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)

1. A simple procedure for the isolation of morphologically intact, metabolically viable sheep liver parenchymal cells is described in detail. 2. The method is based on the initial treatment of fresh liver slices with collagenase and hyaluronidase. 3. The cell preparation was studied with respect to membrane permeability, potassium content, ATP/ADP ratio, adenylate content, and gluconeogenic capacity with respect to various substrates. 4. Data are present with respect to the distribution of phosphoenolpyruvate carboxykinase in isolated cells and whole sheep liver. 5. The results are compared, where possible, with data currently available from isolated perfused sheep liver and multi-catheterised animals.
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PMID:Preparation and biochemical characterisation of isolated parenchymal cells from adult sheep liver. 83 5

The metabolic pathways for the interconversion of oxalacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis form an interlocking system (Scheme 1) that would appear to require complex regulatory mechanisms to permit a proper flow of metabolites through the pathways and to prevent futile cycling. Oxalacetate decarboxylase (I in Scheme 1), P-enolpyruvate synthase (II), P-enolpyruvate carboxylase (III), and pyruvate kinase (V) are constitutive enzymes in this organism. Pyruvate carboxylase (VI) is inducible and has its highest activity in cells grown on glucose or lactate, moderate activity in cells grown on acetate, citrate, or glutamate, and virtually no activity in aspartate-grown cells. P-enolpyruvate carboxykinase (IV) was not detected. The presence of these five enzymes in a single cell has not been previously reported. In Scheme 1, three futile cycles are possible: the simultaneous operation of Reactions I and VI; of Reactions II and V; or of I, II, and III. An examination of the regulatory properties of the individual enzymes after partial purification offers support for the hypothesis of an intricate regulatory system. Oxalacetate decarboxylase (I) is inhibited by acetyl-CoA; phosphoenolpyruvate carboxylase (III) is activated by acetyl-CoA and ADP and inhibited by aspartate; phosphoenolpyruvate synthase (II) is inhibited by 5'-AMP and phosphoenolpyruvate; and pyruvate kinase (V) is activated by 5'-AMP and 2 keto, 3-deoxy,6-phosphogluconate and inhibited by ATP. The presence of metabolites with reciprocal but reinforcing functions is noteworthy. As an example, acetyl-CoA both inhibits the breakdown of oxalacetate and stimulates its formation. Only pyruvate carboxylase appears to be regulated by the carbon substrates of the growth medium.
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PMID:Novel enzymic machinery for the metabolism of oxalacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis. 83 16

Electrophoretically homogeneous phosphoenolpyruvate carboxykinase (EC 4.1.1.49) from Saccharomyces cerevisiae was obtained in high yields by means of a two-step purification procedure consisting of ion-exchange chromatography and affinity chromatography on adenosine 5'-monophosphate-Sepharose 4B. In the latter step the binding of the enzyme to the resin specifically required the presence of Mn2+. The enzyme was eluted when Mn2+ was removed by addition of ethylenediaminetetraacetate to the elution buffer. Homogeneity, molecular weight, and subunit composition of phosphoenolpyruvate carboxykinase were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. A factor which caused an underestimation of the enzyme activity in crude extracts was identified as adenylate kinase. Finally, a method is proposed for the enzymatic assay of bicarbonate using a purified phosphoenolpyruvate carboxykinase preparation.
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PMID:Purification of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae and its use for bicarbonate assay. 388 83

Pyruvate kinase (EC 2.7.1.40) from Azotobacter vinelandii responds sharply to the adenylate energy charge, with a decrease in activity at high values of charge, as expected for an enzyme of an adenosine triphosphate-regenerating sequence. Glycolytic intermediates, especially glucose 6-phosphate, fructose 6-phosphate, and fructose-1,6-diphosphate, strongly stimulate the reaction and overcome the inhibition caused by high values of energy charge. Thus, the properties of this enzyme depend on interaction between energy charge and the concentrations of hexose phosphates. The properties of pyruvate kinase, together with those of phosphoenolpyruvate carboxylase, aspartokinase, and citrate synthase, seem adapted to provide appropriate partitioning of phosphoenolpyruvate between competing pathways in response to metabolic need.
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PMID:Regulation at the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: pyruvate kinase. 555 41

Unusual guanosine nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp, also known as MSI) and guanosine 5'-diphosphate 3'-monophosphate (ppGp, also known as MSIII) accumulate to high concentrations in wild-type cells of Escherichia coli during amino acid starvation. We reported here that both nucleotides strongly inhibit the activity of enzymes IMP dehydrogenase and adenylosuccinate synthetase, the first enzymes of the guanylate and adenylate biosynthetic pathways. In both cases, ppGP (MSII) is a stronger inhibitor than ppGpp (MSI). On the other hand, these two nucleotides exhibited opposite effects on the activity of phosphoenolpyruvate carboxylase, the enzyme that utilizes phosphoenolpyruvate. At their respective physiological concentrations, the activity of phosphoenolpyruvate carboxylase is activated by ppGpp and inhibited by ppGp.
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PMID:Effect of unusual guanosine nucleotides on the activities of some Escherichia coli cellular enzymes. 611 28

Clostridium symbiosum pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP). The nucleotide binding site of this enzyme was labeled using the photoaffinity reagent [32P]-8-azidoadenosine 5'-triphosphate ([32P]-8-azidoATP). Subtilisin cleavage of the [alpha-32P]-8-azidoATP-photolabeled PPDK into domain-sized fragments, prior to SDS-PAGE analysis, allowed us to identify two sites of modification: one between residues 1 and 226 and the other between residues 227 and 334. Saturation of the ATP binding site with adenylyl imidodiphosphate afforded protection against photolabeling. Next, small peptide fragments of [gamma-32P]- 8-azidoATP-photolabeled PPDK were generated by treating the denatured protein with trypsin or alpha-chymotrypsin. A pair of overlapping radiolabeled peptide fragments were separated from the two digests, DMQDMEFTIEEGK (positions 318-330 in trypsin-treated PPDK) and RDMQDMEFTIEEGKL (positions 317-331 in alpha-chymotrypsin-treated PPDK), thus locating one of the positions of covalent modification. Next, catalysis by site-directed mutants generated by amino acid replacement of invariant residues of the PPDK N-terminal domain was tested. K163L, D168A, D170A, D175A, K177L, and G248I PPDK mutants retained substantial catalytic activity while G254I, R337L, and E323L PPDK mutants were inhibited. Comparison of the steady-state kinetic constants measured (at pH 6.8, 25 degrees C) for wild-type PPDK (kcat = 36 s-1, AMPK(m) = 7 microM, PP(i)K(m) = 70 microM, PEPK(m) = 27 microM) to those of R337L PPDK (kcat = 2 s-1, AMPK(m) = 85 microM, PP(i)K(m) = 3700 microM, PEPK(m) = 6 microM) and G254I PPDK (kcat = 0.1 s-1, AMPK(m) = 1300 microM, PP(i)K(m) = 1200 microM, PEPK(m) = 12 microM) indicated impaired catalysis of the nucleotide partial reaction (E.ATP.P(i) --> E-PP.AMP.P(i) --> E-P.AMP.PP(i) in these mutants. The single turnover reactions of [32P]PEP to [32P]E-P.pyruvate catalyzed by the PPDK mutants were shown to be comparable to those of wild-type PPDK. In contrast, the formation of [32P]E-PP/[32P]E-P in single turnover reactions of [beta-32P]ATP/P(i) was significantly inhibited. Finally, the location of the adenosine 5'-diphosphate binding site within the nucleotide binding domain of D-alanine-D-alanine ligase, a structural homologue of the PPDK N-terminal domain [Herzberg, O. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 2652-2657] indicates, by analogy, the location of the nucleotide binding site in PPDK. Residues G254, R337, and E323 as well as the site of photoaffinity labeling are located within this region.
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PMID:Determination of the nucleotide binding site within Clostridium symbiosum pyruvate phosphate dikinase by photoaffinity labeling, site-directed mutagenesis, and structural analysis. 867 15

In 1988, insulin-like growth factor-binding protein-1 (IGFBP-1) became the first characterized member of a group of structurally related soluble proteins which specifically bind and modulate the actions of the IGFs. Since then, a wealth of information has accumulated regarding the physiology of this dynamic serum protein. In this review, we update our 1993 summary (Lee PDK et al. Proc Soc Exp Biol Med 204:4-29) of the status of IGFBP-1 research. The IGFBP-1 protein sequence contains 12 N-terminal and 6 C-terminal cysteine residues which are conserved in other mammalian IGFBP-1 sequences and amongst other IGFBPs; both of the cysteine-rich regions are required for optimal IGF binding. The nonconserved IGFBP-1 midregion may act as both a hinge which defines ligand binding characteristics and as a specific target for protease activity. Integrin-binding and phosphorylation sites within the IGFBP-1 sequence have functional significance in vitro, but their physiologic relevance in vivo have not been defined. The human IGFBP-1 and IGFBP-3 genes are contiguous and located in close proximity to the homeobox A (HOXA) gene cluster on chromosome 7. The other IGFBP genes, located on chromosomes 2, 12, and 17, are also associated with HOX clusters, suggesting evolutionary linkage of the IGFBP and HOX gene families. Similarities between the hIGFBP-1 and phosphoenolpyruvate kinase (PEPCK) promoters, including regions conferring insulin, glucocorticoid, and cyclic adenosine-monophosphate responses, are consistent with our previous hypothesis that IGFBP-1 is involved in regulation of glucose metabolism. The tissue-specific patterns of IGFBP-1 gene expression in liver, kidney, decidua, and ovary may be due to stimulation of IGFBP-1 transcription by hepatic nuclear factor 1 (HNF1) proteins. Clinical and basic studies of IGFBP-1 physiology have been aided by several recently developed assay methods. Numerous investigations have confirmed that insulin, via inhibition of IGFBP-1 transcription, is the primary determinant of IGFBP-1 expression both in vitro and in vivo. IGF-I and IGF-II also have specific inhibitory effects on IGFBP-1 expression. Glucocorticoids and cAMP stimulate IGFBP-1 transcription, but these effects are observed only in conditions of low or absent insulin effect. Other stimulants of IGFBP-1 expression include thyroid hormones and epidermal growth factor. Phorbol ester stimulation of IGFBP-1 expression can supersede the effects of insulin in vitro;however, the mechanism and in vivo correlates of this effect have not been determined. Cytokines and, perhaps, growth hormones may affect IGFBP-1 expression, perhaps by altering the regulatory actions of insulin; this effect may have important clinical relevance. IGFBP-1 expression is upregulated in liver and (nonhuman) kidney during postinjury regeneration. The IGF-inhibitory actions of IGFBP-1 has been confirmed by numerous in vitro studies and several in vivo animal investigations, including administration of recombinant IGFBP-1 and IGFBP-1 transgenic models. IGFBP-1 has been shown to inhibit somatic linear growth, weight gain, tissue growth, and glucose metabolism. Moreover, IGFBP-1 appears to be a primary determinant of free IGF-I levels in serum. Excess levels of IGFBP-1 may contribute to growth failure in intrauterine growth restriction and in pediatric chronic renal failure, while low IGFBP-1 levels are associated with obesity and with cardiovascular risk factors in insulin resistance syndromes. Serum IGFBP-1 measurements may be useful biochemical marker in these pathologic conditions. IGFBP-1 is expressed in decidualized stromal cells of the uterine endometrium and in ovarian granulosa cells. IGFBP-1, together with IGFs, insulin, ovarian steroids, cytokines, and other factors, is involved in a complex system which regulates menstrual cycles, ovulation, decidualization, blastocyst implantation, and fetal growth. (ABSTRACT TRUNCATED)
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PMID:Insulin-like growth factor binding protein-1: recent findings and new directions. 940 39

The histidine containing phospho carrier protein (HPr) kinase/phosphatase is involved in carbon catabolite repression, mainly in Gram-positive bacteria. It is a bifunctional enzyme that phosphorylates Ser-46-HPr in an ATP-dependent reaction and dephosphorylates P-Ser-46-HPr. X-ray analysis of the full-length crystalline enzyme from Staphylococcus xylosus at a resolution of 1.95 A shows the enzyme to consist of two clearly separated domains that are assembled in a hexameric structure resembling a three-bladed propeller. The N-terminal domain has a betaalphabeta fold similar to a segment from enzyme I of the sugar phosphotransferase system and to the uridyl-binding portion of MurF; it is structurally organized in three dimeric modules exposed to form the propeller blades. Two unexpected phosphate ions associated with highly conserved residues were found in the N-terminal dimeric interface. The C-terminal kinase domain is similar to that of the Lactobacillus casei enzyme and is assembled in six copies to form the compact central hub of the propeller. Beyond previously reported similarity with adenylate kinase, we suggest evolutionary relationship with phosphoenolpyruvate carboxykinase. In addition to a phosphate ion in the phosphate-binding loop of the kinase domain, we have identified a second phosphate-binding site that, by comparison with adenylate kinases, we believe accommodates a product/substrate phosphate, normally covalently linked to Ser-46 of HPr. Thus, we propose that our structure represents a product/substrate mimic of the kinase/phosphatase reaction.
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PMID:Structure of the full-length HPr kinase/phosphatase from Staphylococcus xylosus at 1.95 A resolution: Mimicking the product/substrate of the phospho transfer reactions. 1190 9

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

The effects of adenine nucleotides on phosphoenolypyruvate carboxylase were investigated using purified enzyme from the CAM plant, Crassula argentea. At 1 millimolar total concentration and with limiting phosphoenolpyruvate, AMP had a stimulatory effect, lowering the K(m) for phosphoenolpyruvate, ADP caused less stimulation, and ATP decreased the activity by increasing the K(m) for phosphoenolpyruvate. Activation by AMP was not additive to the stimulation by glucose 6-phosphate. Furthermore, AMP increased the K(a) for glucose 6-phosphate. Inhibition by ATP was competitive with phosphoenolpyruvate. In support of the kinetic data, fluorescence binding studies indicated that ATP had a stronger effect than AMP on phosphoenolpyruvate binding, while AMP was more efficient in reducing glucose 6-phosphate binding. As free Mg(2+) was held constant and saturating, these effects cannot be ascribed to Mg(2+) chelation. Accordingly, the enzyme response to the adenylate energy charge was basically of the "R" type (involving enzymes of ATP regenerating sequences) according to D. E. Atkinson's (1968 Biochemistry 7: 4030-4034) concept of energy charge regulation. The effect of energy charge was abolished by 1 millimolar glucose 6-phosphate. Levels of glucose 6-phosphate and of other putative regulatory compounds of phosphoenolpyruvate carboxylase were determined in total leaf extracts during a day-night cycle. The level of glucose 6-phosphate rose at night and dropped sharply during the day. Such a decrease in glucose 6-phosphate concentration could permit an increased control of phosphoenolpyruvate carboxylase by energy charge during the day.
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PMID:The Effect of Adenine Nucleotides on Purified Phosphoenolpyruvate Carboxylase from the CAM Plant Crassula argentea. 1666 57


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