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Acta Crystallographica. Section F,... Oct 2011Phosphoglycerate kinase (PGK) is a widespread two-domain enzyme that plays a critical role in the glycolytic pathway. Several glycolytic enzymes from streptococci have...
Phosphoglycerate kinase (PGK) is a widespread two-domain enzyme that plays a critical role in the glycolytic pathway. Several glycolytic enzymes from streptococci have been identified as surface-exposed proteins that are involved in streptococcal virulence by their ability to bind host proteins. This binding allows pneumococcal cells to disseminate through the epithelial and endothelial layers. Crystallization of PGK from Streptococcus pneumoniae yielded orthorhombic crystals (space group I222, unit-cell parameters a = 62.73, b = 75.38, c = 83.63 Å). However, the unit cell of these crystals was not compatible with the presence of full-length PGK. Various analytical methods showed that only the N-terminal domain of PGK was present in the I222 crystals. The ternary complex of PGK with adenylyl imidodiphosphate (AMP-PNP) and 3-phospho-D-glycerate (3PGA) produced monoclinic crystals (space group P2(1), unit-cell parameters a = 40.35, b = 78.23, c = 59.03 Å, β = 96.34°). Molecular replacement showed that this new crystal form contained full-length PGK, thereby indicating the relevance of including substrates in order to avoid proteolysis during the crystallization process.
Topics: Crystallization; Crystallography, X-Ray; Phosphoglycerate Kinase; Streptococcus pneumoniae
PubMed: 22102049
DOI: 10.1107/S1744309111030922 -
Cellular & Molecular Biology Letters Jan 2022Osteosarcoma (OS) is a common primary bone malignancy. Long noncoding RNA HCG18 is known to play an important role in a variety of cancers. However, its role in OS and...
BACKGROUND
Osteosarcoma (OS) is a common primary bone malignancy. Long noncoding RNA HCG18 is known to play an important role in a variety of cancers. However, its role in OS and relevant molecular mechanisms are unclear.
METHODS
Real-time quantitative PCR was performed to determine the expression of target genes. Function experiments showed the effects of HCG18 and miR-365a-3p on OS cell growth.
RESULTS
HCG18 expression was increased in OS cell lines. Moreover, in vitro and in vivo experiments demonstrated that HCG18 knockdown inhibited OS cell proliferation. Mechanistically, HCG18 was defined as a competing endogenous RNA by sponging miR-365a-3p, thus elevating phosphoglycerate kinase 1 (PGK1) expression by directly targeting its 3'UTR to increase aerobic glycolysis.
CONCLUSION
HCG18 promoted OS cell proliferation via enhancing aerobic glycolysis by regulating the miR-365a-3p/PGK1 axis. Therefore, HCG18 may be a potential target for OS treatment.
Topics: Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; MicroRNAs; Osteosarcoma; Phosphoglycerate Kinase; RNA, Long Noncoding
PubMed: 34991445
DOI: 10.1186/s11658-021-00304-6 -
Proceedings of the National Academy of... Dec 2021Glycolysis plays a fundamental role in energy production and metabolic homeostasis. The intracellular [adenosine triphosphate]/[adenosine diphosphate] ([ATP]/[ADP])...
Glycolysis plays a fundamental role in energy production and metabolic homeostasis. The intracellular [adenosine triphosphate]/[adenosine diphosphate] ([ATP]/[ADP]) ratio controls glycolytic flux; however, the regulatory mechanism underlying reactions catalyzed by individual glycolytic enzymes enabling flux adaptation remains incompletely understood. Phosphoglycerate kinase (PGK) catalyzes the reversible phosphotransfer reaction, which directly produces ATP in a near-equilibrium step of glycolysis. Despite extensive studies on the transcriptional regulation of PGK expression, the mechanism in response to changes in the [ATP]/[ADP] ratio remains obscure. Here, we report a protein-level regulation of human PGK (hPGK) by utilizing the switching ligand-binding cooperativities between adenine nucleotides and 3-phosphoglycerate (3PG). This was revealed by nuclear magnetic resonance (NMR) spectroscopy at physiological salt concentrations. MgADP and 3PG bind to hPGK with negative cooperativity, whereas MgAMPPNP (a nonhydrolyzable ATP analog) and 3PG bind to hPGK with positive cooperativity. These opposite cooperativities enable a shift between different ligand-bound states depending on the intracellular [ATP]/[ADP] ratio. Based on these findings, we present an atomic-scale description of the reaction scheme for hPGK under physiological conditions. Our results indicate that hPGK intrinsically modulates its function via ligand-binding cooperativities that are finely tuned to respond to changes in the [ATP]/[ADP] ratio. The alteration of ligand-binding cooperativities could be one of the self-regulatory mechanisms for enzymes in bidirectional pathways, which enables rapid adaptation to changes in the intracellular environment.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Catalysis; Catalytic Domain; Escherichia coli; Gene Expression Regulation, Enzymologic; Glyceric Acids; Glycolysis; Humans; Models, Molecular; Phosphoglycerate Kinase; Protein Binding; Protein Conformation
PubMed: 34893542
DOI: 10.1073/pnas.2112986118 -
Molecules (Basel, Switzerland) Jun 2017Inhibition of apoptosis is a potential therapy to treat human diseases such as neurodegenerative disorders (e.g., Parkinson's disease), stroke, and sepsis. Due to the...
Inhibition of apoptosis is a potential therapy to treat human diseases such as neurodegenerative disorders (e.g., Parkinson's disease), stroke, and sepsis. Due to the lack of druggable targets, it remains a major challenge to discover apoptosis inhibitors. The recent repositioning of a marketed drug (i.e., terazosin) as an anti-apoptotic agent uncovered a novel target (i.e., human phosphoglycerate kinase 1 (hPgk1)). In this study, we developed a virtual screening (VS) pipeline based on the X-ray structure of Pgk1/terazosin complex and applied it to a screening campaign for potential anti-apoptotic agents. The hierarchical filters in the pipeline (i.e., similarity search, a pharmacophore model, a shape-based model, and molecular docking) rendered 13 potential hits from Specs chemical library. By using PC12 cells (exposed to rotenone) as a cell model for bioassay, we first identified that AK-918/42829299, AN-465/41520984, and AT-051/43421517 were able to protect PC12 cells from rotenone-induced cell death. Molecular docking suggested these hit compounds were likely to bind to hPgk1 in a similar mode to terazosin. In summary, we not only present a versatile VS pipeline for potential apoptosis inhibitors discovery, but also provide three novel-scaffold hit compounds that are worthy of further development and biological study.
Topics: Animals; Apoptosis; Cell Survival; Databases, Chemical; Drug Evaluation, Preclinical; Humans; Models, Molecular; Molecular Docking Simulation; PC12 Cells; Phosphoglycerate Kinase; Prazosin; Protein Kinase Inhibitors; Rats; Small Molecule Libraries
PubMed: 28635653
DOI: 10.3390/molecules22061029 -
European Journal of Biochemistry Oct 2001Yeast phosphoglycerate kinase (yPGK) is a monomeric two domain protein used as folding model representative of large proteins. We inserted short unstructured sequences...
Yeast phosphoglycerate kinase (yPGK) is a monomeric two domain protein used as folding model representative of large proteins. We inserted short unstructured sequences (four Gly or four Thr) into the connections between secondary structure elements and studied the consequences of these insertions on the folding process and stability of yPGK. All the mutated proteins can refold efficiently. The effect per residue on stability is larger for the first inserted residue. Insertion in two long betaalpha loops (at residue positions 71 and 129) is more destabilizing than an insertion in a short alphabeta loop (at residue position 89) located on the opposite side of the N-terminal domain. The effect on stability is mainly due to a large increase of the unfolding rate rather than a decrease of the folding rate. This suggests that these connections between secondary structure elements do not play an active role in directing the folding process. Insertion into the short alphabeta loop (position 89) has limited effects on stability and results in the detection of a kinetic phase not previously seen with the wild-type protein, suggesting that insertions in this particular loop do qualitatively affect the folding process without a large effect on folding efficiency. For the two long betaalpha loops (positions 71 and 129) located in the inner surface of the N-terminal domain, the effects on stability are possibly associated with decoupling of the two domains as observed by differential scanning calorimetry during thermal unfolding.
Topics: Enzyme Stability; Hot Temperature; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Phosphoglycerate Kinase; Protein Conformation; Protein Denaturation; Protein Folding; Saccharomyces cerevisiae; Thermodynamics
PubMed: 11589702
DOI: 10.1046/j.0014-2956.2001.02439.x -
The Journal of Biological Chemistry Apr 2000The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for...
The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25 degrees C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heat-labile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.
Topics: Amino Acid Sequence; Antarctic Regions; Calorimetry, Differential Scanning; Cloning, Molecular; Cold Temperature; Escherichia coli; Kinetics; Molecular Sequence Data; Phosphoglycerate Kinase; Protein Conformation; Protein Folding; Pseudomonas; Thermodynamics
PubMed: 10753921
DOI: 10.1074/jbc.275.15.11147 -
European Journal of Biochemistry Apr 1992Site-directed mutagenesis has been used to produce two mutant forms of yeast phosphoglycerate kinase in which the interdomain residue Phe194 has been replaced by a...
Site-directed mutagenesis has been used to produce two mutant forms of yeast phosphoglycerate kinase in which the interdomain residue Phe194 has been replaced by a leucine or tryptophan residue. Using 1H-NMR spectroscopy, it was found that the mutations at position 194 induce both local and long-range conformational changes in the protein. It was also found that 3-phosphoglycerate binding to the mutant proteins induces somewhat different conformational effects to those observed for wild-type phosphoglycerate kinase. The affinity of mutant Phe194----Trp for 3-phosphoglycerate was found by NMR studies to be unaffected, while the affinity of Phe194----Leu mutant is reduced by about threefold relative to the wild-type enzyme. The binding of ATP at the electrostatic site of the mutant proteins is also seen to be about three times weaker for the Phe194----Leu mutant when compared to wild-type or Phe194----Leu mutant. These results are discussed in the light of the kinetic studies on the mutants which show that for Phe194----Leu mutant the Km values for both 3-phosphoglycerate and ATP, as well as the Vmax, are decreased relative to the wild-type enzyme, while for mutant Phe194----Trp, the Km values for 3-phosphoglycerate and ATP are unaffected and the Vmax is decreased when compared to wild-type enzyme. Kinetic studies in the presence of sulphate reveal that the anion activation is greater for mutant Phe194----Trp and less for mutant Phe194----Leu, relative to that observed for wild-type phosphoglycerate kinase. The NMR data, taken together with the kinetic data, are consistent with the on and off rates of 3-phosphoglycerate being affected by the mutations at position 194. It is suggested that Phe194 is important for the mobility of the interdomain region and the relative movement of the 3-phosphoglycerate binding site which allows the optimum conformation for catalysis to be attained. Apparently Trp194 reduces the mobility of the interdomain region of the protein, while Leu194 increases it.
Topics: Adenosine Triphosphate; Cations, Divalent; Kinetics; Leucine; Magnesium; Magnetic Resonance Spectroscopy; Mutation; Phenylalanine; Phosphoglycerate Kinase; Protein Conformation; Saccharomyces cerevisiae; Tryptophan
PubMed: 1555608
DOI: 10.1111/j.1432-1033.1992.tb16755.x -
European Journal of Biochemistry Apr 19761. Phosphoglycerate kinase has been isolated from a photosynthetic plant tissue, Beta vulgaris leaves. The purification procedure is described. 2. The best preparation...
1. Phosphoglycerate kinase has been isolated from a photosynthetic plant tissue, Beta vulgaris leaves. The purification procedure is described. 2. The best preparation had no detectable impurity on electrophoresis, and had a specific activity comparable with the same enzyme from other sources. 3. The molecular weight was not distinguishably different from that of the yeast or muscle enzyme, as measured by polyacrylamide-dodecylsulphate electrophoresis. Measurement of aromatic and sulphydryl residues indicated a close similarity with the yeast enzyme. The enzyme appears to have substantially lower isoelectric point than phosphoglycerate kinases from other sources. 4. Kinetic studies indicated that the affinities for the substrates MgATP2- and 3-phosphoglycerate were not significantly different from those of the 'glycolytic' yeast enzyme. There was no evidence that the B. vulgaris enzyme had specific properties making it more suitable for its gluconeogenic rather than glycolytic role.
Topics: Adenosine Triphosphate; Cations, Divalent; Chromatography, Affinity; Kinetics; Magnesium; Molecular Weight; Phosphoglycerate Kinase; Photosynthesis; Plants; Species Specificity
PubMed: 1261557
DOI: 10.1111/j.1432-1033.1976.tb10251.x -
The EMBO Journal 1982The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies....
The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies. The substrate binding sites, as deduced from electron density maps, are compatible with known substrate specificity and the stereochemical requirements for the enzymic reaction. A carboxyl-imidazole interaction appears to be involved in controlling the transition between the open and closed forms of the enzyme.
Topics: Amino Acid Sequence; Models, Molecular; Phosphoglycerate Kinase; Protein Conformation; Saccharomyces cerevisiae; Substrate Specificity; X-Ray Diffraction
PubMed: 6765200
DOI: 10.1002/j.1460-2075.1982.tb01366.x -
Journal of Biochemistry Nov 1980Blue dextran or Cibacron Blue F3GA has been shown to inhibit yeast phosphoglycerate kinase [EC 2.7.2.3] competitively with respect to ATP (Thompson et al. (1975) Proc....
Blue dextran or Cibacron Blue F3GA has been shown to inhibit yeast phosphoglycerate kinase [EC 2.7.2.3] competitively with respect to ATP (Thompson et al. (1975) Proc. Natl. Acad. Sci. U.S. 72, 663--667; Beissner and Rudolph (1979) J. Biol. Chem. 254, 6273--6277). However, we have found that phosphoglycerate kinase of Lactobacillus plantarum was inhibited by Cibacron Blue F3GA, the blue chromophore of blue dextran, noncompetitively with respect to ATP, but competitively with respect to 3-phosphoglycerate. Further inhibition studies with Cibacron Blue F3GA suggest that one molecule of the dye was bound per molecule of phosphoglycerate kinase at a saturated level of either substrate, but two molecules of the dye were bound per molecule of the kinase with an unsaturated level of either substrate used as a fixed substrate. Furthermore, phosphoglycerate mutase [EC 2.7.5.3] of Leuconostoc dextranicum was also inhibited by Cibacron Blue F3GA competitively with respect to 3-phosphoglycerate and noncompetitively with respect to 2,3-bisphosphoglycerate. These results suggest that the 3-phosphoglycerate-binding site on both phosphoglycerate kinase and phosphoglycerate mutase can interact with Cibacron Blue F3GA.
Topics: Adenosine Triphosphate; Anthracenes; Binding Sites; Coloring Agents; Glyceric Acids; Lactobacillus; Leuconostoc; Organophosphorus Compounds; Phosphoglycerate Kinase; Phosphoglycerate Mutase; Phosphotransferases; Protein Binding; Triazines
PubMed: 6257659
DOI: 10.1093/oxfordjournals.jbchem.a133090