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Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells.The Journal of Biological Chemistry May 2020Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell...
Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.
Topics: A549 Cells; Diphosphoglyceric Acids; Glucose; Glyceric Acids; Glycolysis; HeLa Cells; Humans; Kinetics; Lactic Acid; Neoplasm Proteins; Neoplasms; Phosphoglycerate Kinase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 32217690
DOI: 10.1074/jbc.RA119.012312 -
International Journal of Molecular... Aug 2021Restenosis is a common vascular complication after balloon angioplasty. Catheter balloon inflation-induced transient ischemia (hypoxia) of local arterial tissues plays a...
Restenosis is a common vascular complication after balloon angioplasty. Catheter balloon inflation-induced transient ischemia (hypoxia) of local arterial tissues plays a pathological role in neointima formation. Phosphoglycerate kinase 1 (PGK1), an adenosine triphosphate (ATP)-generating glycolytic enzyme, has been reported to associate with cell survival and can be triggered under hypoxia. The purposes of this study were to investigate the possible role and regulation of PGK1 in vascular smooth muscle cells (VSMCs) and balloon-injured arteries under hypoxia. Neointimal hyperplasia was induced by a rat carotid artery injury model. The cellular functions and regulatory mechanisms of PGK1 in VSMCs were investigated using small interfering RNAs (siRNAs), chemical inhibitors, or anaerobic cultivation. Our data indicated that protein expression of PGK1 can be rapidly induced at a very early stage after balloon angioplasty, and the silencing PGK1-induced low cellular energy circumstance resulted in the suppressions of VSMC proliferation and migration. Moreover, the experimental results demonstrated that blockage of PDGF receptor-β (PDGFRB) or its downstream pathway, the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) axis, effectively reduced hypoxia-induced factor-1 (HIF-1α) and PGK1 expressions in VSMCs. In vivo study evidenced that PGK1 knockdown significantly reduced neointima hyperplasia. PGK1 was expressed at the early stage of neointimal formation, and suppressing PGK1 has a potential beneficial effect for preventing restenosis.
Topics: Angioplasty, Balloon; Animals; Carotid Artery Injuries; Cell Movement; Cells, Cultured; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Phosphoglycerate Kinase; Rats; Rats, Sprague-Dawley; Signal Transduction
PubMed: 34445528
DOI: 10.3390/ijms22168822 -
Proceedings of the National Academy of... Feb 2024The drug terazosin (TZ) binds to and can enhance the activity of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and can increase ATP levels. That finding...
The drug terazosin (TZ) binds to and can enhance the activity of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and can increase ATP levels. That finding prompted studies of TZ in Parkinson's disease (PD) in which decreased neuronal energy metabolism is a hallmark feature. TZ was neuroprotective in cell-based and animal PD models and in large epidemiological studies of humans. However, how TZ might increase PGK1 activity has remained a perplexing question because structural data revealed that the site of TZ binding to PGK1 overlaps with the site of substrate binding, predicting that TZ would competitively inhibit activity. Functional data also indicate that TZ is a competitive inhibitor. To explore the paradoxical observation of a competitive inhibitor increasing enzyme activity under some conditions, we developed a mass action model of TZ and PGK1 interactions using published data on PGK1 kinetics and the effect of varying TZ concentrations. The model indicated that TZ-binding introduces a bypass pathway that accelerates product release. At low concentrations, TZ binding circumvents slow product release and increases the rate of enzymatic phosphotransfer. However, at high concentrations, TZ inhibits PGK1 activity. The model explains stimulation of enzyme activity by a competitive inhibitor and the biphasic dose-response relationship for TZ and PGK1 activity. By providing a plausible mechanism for interactions between TZ and PGK1, these findings may aid development of TZ or other agents as potential therapeutics for neurodegenerative diseases. The results may also have implications for agents that interact with the active site of other enzymes.
Topics: Humans; Animals; Phosphoglycerate Kinase; Prazosin; Parkinson Disease; Glycolysis
PubMed: 38377207
DOI: 10.1073/pnas.2318956121 -
Microbiology (Reading, England) Nov 2017In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of...
In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 °C and at 70 °C. At 30 °C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 °C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 °C and at 70 °C, however, at 70 °C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.
Topics: Enzyme Stability; Gluconeogenesis; Glyceraldehyde 3-Phosphate; Glyceraldehyde-3-Phosphate Dehydrogenases; Glyceric Acids; Half-Life; Hot Temperature; Kinetics; Models, Statistical; Phosphoglycerate Kinase; Recombinant Proteins; Saccharomyces cerevisiae; Substrate Cycling; Sulfolobus solfataricus; Thermodynamics
PubMed: 28982396
DOI: 10.1099/mic.0.000542 -
Plant Physiology Feb 2018In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in...
In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in gluconeogenesis and the Calvin-Benson cycle. In the databases, we found three genes that encode putative PGKs. Arabidopsis () PGK1 was localized exclusively in the chloroplasts of photosynthetic tissues, while PGK2 was expressed in the chloroplast/plastid of photosynthetic and nonphotosynthetic cells. PGK3 was expressed ubiquitously in the cytosol of all studied cell types. Measurements of carbohydrate content and photosynthetic activities in PGK mutants and silenced lines corroborated that PGK1 was the photosynthetic isoform, while PGK2 and PGK3 were the plastidial and cytosolic glycolytic isoforms, respectively. The knockdown mutant displayed reduced growth, lower photosynthetic capacity, and starch content. The knockout mutant was characterized by reduced growth but higher starch levels than the wild type. The double mutant was bigger than and displayed an intermediate phenotype between the two single mutants in all measured biochemical and physiological parameters. Expression studies in mutants showed that and were down-regulated in and , respectively. These results indicate that the down-regulation of photosynthetic activity could be a plant strategy when glycolysis is impaired to achieve metabolic adjustment and optimize growth. The double mutants of and the triose-phosphate transporter ( displayed a drastic growth phenotype, but they were viable. This implies that other enzymes or nonspecific chloroplast transporters could provide 3-phosphoglycerate to the cytosol. Our results highlight both the complexity and the plasticity of the plant primary metabolic network.
Topics: Arabidopsis; Arabidopsis Proteins; Cytosol; Gene Expression Regulation, Plant; Glyceric Acids; Metabolomics; Multigene Family; Mutation; Phosphoglycerate Kinase; Plant Components, Aerial; Plant Roots; Plants, Genetically Modified; Plastics
PubMed: 28951489
DOI: 10.1104/pp.17.01227 -
Journal of Biochemistry Jun 1979(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was... (Comparative Study)
Comparative Study
(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.
Topics: Amino Acids; Circular Dichroism; Geobacillus stearothermophilus; Kinetics; Molecular Weight; Phosphoglycerate Kinase; Protein Conformation; Species Specificity; Spectrometry, Fluorescence; Substrate Specificity; Thermus
PubMed: 457645
DOI: 10.1093/oxfordjournals.jbchem.a132480 -
British Journal of Cancer Apr 2015Phosphoglycerate kinase-1 (PGK1) has been recently documented in various malignancies; however, the molecular mechanisms of the variable PGK1 expression and its clinical...
BACKGROUND
Phosphoglycerate kinase-1 (PGK1) has been recently documented in various malignancies; however, the molecular mechanisms of the variable PGK1 expression and its clinical significance in terms of survival status remain unclear.
METHODS
Real-time quantitative PCR (real-time qPCR) and western blotting were used to verify PGK1 expression in 46 fresh breast cancer tissues and matched normal tissues. A tissue microarray (TMA) comprising 401 breast cancer tissues and 123 matched normal tissues was investigated by immunohistochemistry for PGK1 expression. Then, the correlation between PGK1 expression and the clinicopathologic features was analysed.
RESULTS
PGK1 mRNA and protein expression were significantly increased in breast cancer tissues compared with that in normal breast tissues. High PGK1 expression was significantly associated with higher histologic grade (P=0.009) and positive status of ER (P=0.004), Her-2 (P=0.026) and P53 (P=0.012). High levels of PGK1 expression were associated with worse overall survival (OS, P=0.02). Furthermore, patients who underwent paclitaxel chemotherapy with high levels PGK1 expression had shorter OS than did those with low levels of PGK1 expression (P<0.001). Multivariate analysis indicated that PGK1 (P=0.001) was an independent predictor in the patients treated with paclitaxel.
CONCLUSIONS
PGK1 is a prognostic biomarker of chemoresistance to paclitaxel treatment in breast cancer.
Topics: Adult; Aged; Antineoplastic Agents, Phytogenic; Biomarkers, Tumor; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Paclitaxel; Phosphoglycerate Kinase; Prognosis
PubMed: 25867275
DOI: 10.1038/bjc.2015.114 -
Cell Death & Disease Feb 2023Glycolysis is the most predominant metabolic reprogramming of pancreatic cancer (PC), the underlying mechanism of which in PC cells remains unclear. In this study, we...
Glycolysis is the most predominant metabolic reprogramming of pancreatic cancer (PC), the underlying mechanism of which in PC cells remains unclear. In this study, we found for the first time that KIF15 promotes the glycolytic capacity of PC cells and PC tumor growth. Moreover, the expression of KIF15 was negatively correlated with the prognosis of PC patients. The ECAR and OCR measurements indicated that KIF15 knockdown significantly impaired the glycolytic capacity of PC cells. Western blotting demonstrated that the expression of glycolysis molecular markers decreased rapidly after the knockdown of KIF15. Further experiments revealed that KIF15 promoted the stability of PGK1 and its effect on PC cell glycolysis. Interestingly, the overexpression of KIF15 impaired the ubiquitination level of PGK1. To investigate the underlying mechanism by which KIF15 regulates the function of PGK1, we performed mass spectrometry (MS). The MS and Co-IP assay indicated that KIF15 recruited and enhanced the binding between PGK1 and USP10. The ubiquitination assay verified that KIF15 recruited and promoted the effect of USP10 on PGK1, thereby deubiquitinating PGK1. Through the construction of KIF15 truncators, we found that KIF15 is bound to PGK1 and USP10 through its coil2 domain. Together, our study demonstrated for the first time that KIF15 enhances the glycolytic capacity of PC through the recruitment of USP10 and PGK1, and that the KIF15/USP10/PGK1 axis may serve as an effective therapeutic agent for PC.
Topics: Humans; Pancreatic Neoplasms; Ubiquitination; Glycolysis; Cell Line, Tumor; Cell Proliferation; Kinesins; Ubiquitin Thiolesterase; Phosphoglycerate Kinase
PubMed: 36807568
DOI: 10.1038/s41419-023-05679-2 -
Angewandte Chemie (International Ed. in... Apr 2017The 1994 structure of a transition-state analogue with AlF and GDP complexed to G1α, a small G protein, heralded a new field of research into the structure and... (Review)
Review
The 1994 structure of a transition-state analogue with AlF and GDP complexed to G1α, a small G protein, heralded a new field of research into the structure and mechanism of enzymes that manipulate the transfer of phosphoryl (PO ) groups. The number of enzyme structures in the PDB containing metal fluorides (MF ) as ligands that imitate either a phosphoryl or a phosphate group was 357 at the end of 2016. They fall into three distinct geometrical classes: 1) Tetrahedral complexes based on BeF that mimic ground-state phosphates; 2) octahedral complexes, primarily based on AlF , which mimic "in-line" anionic transition states for phosphoryl transfer; and 3) trigonal bipyramidal complexes, represented by MgF and putative AlF moieties, which mimic the geometry of the transition state. The interpretation of these structures provides a deeper mechanistic understanding into the behavior and manipulation of phosphate monoesters in molecular biology. This Review provides a comprehensive overview of these structures, their uses, and their computational development.
Topics: Aluminum Compounds; Animals; Fluorides; Humans; Models, Molecular; Phosphates; Phosphoglycerate Kinase; Phosphoric Monoester Hydrolases
PubMed: 27862756
DOI: 10.1002/anie.201606474 -
Proceedings of the National Academy of... Oct 2010We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function, and folding landscape of phosphoglycerate...
We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function, and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal), and Sph (spherical compact). With an adjustment for viscosity, crowded wild-type PGK and fluorescent PGK are about 15 times or more active in 200 mg/ml Ficoll than in aqueous solution. Our results suggest a previously undescribed solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: Rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates. We also examine T-jump unfolding of PGK as a function of crowding experimentally. We uncover a nonmonotonic folding relaxation time vs. Ficoll concentration. Theory and modeling explain why an optimum concentration exists for fastest folding. Below the optimum, folding slows down because the unfolded state is stabilized relative to the transition state. Above the optimum, folding slows down because of increased viscosity.
Topics: Binding Sites; Computer Simulation; Ficoll; Fluorescence Resonance Energy Transfer; Kinetics; Models, Chemical; Models, Molecular; Phosphoglycerate Kinase; Protein Conformation; Protein Folding; Temperature; Viscosity; Yeasts
PubMed: 20921368
DOI: 10.1073/pnas.1006760107