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The Journal of Biological Chemistry Jun 1990A cluster of conserved histidines and arginines (His-62, His-167, Arg-21, Arg-38, and Arg-168) in 3-phosphoglycerate kinase (PGK) has been implicated as possibly...
A cluster of conserved histidines and arginines (His-62, His-167, Arg-21, Arg-38, and Arg-168) in 3-phosphoglycerate kinase (PGK) has been implicated as possibly involved in the binding of 3-phosphoglycerate (3-PG) and/or stabilization of the negatively charged transition state. The role of these residues in the catalytic function of yeast PGK and in the substrate- and sulfate-dependent activation was investigated by site-directed mutagenesis. The following substitutions, R21A, R21Q, H62Q, H167S, and R168Q, produced functional enzymes. In contrast, the R38A and R38Q mutations resulted in a complete loss of catalytic activity. These results demonstrate that of the basic residues studied, only arginine 38 is essential for the catalytic function of PGK. A moderate decrease in the catalytic efficiency as the result of the R21A, H167S, and R168Q mutations and an increased catalytic efficiency of the H62Q mutant rule out a possible role of a positive charge at these positions in the mechanism of phosphoryl transfer reaction. In contrast to the wild type PGK and the H62Q mutant, both of which are activated at low and inhibited at high sulfate concentration, the H167S, R168Q, and R21A mutants exhibited a progressive inhibition with increased concentration of sulfate. The activation observed at high concentration of either ATP or 3-PG as a variable substrate in the steady-state kinetics of wild type PGK was abolished as the result of the latter three mutations. The results of this work support the hypothesis that PGK has two binding sites for anionic ligands, the catalytic and regulatory sites for each substrate and the activatory and inhibitory sites for sulfate, and suggest that arginine 21, arginine 168, and histidine 167 are located in the activatory anion binding site, common for sulfate, 3-PG, and ATP. The increased Km values for both substrates and decreased specific activities of the mutants suggest that this regulatory site is close to the catalytic site.
Topics: Amino Acid Sequence; Arginine; Base Sequence; Binding Sites; Enzyme Activation; Histidine; Kinetics; Molecular Sequence Data; Molecular Weight; Mutation; Phosphoglycerate Kinase; Protein Conformation; Saccharomyces cerevisiae
PubMed: 2191956
DOI: No ID Found -
The Journal of Physical Chemistry... Mar 2021Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves...
Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.
Topics: Adenosine Triphosphate; Allosteric Regulation; Antibodies; Antigen-Antibody Reactions; Histidine; Oligopeptides; Phosphoglycerate Kinase; Protein Conformation; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 33710900
DOI: 10.1021/acs.jpclett.1c00437 -
The Journal of Biological Chemistry Feb 2008Isoenzymes of phosphoglycerate kinase in Trypanosoma brucei are differentially expressed in its two main life stages. This study addresses how the organism manages to...
Isoenzymes of phosphoglycerate kinase in Trypanosoma brucei are differentially expressed in its two main life stages. This study addresses how the organism manages to make sufficient amounts of the isoenzyme with the correct localization, which processes (transcription, splicing, and RNA degradation) control the levels of mRNAs, and how the organism regulates the switch in isoform expression. For this, we combined new quantitative measurements of phosphoglycerate kinase mRNA abundance, RNA precursor stability, trans splicing, and ribosome loading with published data and made a kinetic computer model. For the analysis of regulation we extended regulation analysis. Although phosphoglycerate kinase mRNAs are present at surprisingly low concentrations (e.g. 12 molecules per cell), its protein is highly abundant. Substantial control of mRNA and protein levels was exerted by both mRNA synthesis and degradation, whereas splicing and precursor degradation had little control on mRNA and protein concentrations. Yet regulation of mRNA levels does not occur by transcription, but by adjusting mRNA degradation. The contribution of splicing to regulation is negligible, as for all cases where splicing is faster than RNA precursor degradation.
Topics: Animals; Computer Simulation; Gene Expression Regulation; Genes, Protozoan; Isoenzymes; Kinetics; Models, Biological; Phosphoglycerate Kinase; Protozoan Proteins; RNA Splicing; RNA, Messenger; RNA, Protozoan; Ribosomes; Transcription, Genetic; Trypanosoma brucei brucei
PubMed: 17991737
DOI: 10.1074/jbc.M705782200 -
Biomolecular NMR Assignments Oct 2017Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP...
Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the H, N and C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF:ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the H-N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex.
Topics: Adenosine Diphosphate; Fluorides; Glyceraldehyde 3-Phosphate; Humans; Magnesium Compounds; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Phosphoglycerate Kinase; Protein Binding; Protein Conformation, alpha-Helical
PubMed: 28866776
DOI: 10.1007/s12104-017-9758-3 -
Biomedicine & Pharmacotherapy =... May 2023Inter-α-trypsin inhibitor heavy chain 4 (ITI-H4) is one of the acute phase proteins and is mainly related with inflammatory diseases such as bacterial bloodstream...
Inter-α-trypsin inhibitor heavy chain 4 (ITI-H4) is one of the acute phase proteins and is mainly related with inflammatory diseases such as bacterial bloodstream infection and recurrent pregnancy loss (RPL). In a previous study, ITI-H4 was reported to be cleaved by kallikrein B1 (KLKB1) and its cleaved form induces the imbalance between pro- and anti-inflammatory cytokines. Therefore, in this study, putative substrates of ITI-H4 were isolated by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. Of those, phosphoglycerate kinase 1 (PGK1) was found to be a binding protein of ITI-H4. PGK1 increases the level of ITI-H4 expression and blocks the cleavage of ITI-H4 mediated by KLKB1. It also inhibits pro-inflammatory response by inhibiting the JAK2/STAT3 signaling pathway. Therefore, PGK1, a novel binding partner of ITI-H4, is expected to have cellular functions in the pathogenesis of ITI-H4-related inflammatory diseases.
Topics: Pregnancy; Female; Humans; Cytokines; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Phosphoglycerate Kinase
PubMed: 36841032
DOI: 10.1016/j.biopha.2023.114437 -
European Journal of Biochemistry Apr 19781. The testis-specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from... (Comparative Study)
Comparative Study
1. The testis-specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from 'normal' phosphoglycerate kinase which is also present in testis tissue. The purification procedure is described. 2. The best preparations had no detectable impurity on electrophoresis, and had specific activities comparable with the same enzyme from other sources. 3. Kinetic studies indicated that the two isoenzymes have identical properties, within experimental error, for substrate affinity (for MgATP, 3-phosphoglycerate and MgADP), energy of activation and thermal denaturation. 4. The molecular weights of both isoenzymes were not distinguishably different from those previously reported, as measured by polyacrylamide/dodecylsulphate electrophoresis. The amino acid compositions showed only slight differences, and tryptic peptide maps showed that there was close homology of sequence. Starch gel electrophoresis at pH 6.5 indicates that the B isoenzyme has 1--2 more positive charges than the A. 5. Phosphoglycerate kinase A isolated from sheep muscle was shown, within experimental error, to be identical to the phosphoglycerate kinase A isolated from testis. 6. The results further substantiate the suggestion that the B isoenzyme is coded by a gene which was duplicated from the phosphoglycerate kinase A gene.
Topics: Amino Acids; Animals; Isoenzymes; Kinetics; Male; Molecular Weight; Phosphoglycerate Kinase; Sheep; Testis
PubMed: 639826
DOI: 10.1111/j.1432-1033.1978.tb12215.x -
Cell Cycle (Georgetown, Tex.) Jul 2016
Topics: Phosphoglycerate Kinase
PubMed: 27105392
DOI: 10.1080/15384101.2016.1179037 -
The Journal of Biological Chemistry Aug 2012Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen....
Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen. The actin and plasminogen binding sites of GBS-PGK were identified using truncated GBS-PGK molecules, followed by peptide mapping. These experiments identified two actin and plasminogen binding sites located between amino acids 126-134 and 204-208 of the 398-amino acid-long GBS-PGK molecule. Substitution of the lysine residues within these regions with alanine resulted in significantly reduced binding to both actin and plasminogen. In addition, conversion of the glutamic acid residue at amino acid 133 to proline, the amino acid found at this position for the PGK protein of Streptococcus pneumoniae, also resulted in significantly reduced binding to actin and plasminogen. These results demonstrate that the lysine residues at amino acid positions 126, 127, 130, 204, and 208 along with the glutamic acid residue at amino acid position 133 are necessary for actin and plasminogen binding by GBS-PGK.
Topics: Actins; Bacterial Proteins; Humans; Peptide Mapping; Phosphoglycerate Kinase; Plasminogen; Protein Binding; Streptococcus agalactiae; Streptococcus pneumoniae
PubMed: 22761440
DOI: 10.1074/jbc.M112.361261 -
Aging Jul 2020The neurotoxin MPP (1-methyl-4-phenylpyridinium ion) disrupts mitochondrial function leading to oxidative stress and neuronal death. Here we examine whether activation...
The neurotoxin MPP (1-methyl-4-phenylpyridinium ion) disrupts mitochondrial function leading to oxidative stress and neuronal death. Here we examine whether activation of the Keap1-Nrf2 cascade can protect SH-SY5Y neuroblastoma cells from MPP-induced cytotoxicity. Treatment of SH-SY5Y cells with CBR-470-1, an inhibitor of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1), leads to methylglyoxal modification of Keap1, Keap1-Nrf2 disassociation, and increased expression of Nrf2 responsive genes. Pretreatment with CBR-470-1 potently attenuated MPP-induced oxidative injury and SH-SY5Y cell apoptosis. CBR-470-1 neuroprotection is dependent upon Nrf2, as Nrf2 shRNA or CRISPR/Cas9-mediated Nrf2 knockout, abolished CBR-470-1-induced SH-SY5Y cytoprotection against MPP. Consistent with these findings, PGK1 depletion or knockout mimicked CBR-470-1-induced actions and rendered SH-SY5Y cells resistant to MPP-induced cytotoxicity. Furthermore, activation of the Nrf2 cascade by CRISPR/Cas9-induced Keap1 knockout protected SH-SY5Y cells from MPP. In Keap1 or PGK1 knockout SH-SY5Y cells,CBR-470-1 failed to offer further cytoprotection against MPP. Collectively PGK1 inhibition by CBR-470-1 protects SH-SY5Y cells from MPP via activation of the Keap1-Nrf2 cascade.
Topics: 1-Methyl-4-phenylpyridinium; Cell Line, Tumor; Gene Knockout Techniques; Humans; Kelch-Like ECH-Associated Protein 1; Mitochondria; NF-E2-Related Factor 2; Neuroprotective Agents; Neurotoxicity Syndromes; Phosphoglycerate Kinase; RNA, Small Interfering; Signal Transduction
PubMed: 32649311
DOI: 10.18632/aging.103443 -
FEBS Letters Feb 1989It has been shown that the denaturation of phosphoglycerate kinase (PGK) can be observed not only when the solution is heated above 30 degrees C, but also when it is...
It has been shown that the denaturation of phosphoglycerate kinase (PGK) can be observed not only when the solution is heated above 30 degrees C, but also when it is cooled below this temperature. The disruption of the native PGK structure upon cooling and the subsequent formation of this structure upon heating both proceed in two distinct stages which correspond to the independent disruption or reformation of each of its domains. In contrast, the heat denaturation of PGK proceeds in one stage, showing that the two domains of the molecule are associated into a single complex which figures in the denaturation process as a cooperative unit. It follows that, at elevated temperature, there is a positive interaction between the domains, which disappears at lower temperatures. This might be due to hydrophobic interactions, which are known to be temperature dependent. The temperature decrease leads to a decrease in inter- and intradomain interactions, which results in an increase of the independence of the domains and a decrease in their stability.
Topics: Cold Temperature; Hot Temperature; Phosphoglycerate Kinase; Protein Denaturation; Saccharomyces cerevisiae; Thermodynamics
PubMed: 2646151
DOI: 10.1016/0014-5793(89)80544-7