of Saccharomyces cerevisiae (70% homology), but not to GTP-dependent Pck1 proteins. PCK1 transcript levels were efficiently repressed by glucose and derepressed (induced) on gluconeogenetic carbon sources. PCK1 regulation occurs on the level of transcription, as demonstrated by a fusion of the PCK1 promoter to the LAC4 reporter gene, yielding derepressed/repressed expression ratios of > 100. Homologous sequences in the PCK1 promoters of C. albicans and S. cerevisiae were identified. The PCK1 promoter may be useful to efficiently regulate expression and thereby test the function of genes in C. albicans.
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PMID:Sequence and promoter regulation of the PCK1 gene encoding phosphoenolpyruvate carboxykinase of the fungal pathogen Candida albicans. 922 95

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 7.4. Omitting ascorbate or replacing the Fe2+ with Mn2+ or Mg2+ gives no inactivation. Mn2+, Mg2+, or Co2+ at 100-fold molar excess over Fe2+ offered complete protection from Fe2+/ascorbate-induced inactivation. The substrates PEP and GTP, but not OAA, GDP, or CO2, offered full protection from inactivation. The addition of 5 mM EDTA stopped further inactivation of the enzyme. Thermodynamic studies indicate that the inactive enzyme no longer binds Mn2+ but still had high affinity for GTP indicating that the inactivation process was specific for the metal site. A decrease in cysteine content was observed over time following PEPCK treatment with Fe2+ and ascorbate. The apparent first-order rate constant for free sulfhydryl loss (0.085 +/- 0.005 min-1) is similar to the apparent first-order rate constant for inactivation (0.067 +/- 0.005 min-1). Amino acid composition analysis revealed that cysteic acid was generated upon Fe2+/ascorbate addition to PEPCK. Native chicken liver PEPCK has an Mr of 67 kDa. SDS-PAGE of the inactivated enzyme showed the presence of two new bands at 31.7 and 35.3 kDa indicating that PEPCK was specifically cleaved at a single site. The rate of cleavage was slower than the rate of inactivation and fully inactivated enzyme was only 50% cleaved. The Fe2+/ascorbate-catalyzed inactivation was not solely due to protein cleavage. The protein fragments generated by cleavage were separated by C4 reverse phase HPLC. The cleavage exposed a new N-terminus which was identified to be the 35.3 kDa C-terminal half of PEPCK. Sequencing of the fragments indicated that the site of cleavage was between Asp296 and Ile297. These results indicate that Asp296 is involved in metal chelation. This agrees with previous studies [Hlavaty, J. J., & Nowak, T. (1997) Biochemistry 36, 3389-3403] that suggested that Asp295 and Asp296 are involved in metal binding.
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PMID:Affinity cleavage at the metal-binding site of phosphoenolpyruvate carboxykinase. 939 80

This presentation gives an overview about the factors involved in the regulation of gluconeogenesis. Then, based on these regulatory principles, the changes seen in impaired liver function are discussed. Gluconeogenesis from lactate and pyruvate is mediated through pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) activity. The PC mediated pathway depends on substrate supply and on the downregulation of the oxidative pathway for pyruvate. Both enzymes need ATP or GTP and, thus, depend on the cellular energy charge. Tissue anoxia can reduce the energy charge and limit the flow through the PEPCK pathway. Thus, one expects a coupling between reduced splanchnic blood flow, limited oxygen supply to the liver, resulting tissue anoxia, and reduced gluconeogenesis. Conditions are shown, where this coupling exists. Since gluconeogenesis is concentrated in the periportal region of the liver, the local oxygen tension is sufficient under many circumstances to maintain a high glucose production level. Also, the enzyme activity of PEPCK can compensate for long term anoxia. Thus, gluconeogenesis is sufficient in most cases, as seen in critically ill patients. However, this could be associated with a reduction in the perivenous oxygen tension, possibly below critical levels. Beta-adrenergic stimulation increases gluconeogenesis. Examples are shown where this stimulation can overlay the dependency on the oxygen tension and substrate supply. Catecholamines are generally used to stabilize the hemodynamic system. This treatment could limit splanchnic bloodflow and, as a consequence, the oxygen supply to the liver with a simultaneous stimulation of gluconeogenesis and can cause severe anoxia in the perivenous region. These negative side effects of catecholamine treatment should be avoided and the ideal treatment should aim at improving splanchnic flow without stimulation of gluconeogenesis.
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PMID:Gluconeogenesis in patients with impaired liver function. 946 36

Enterocytes from fasted rabbits make glucose from exogenous fructose and dihydroxyacetone at rates of 180 and 91 nmol/min/10(8) cells but do not make glucose from glycerol, aspartate, malate, lactate, alpha-ketoglutarate, glutamate or glutamine. Total activities of phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase in isolated enterocytes are 0.44, 0.60 and 1.90 mumol/min/10(8) cells, and > or = 95% of carboxykinase activity is intramitochondrial. Enterocytes contain marginal glycerol kinase (0.05 mumol/ min/10(8) cells) and essentially no pyruvate carboxylase activities. Enterocyte mitochondria synthesize citrate from exogenous phosphoenolpyruvate and acetylcarnitine at a rate of 2.40 nmol/min/mg protein. Citrate formation is highly dependent on exogenous HCO3 and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricarboxylate. Citrate synthesis is stimulated consistently by GDP and significantly so by GTP. Citrate production is unaffected by ADP or ATP. Enterocytes from fasted-refed rabbits contain activities of 0.05, 0.12, 0.39 and 0.56 mumol/min/mg cytosolic protein of ATP:citrate lyase, NADP:malate dehydrogenase, glucose 6-phosphate dehydrogenase and NADP:isocitrate dehydrogenase. Activities of NADP:malate dehydrogenase, glucose 6-phosphate dehydrogenase and NADP:isocitrate dehydrogenase are significantly higher in enterocytes from fasted-refed rabbits than those from fasted rabbits. Mitochondrial phosphoenolpyruvate carboxykinase in enterocytes in vivo could convert glycolysis-derived phosphoenolpyruvate to oxaloacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA carboxylase, a source of carbon via ATP:citrate lyase and of NADPH via NADP:malate dehydrogenase or NADP:isocitrate dehydrogenase.
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PMID:Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit enterocytes: implications for lipogenesis. 946 72


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