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Methods in Enzymology 1975
Topics: Acetone; Ammonium Sulfate; Animals; Chemical Phenomena; Chemistry, Physical; Chickens; Chromatography, DEAE-Cellulose; Chromatography, Gel; Diphosphoglyceric Acids; Ethanol; Fractional Precipitation; Hot Temperature; Kidney; Kinetics; Methods; Molecular Weight; Muscles; Phosphoglycerate Mutase; Phosphotransferases; Saccharomyces cerevisiae; Thermodynamics
PubMed: 166275
DOI: 10.1016/0076-6879(75)42149-8 -
PLoS Neglected Tropical Diseases 2014Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in...
Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF). iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS) was conducted at two different locations: ∼220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ∼160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z'-factor >0.50) and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 µM) and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a "druggability paradox" of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data.
Topics: Animals; Brugia malayi; Enzyme Inhibitors; Filaricides; High-Throughput Screening Assays; Inhibitory Concentration 50; Phosphoglycerate Mutase
PubMed: 24416464
DOI: 10.1371/journal.pntd.0002628 -
British Journal of Cancer 1997The distribution of phosphoglycerate mutase (EC 5.4.2.1, PGM), 2,3-bisphosphoglycerate phosphatase (EC 3.1.3.13, BPGP) and creatine kinase (EC 2.7.3.2, CK) activity and...
The distribution of phosphoglycerate mutase (EC 5.4.2.1, PGM), 2,3-bisphosphoglycerate phosphatase (EC 3.1.3.13, BPGP) and creatine kinase (EC 2.7.3.2, CK) activity and isoenzymes in various regions of adult human brain and in brain tumours (astrocytomas, anaplastic astrocytomas, glioblastomas and meningiomas) has been determined using electrophoresis. PGM and cytosolic CK exist in mammalian tissues as three isoenzymes that result from the homodimeric and heterodimeric combinations of two subunits [types M (muscle) and B (brain)] coded by separated genes. In addition, a dimeric form and an octameric form of mitochondrial CK exist in mammals. Type BB-PGM was the major PGM isoenzyme found in normal brain, although type MB-PGM and type MM-PGM were also detected. All brain tumours possessed lower PGM activity than normal brain, and meningiomas showed higher BPGP activity. In astrocytic tumours, the proportion of type MB- and type MM-PGM decreased, and in meningiomas these isoenzymes were not detected. Type BB-CK and mitochondrial CK were the only CK isoenzymes detected in normal brain. Astrocytomas possessed lower CK activity than anaplastic astrocytomas and glioblastomas and, in addition, tended to possess lower CK content than normal brain. No qualitative changes of the normal CK isoenzyme pattern were observed in the tumours.
Topics: Adult; Aged; Aged, 80 and over; Aging; Brain Neoplasms; Caudate Nucleus; Cerebellum; Cerebral Cortex; Creatine Kinase; Electrophoresis; Female; Humans; Isoenzymes; Male; Middle Aged; Phosphoglycerate Mutase; Phosphoric Monoester Hydrolases
PubMed: 9365161
DOI: 10.1038/bjc.1997.525 -
The Journal of Biological Chemistry Aug 2004The putative tumor metastasis suppressor protein Nm23-H1 is a nucleoside diphosphate kinase that exhibits a novel protein kinase activity when bound to...
The putative tumor metastasis suppressor protein Nm23-H1 is a nucleoside diphosphate kinase that exhibits a novel protein kinase activity when bound to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In this study we show that the glycolytic enzyme phosphoglycerate mutase B (PGM) becomes phosphorylated in the presence of the Nm23-H1.GAPDH complex in vitro. Mutation of His-10 in PGM abolishes the Nm23-H1.GAPDH complex-induced phosphorylation. Nm23-H1, GAPDH, and PGM are known to co-localize as shown by free flow isoelectric focusing. In association with Nm23-H1 and GAPDH, PGM could be activated by dCTP, which is a substrate of Nm23-H1, in addition to the well known PGM activator 2,3-bisphosphoglycerate. A synthetic cell-penetrating peptide (PGMtide) encompassing the phosphorylated histidine and several residues from PGM (LIRHGE) promoted growth arrest of several tumor cell lines, whereas proliferation of tested non-tumor cells was not influenced. Analysis of metabolic activity of one of the tumor cell lines, MCF-7, indicated that PGMtide inhibited glycolytic flux, consistent with in vivo inhibition of PGM. The specificity of the observed effect was further determined experimentally by testing the effect of PGMtide on cells growing in the presence of pyruvate, which helps to compensate PGM inhibition in the glycolytic pathway. Thus, growth of MCF-7 cells was not arrested by PGMtide in the presence of pyruvate. The data presented here provide evidence that inhibition of PGM activity can be achieved by exogenous addition of a polypeptide, resulting in inhibition of glycolysis and cell growth arrest in cell culture.
Topics: Cell Line, Tumor; Cell Proliferation; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycolysis; Growth Inhibitors; Humans; Nucleoside-Diphosphate Kinase; Peptides; Phosphoglycerate Mutase; Phosphorylation; Protein Binding; Tumor Suppressor Proteins
PubMed: 15181008
DOI: 10.1074/jbc.M402768200 -
International Journal of Biological... May 2021Entamoeba histolytica phosphoserine phosphatase (EhPSP), a regulatory enzyme in the serine biosynthetic pathway, is also a structural homolog of cofactor-dependent...
Entamoeba histolytica phosphoserine phosphatase (EhPSP), a regulatory enzyme in the serine biosynthetic pathway, is also a structural homolog of cofactor-dependent phosphoglycerate mutase (dPGM). However, despite sharing many of its catalytic residues with dPGM, EhPSP displays no significant mutase activity. In the current work, we determined a crystal structure of EhPSP in complex with 3-PGA to 2.5 Å resolution and observed striking differences between the orientation of 3-PGA bound to EhPSP and that to its other homologous structures. We also performed computational modeling and simulations of the intermediate 2,3-bisphosphoglyceric acid into the active site of EhPSP to better understand its mechanistic details. Based on these results and those of a similar study with the dPGMs from E. coli and B. pseudomallei, the affinity of EhPSP for 2,3-BPG was concluded to be lower than those of the other proteins. Moreover, a different set of 2,3-BPG interacting residues was observed in EhPSP compared to dPGMs, with all of the crucial interacting residues of dPGMs either missing or substituted with weakly interacting residues. This study has expanded our understanding, at the structural level, of the inability of EhPSP to catalyze the mutase reaction and has strengthened earlier conclusions indicating it to be a true phosphatase.
Topics: Catalytic Domain; Entamoeba histolytica; Glyceric Acids; Models, Molecular; Phosphoglycerate Mutase; Phosphoric Monoester Hydrolases; Protein Conformation; Protozoan Proteins; Sequence Alignment
PubMed: 33631257
DOI: 10.1016/j.ijbiomac.2021.02.153 -
Acta Biochimica Polonica Nov 2021Although many atypical proteinaceous cell wall components that belong to a group of multitasking, "moonlighting" proteins, have been repeatedly identified in numerous...
Towards understanding the novel adhesin function of Candida albicans phosphoglycerate mutase at the pathogen cell surface: some structural analysis of the interactions with human host extracellular matrix proteins.
Although many atypical proteinaceous cell wall components that belong to a group of multitasking, "moonlighting" proteins, have been repeatedly identified in numerous pathogenic microorganisms, their novel extracellular functions and secretion mechanisms remain largely unrecognized. In Candida albicans, one of the most common fungal pathogens in humans, phosphoglycerate mutase (Gpm1) - a cytoplasmic enzyme involved in the glycolysis pathway - has been shown to occur on the cell surface and has been identified as a potentially important virulence factor. In this study, we demonstrated tight binding of C. albicans Gpm1 to the candidal cell surface, thus suggesting that the readsorption of soluble Gpm1 from the external environment could be a likely mechanism leading to the presence of this moonlighting protein on the pathogen surface. Several putative Gpm1-binding receptors on the yeast surface were identified. The affinities of Gpm1 to human vitronectin (VTR) and fibronectin (FN) were characterized with surface plasmon resonance measurements, and the dissociation constants of the complexes formed were determined to be in the order of 10-8 M. The internal Gpm1 sequence motifs, directly interacting with VTR (aa 116-158) and FN (aa 138-175) were mapped using chemical crosslinking and mass spectrometry. Synthetic peptides with matching sequences significantly inhibited formation of the Gpm1-VTR and Gpm1-FN complexes. A molecular model of the Gpm1-VTR complex was developed. These results provide the first structural insights into the adhesin function of candidal surface-exposed Gpm1.
Topics: Candida albicans; Cell Membrane; Cell Wall; Extracellular Matrix Proteins; Fibronectins; Fungal Proteins; Humans; Models, Molecular; Phosphoglycerate Mutase; Protein Binding; Surface Plasmon Resonance; Virulence Factors; Vitronectin
PubMed: 34773933
DOI: 10.18388/abp.2020_5959 -
Cancer Research Jul 2014Glycolytic enzyme phosphoglycerate mutase (PGAM) plays an important role in coordinating energy production with generation of reducing power and the biosynthesis of...
Glycolytic enzyme phosphoglycerate mutase (PGAM) plays an important role in coordinating energy production with generation of reducing power and the biosynthesis of nucleotide precursors and amino acids. Inhibition of PGAM by small RNAi or small molecule attenuates cell proliferation and tumor growth. PGAM activity is commonly upregulated in tumor cells, but how PGAM activity is regulated in vivo remains poorly understood. Here we report that PGAM is acetylated at lysine 100 (K100), an active site residue that is invariably conserved from bacteria, to yeast, plant, and mammals. K100 acetylation is detected in fly, mouse, and human cells and in multiple tissues and decreases PGAM2 activity. The cytosolic protein deacetylase sirtuin 2 (SIRT2) deacetylates and activates PGAM2. Increased levels of reactive oxygen species stimulate PGAM2 deacetylation and activity by promoting its interaction with SIRT2. Substitution of endogenous PGAM2 with an acetylation mimetic mutant K100Q reduces cellular NADPH production and inhibits cell proliferation and tumor growth. These results reveal a mechanism of PGAM2 regulation and NADPH homeostasis in response to oxidative stress that impacts cell proliferation and tumor growth.
Topics: Acetylation; Animals; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glycolysis; HEK293 Cells; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; Mutation; NADP; Oxidative Stress; Phosphoglycerate Mutase; Protein Binding; Reactive Oxygen Species; Sirtuin 2
PubMed: 24786789
DOI: 10.1158/0008-5472.CAN-13-3615 -
Molecules (Basel, Switzerland) Jun 2018Upregulation of phosphoglycerate mutase 1 (PGAM1) has been identified as one common phenomenon in a variety of cancers. Inhibition of PGAM1 provides a new promising...
Upregulation of phosphoglycerate mutase 1 (PGAM1) has been identified as one common phenomenon in a variety of cancers. Inhibition of PGAM1 provides a new promising therapeutic strategy for cancer treatment. Herein, based on our previous work, a series of new -xanthone benzenesulfonamides were discovered as novel PGAM1 inhibitors. The representative molecule , with an IC of 2.1 μM, showed an enhanced PGAM1 inhibitory activity and higher enzyme inhibitory specificity compared to PGMI-004A, as well as a slightly improved antiproliferative activity.
Topics: Carbon-13 Magnetic Resonance Spectroscopy; Cell Line; Cell Proliferation; Drug Design; Enzyme Inhibitors; Humans; Phosphoglycerate Mutase; Proton Magnetic Resonance Spectroscopy; Spectrometry, Mass, Electrospray Ionization; Sulfonamides; Xanthones; Benzenesulfonamides
PubMed: 29890679
DOI: 10.3390/molecules23061396 -
The Journal of Biological Chemistry Dec 1978The steady state kinetics and effects of salts on chicken breast phosphoglycerate mutase have been examined. The enzyme can catalyze three phosphoryl transfer reactions:...
The steady state kinetics and effects of salts on chicken breast phosphoglycerate mutase have been examined. The enzyme can catalyze three phosphoryl transfer reactions: mutase, bisphosphoglycerate phosphatase, and bisphosphoglycerate synthase. The mutase rate was measured in the favorable direction (Keq = glycerate-3-P/glycerate-2-P approximately equal to 12) using [2T]glycerate-2-P as substrate. The bisphosphoglycerate phosphatase activity was studied in the presence of the activator, glycolate-2-P. The latter is an analog of the glycerate-P's and appears to act as an abortive mutase substrate. The kinetic pattern obtained with both activities is that of a ping-pong mechanism with inhibition by the second substrate occurring at a lower concentration than the Km value for that substrate. The kinetic parameters for the mutase determined in 50 mM N-[tris(hydroxymethyl)methyl-2-amino]ethanesulfonate (TES)/sodium buffer containing 0.1 M KCl, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.069 micron; Km glycerate-2-P, 14 micron; Km glycerate-3-P approximately 200 micron; Ki glycerate-2-P, 4 micron. The kinetic parameters for the phosphatase reaction in 50 mM triethanolamine/Cl- buffer, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.065 micron:Km glycolate-2P, 479 micron; Ki glycolate-2-P, 135 micron. The enzyme is sensitive to changes in the ionic environment. Increasing salt concentrations activate the phosphatase in the presence of glycolate-2-P by decreasing the apparent Km of glycerate-2,3-P2. The effects are due to the anionic component and Cl- greater than acetate greater than TES. The same salts are competitive inhibitors with respect to glycolate-2-P. With high levels of KCl that produce a 30-fold decrease in the apparent maximal velocity due to competition with glycolate-2-P, the Km of glycerate-2,3-P2 remains low. These observations lead us to postulate that each monophosphoglycerate substrate has a separate site on the enzyme and that glycerate-2,3-P2 can bind to either site. The binding of anions to one site of the nonphosphorylated enzyme allows an increase in the on and off rates of glycerate-2,3-P2 at the alternate site. Salts inhibit the mutase reaction. The Km of glycerate-2,3-P2 is increased as is that of glycerate-2-P. The effect on the Km of glycerate-2,3-P2 is attributed to an increase in the off rate/on rate ratio for glycerate-2,3-P2. The bisphosphoglycerate synthase reaction is shown to require added glycerate-3-P. The equilibrium between enzyme and glycerate-1,3-P2 is favorable (Kdiss less than or equal 7 X 10(-8) M) and suggests that in the absence of a separate synthase this reaction may have functional significance.
Topics: Animals; Diphosphoglyceric Acids; Kinetics; Mathematics; Muscles; Osmolar Concentration; Phosphoglycerate Mutase; Phosphoric Monoester Hydrolases; Phosphotransferases; Potassium Chloride
PubMed: 213437
DOI: No ID Found -
Biochemical and Biophysical Research... May 1978
Topics: Bacillus megaterium; Calcium; Kinetics; Lasalocid; Magnesium; Manganese; Phosphoglycerate Mutase; Phosphotransferases; Spores, Bacterial
PubMed: 208551
DOI: 10.1016/0006-291x(78)90567-3