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Biochimica Et Biophysica Acta Sep 2004Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus...
Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus transcription and an extracellular disulfide reductase in angiogenesis. Probing of a developmental expression library from Dictyostelium discoideum with radiolabelled calmodulin led to the isolation of a cDNA encoding a putative calmodulin-binding protein (DdPGK) with 68% sequence similarity to human PGK. Dictyostelium, rabbit and yeast PGKs bound to calmodulin-agarose in a calcium-dependent manner while DdPGK constructs lacking the calmodulin-binding domain (209KPFLAILGGAKVSDKIKLIE228) failed to bind. The calmodulin-binding domain shows 80% identity between diverse organisms and is situated beside the hinge and within the ATP binding domain adjacent to nine mutations associated with PGK deficiency. Calmodulin addition inhibits yeast PGK activity in vitro while the calmodulin antagonist W-7 abrogates this inhibition. Together, these data suggest that PGK activity may be negatively regulated by calcium and calmodulin signalling in eukaryotic cells.
Topics: Amino Acid Sequence; Animals; Calcium; Calmodulin; Calmodulin-Binding Proteins; Conserved Sequence; Dictyostelium; Humans; Molecular Sequence Data; Phosphoglycerate Kinase; Rabbits; Saccharomyces cerevisiae; Sequence Homology, Amino Acid
PubMed: 15363631
DOI: 10.1016/j.bbamcr.2004.08.003 -
Journal of Bacteriology Apr 1988The Zymomonas mobilis gene encoding phosphoglycerate kinase (EC 2.7.3.2), pgk, has been cloned into Escherichia coli and sequenced. It consists of 336 amino acids,...
The Zymomonas mobilis gene encoding phosphoglycerate kinase (EC 2.7.3.2), pgk, has been cloned into Escherichia coli and sequenced. It consists of 336 amino acids, including the N-terminal methionine, with a molecular weight of 41,384. This promoterless gene is located 225 base pairs downstream from the gap gene and is part of the gap operon. Previous studies have shown that the specific activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase do not change coordinately in Z. mobilis, although the two enzymes appear to be under the control of a common promoter. The translated amino acid sequence for the Z. mobilis phosphoglycerate kinase is less conserved than those of eucaryotic genes. A comparison of known sequences for phosphoglycerate kinase revealed a high degree of conservation of structure with 102 amino acid positions being retained by all. In general, the amino acid positions at the boundaries of beta-sheet and alpha-helical regions and those connecting these regions were more highly conserved than the amino acid positions within regions of secondary structure.
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Codon; DNA Restriction Enzymes; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gene Expression Regulation; Genes, Bacterial; Glyceraldehyde-3-Phosphate Dehydrogenases; Gram-Negative Bacteria; Molecular Sequence Data; Operon; Phosphoglycerate Kinase; Sequence Homology, Nucleic Acid; Transcription, Genetic
PubMed: 2832389
DOI: 10.1128/jb.170.4.1926-1933.1988 -
Scientific Reports Oct 2022Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with...
Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype-phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.
Topics: Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Phosphoglycerate Kinase; Protein Denaturation; Protein Folding; Thermodynamics
PubMed: 36229482
DOI: 10.1038/s41598-022-22088-1 -
FEBS Letters Feb 1992E. coli D-glyceraldehyde-3-phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble E. coli 3-phosphoglycerate kinase with a...
E. coli D-glyceraldehyde-3-phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble E. coli 3-phosphoglycerate kinase with a stoichiometry of 1.77 +/- 0.61 kinase molecules per tetramer of the dehydrogenase and an apparent Kd of 1.03 +/- 0.68 microM (10 mM sodium phosphate, 0.15 M NaCl). No interaction was detected between E. coli D-glyceraldehyde-3-phosphate dehydrogenase and rabbit muscle 3-phosphoglycerate kinase. The species-specificity of the bienzyme association made it possible to develop a kinetic approach to demonstrate the functionally significant interaction between E. coli D-glyceraldehyde-3-phosphate dehydrogenase and E. coli 3-phosphoglycerate kinase, which consists of an increase in steady-state rate of the coupled reaction.
Topics: Animals; Enzymes, Immobilized; Glyceraldehyde-3-Phosphate Dehydrogenases; Kinetics; Phosphoglycerate Kinase; Rabbits
PubMed: 1544404
DOI: 10.1016/0014-5793(92)80548-u -
The Biochemical Journal Jul 1993The role of Mg2+ in the binding of ADP and ATP to pig muscle and yeast 3-phosphoglycerate kinases has been studied by equilibrium dialysis. Whereas the Kd of ATP binding...
The role of Mg2+ in the binding of ADP and ATP to pig muscle and yeast 3-phosphoglycerate kinases has been studied by equilibrium dialysis. Whereas the Kd of ATP binding varies between 0.17 and 0.23 mM (S.E.M. 0.03 mM) for both enzymes, independently of the presence of Mg2+, the Kd values for ADP and MgADP binding are in the range 0.18-0.27 mM (S.E.M. 0.04 mM) and 0.05-0.06 mM (S.E.M. 0.01 mM) respectively. Thus Mg2+ exclusively tightens the interaction of ADP, but not of ATP, with the protein molecule. Although the equilibrium dialysis data are consistent with a model possessing a single site for nucleotides, the existence of a much weaker secondary site (with a Kd value at least two orders of magnitude larger) cannot be excluded. The binding of AMP and adenosine to pig muscle 3-phosphoglycerate kinase is weaker than binding of MgATP; the respective Kd values are 0.36 +/- 0.05 mM and 0.65 +/- 0.05 mM. Thus, in addition to the interaction of the alpha-phosphate that is detectable by crystallography [Banks, Blake, Evans, Haser, Rice, Hardy, Merrett and Phillips (1979) Nature (London) 279, 773-777], the beta- and/or gamma-phosphate(s) of MgATP may also interact with the enzyme molecule. The fact that MgADP binds more tightly than ADP is consistent with its stronger inhibition of the reaction catalysed by the enzyme between 3-phosphoglycerate and MgATP. MgADP is a product of this reaction, and inhibits it competitively with both substrates; as an inhibitor its KI is comparable with the Kd found in binding studies. At the same time, the Km value for MgADP in the reverse reaction (0.18 +/- 0.05 mM; mean +/- S.E.M.) is higher than these constants; this may be due either to a different kinetic mechanism in this direction of the enzymic reaction, or to different binding modes of MgADP as inhibitor and as substrate. The reason why inhibition by MgADP is competitive with 3-phosphoglycerate may be that its binding prevents the specific change in conformation that the enzyme undergoes [Harlos, Vas and Blake (1992) Proteins 12, 133-144] when it binds 3-phosphoglycerate.
Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Binding Sites; Dialysis; Kinetics; Magnesium; Molecular Conformation; Muscles; Phosphoglycerate Kinase; Swine
PubMed: 8343139
DOI: 10.1042/bj2930595 -
Phytochemistry Jul 2012Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and...
Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.
Topics: Amino Acid Sequence; Cloning, Molecular; Cytosol; DNA, Complementary; Enzyme Stability; Evolution, Molecular; Gene Expression Regulation, Plant; Helianthus; Isoenzymes; Kinetics; Models, Molecular; Molecular Sequence Data; Phosphoglycerate Kinase; Phylogeny; Plastids; Protein Structure, Tertiary; Protein Transport; Seeds; Sequence Analysis, DNA; Temperature
PubMed: 22552275
DOI: 10.1016/j.phytochem.2012.04.001 -
Cancer Science Nov 2010Phosphoglycerate kinase 1 (PGK-1) is a multifunctional protein that is involved in the glycolytic pathway and the generation of the angiogenesis inhibitor angiostatin....
Phosphoglycerate kinase 1 (PGK-1) is a multifunctional protein that is involved in the glycolytic pathway and the generation of the angiogenesis inhibitor angiostatin. In a previous study, we showed that the overexpression of full-length PGK-1 in Lewis lung carcinoma (LLC-1) can reduce tumor growth in vivo by downregulation of COX-2 expression. Phosphoglycerate kinase 1 has two functional domains: a catalytic domain (CD); and a nucleotide-binding domain (NBD). To identify the functional domain of PGK-1 responsible for its antitumor effects, we evaluated the tumorigenicity of LLC-1 cells overexpressing full-length PGK-1 (LLC-1/PGK), CD (LLC-1/CD), and NBD (LLC-1/NBD). Although no difference in tumor cell growth was observed in vitro, the tumor invasiveness was reduced in the LLC-1/PGK, LLC-1/CD, and LLC-1/NBD cells compared to parental LLC-1 cells in vivo. In addition, in vivo tumor growth retardation by LLC-1/CD and LLC-1/NBD cells was observed, similar to that by LLC-1/PGK cells. However, the reduced stability of COX-2 mRNA and downregulation of the COX-2 protein and its metabolite, prostaglandin E2, was only found in LLC-1/PGK and LLC-1/NBD cells. Low levels of COX-2 were also observed in the tumor mass formed by the modified cells when injected into mice. The results indicate that COX-2 suppression by PGK-1 is independent of its catalytic activity. COX-2 targeting by PGK-1 can be attributed to its NBD and is probably a result of the destabilization of COX-2 gene transcripts brought about by the mRNA-binding property of PGK-1.
Topics: Animals; Binding Sites; Blotting, Western; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclooxygenase 2; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Nucleotides; Phosphoglycerate Kinase; RNA Stability; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Transforming Growth Factor beta1; Tumor Burden
PubMed: 20731664
DOI: 10.1111/j.1349-7006.2010.01691.x -
Proceedings of the National Academy of... Jul 1994Production of the glycoprotein hormone erythropoietin (Epo) in response to hypoxic stimuli is almost entirely restricted to particular cells within liver and kidney, yet... (Comparative Study)
Comparative Study
Production of the glycoprotein hormone erythropoietin (Epo) in response to hypoxic stimuli is almost entirely restricted to particular cells within liver and kidney, yet the transcriptional enhancer lying 3' to the Epo gene shows activity inducible by hypoxia after transfection into a wide variety of cultured cells. The implication of this finding is that many cells which do not produce Epo contain a similar, if not identical, oxygen-regulated control system, suggesting that the same system is involved in the regulation of other genes. We report that the human phosphoglycerate kinase 1 and mouse lactate dehydrogenase A genes are induced by hypoxia with characteristics which resemble induction of the Epo gene. In each case expression is induced by cobalt, but not by cyanide, and hypoxic induction is blocked by the protein-synthesis inhibitor cycloheximide. We show that the relevant cis-acting control sequences are located in the 5' flanking regions of the two genes, and we define an 18-bp element in the 5' flanking sequence of the phosphoglycerate kinase 1 gene which is both necessary and sufficient for the hypoxic response, and which has sequence and protein-binding similarities to the hypoxia-inducible factor 1 binding site within the Epo 3' enhancer.
Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line; Cloning, Molecular; Cobalt; Cyanides; Cycloheximide; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Isoenzymes; L Cells; L-Lactate Dehydrogenase; Liver Neoplasms; Mice; Molecular Sequence Data; Oxygen; Phosphoglycerate Kinase; Promoter Regions, Genetic; Sequence Deletion; Sequence Homology, Nucleic Acid; TATA Box; Transfection; Tumor Cells, Cultured
PubMed: 8022811
DOI: 10.1073/pnas.91.14.6496 -
European Journal of Biochemistry Mar 1975Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A 1%,1Cm 280 equals 6.7; Amax/Amin equals 2.26; e280 equals...
Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A 1%,1Cm 280 equals 6.7; Amax/Amin equals 2.26; e280 equals 31.5 mM(-1) X cm(-1). The enzyme consists of about 420 amino-acid residues and is a slightly acidic protein with an isoelectric point of 6.5 as expected from amino-acid analysis. The most notable features of the chemical composition are two tryptophan, 12 methionine and four half-cystine residues per enzyme molecule. Although phosphoglycerate kinases from mammalian tissues are partially similar to each other, clear differences in serine, glutamic acid, glycine, cysteine, valine, leucine, tyrosine, tryptophan and arginine contents were found. Fingerprinting and column chromatography of tryptic digests of the S-carboxymethylated protein confirm the data of amino-acid analysis. Liver phosphoglycerate kinase is inactivated when modified with either p-chloromercuribenzoate or 5,5'dithio-bis(2-nitrobenzoic acid) (Nbs2). The enzyme has two thiol groups available for reaction with Nbs2 under denaturing conditions, one of which is essential for catalysis. After reduction by NaBH4 four cysteine residues per molecule were determined with Nbs2, sugessting the presence of a disulfide bridge. Using sedimentation equilibrium studies, the molecular weight was found to be 49600. Gel filtration yielded values of 43000-50000. By analytical dodecylsulfate-polyacrylamide gel electrophoresis a molecular weight of 45600 was estimated. Inconsistent with these results in the value 37500 obtained by thin-layer gel chromatography in 6 M guanidine-HCl. Sedimentation velocity experiments revealed a sedimentation coefficient s20,w equals 3.4 S. The Stokes radius was 2.77 nm, the partial specific volume v 0.747 ml x g(-1). The diffusion coefficient was found to be 76.9 mum2 x s(-1) by analytical gel filtration. From these data a molecular weight of 44000 was calculated. Other physical constants of bovine-liver phosphoglycerate kinase are: frictional ratio f/f0 equals 1.18, axial ratio equals 3.3, maximal degree of hydration equals 0.1 g per g of protein. Bovine-layer phosphoglycerate kinase could not be dissociated into smaller subunits by treatments which have caused dissociation of various other proteins (8 M urea, 6 M guanidine-HCl, dodecyl sulfate, carboxymethylation, maleylation). All experiments strongly support the lack of subunit structure of the enzyme. Some characteristics of bovine-liver phosphoglycerate kinase are compared with the corresponding proteins from rabbit muscle, yeast and human erythrocytes.
Topics: Amino Acids; Animals; Binding Sites; Cattle; Chloromercuribenzoates; Disulfides; Dithionitrobenzoic Acid; Liver; Macromolecular Substances; Mathematics; Molecular Weight; Organ Specificity; Peptide Fragments; Phosphoglycerate Kinase; Protein Binding; Protein Conformation; Species Specificity; Spectrophotometry, Ultraviolet; Sulfhydryl Compounds
PubMed: 1175587
DOI: 10.1111/j.1432-1033.1975.tb03992.x -
International Journal of Molecular... Oct 2022An investigation of alternatives to immune checkpoint inhibitors for advanced urothelial cancer (aUC), with biologic information, is urgently needed. Clinical data for...
Gemcitabine-Paclitaxel Chemotherapy for Patients with Advanced Urothelial Cancer Refractory to Cisplatin-Based Chemotherapy: Predictive Role of PGK1 for Treatment Response to Cytotoxic Chemotherapy.
An investigation of alternatives to immune checkpoint inhibitors for advanced urothelial cancer (aUC), with biologic information, is urgently needed. Clinical data for 53 patients who received gemcitabine-paclitaxel therapy (GP) as 2nd-line chemotherapy for aUC refractory to platinum-based chemotherapy were retrospectively reviewed. The efficacy and tolerability of GP were evaluated, and the predictive value of phosphoglycerate kinase 1 (PGK1) immunostained in surgical specimens was investigated for treatment outcomes in 1st- and 2nd-line chemotherapy. GP was associated with an objective response rate of 35.8% and a median overall survival duration of 12.3 months. Multivariate analysis showed that PS2 and 1st- and 2nd-line non-response are independent predictors of worse progression-free survival and that PS2 and 1st-line non-response are independent predictors of worse overall survival. Adverse events were manageable, and no therapy-related deaths occurred. Non-response rates to 1st-line chemotherapy were significantly higher in patients with a high expression of PGK1 in the nucleus than in those with low expression ( = 0.006). Our study demonstrates the efficacy and tolerability of 2nd-line GP for patients with aUC who are refractory to platinum-based chemotherapy. Moreover, PGK1 in the nucleus was predictive values for resistance to platinum-based chemotherapy in aUC.
Topics: Humans; Cisplatin; Retrospective Studies; Immune Checkpoint Inhibitors; Phosphoglycerate Kinase; Antineoplastic Combined Chemotherapy Protocols; Paclitaxel; Carcinoma, Transitional Cell; Platinum; Biological Products; Gemcitabine
PubMed: 36292976
DOI: 10.3390/ijms232012119