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European Journal of Biochemistry Dec 1970
Topics: Adenine Nucleotides; Amino Acids; Animals; Chemical Phenomena; Chemistry; Chromatography, Gel; Chromatography, Ion Exchange; Drug Stability; Electrophoresis; Glycolysis; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Muscles; Nucleotides; Peptides; Phosphoglycerate Kinase; Rabbits; Saccharomyces; Sulfhydryl Compounds; Ultracentrifugation; Valine
PubMed: 5493986
DOI: 10.1111/j.1432-1033.1970.tb01202.x -
Journal of Inherited Metabolic Disease Nov 2014Mutations in genes encoding metabolic enzymes are often the cause of inherited diseases. Mutations usually affect the ability of proteins to fold properly, thus leading...
Mutations in genes encoding metabolic enzymes are often the cause of inherited diseases. Mutations usually affect the ability of proteins to fold properly, thus leading to enzyme loss of function. In this work, we explored the relationships between protein stability, aggregation, and degradation in vitro and inside cells in a large set of mutants associated with human phosphoglycerate kinase 1 (hPGK1) deficiency. To this end, we studied a third of the pathogenic alleles reported in the literature using expression analyses and biochemical, biophysical, and computational procedures. Our results show that most pathogenic variants studied had an increased tendency to aggregate when expressed in Escherichia coli, well correlating with the denaturation half-lives measured by thermal denaturation in vitro. Further, the most deleterious mutants show reduced stability toward chemical denaturation and proteolysis, supporting a pivotal role of thermodynamic stability in the propensity toward aggregation and proteolysis of pathogenic hPGK1 mutants in vitro and inside cells. Our strategy allowed us to unravel the complex relationships between protein stability, aggregation, and degradation in hPGK1 deficiency, which might be used to understand disease mechanisms in many inborn errors of metabolism. Our results suggest that pharmacological chaperones and protein homeostasis modulators could be considered as good candidates for therapeutic approaches for hPGK1 deficiency.
Topics: Alleles; Enzyme Stability; Escherichia coli; Genetic Diseases, X-Linked; Homeostasis; Humans; Metabolism, Inborn Errors; Mutation; Phosphoglycerate Kinase; Protein Conformation; Protein Denaturation; Proteolysis
PubMed: 24838780
DOI: 10.1007/s10545-014-9721-8 -
European Journal of Biochemistry Dec 1990Two short analogues of 3-phosphoglycerate, -OOC-CHOH-CH2-O-PO32-, phosphonolactate, (-OOC-CHOH-CH2-PO32-) and arsonolactate (-OOC-CHOH-CH2-AsO32-) have been tested with...
Two short analogues of 3-phosphoglycerate, -OOC-CHOH-CH2-O-PO32-, phosphonolactate, (-OOC-CHOH-CH2-PO32-) and arsonolactate (-OOC-CHOH-CH2-AsO32-) have been tested with 3-phosphoglycerate kinase. None of these served as substrate for the kinase reaction, unlike the previously studied [Orr, G. A. & Knowles, J. R. (1974) Biochem. J. 141, 721-723] analogues -OOC-CHOH-CH2-CH2-PO32- and -OOC-CHOH-CH2-CH2-AsO32-, which are isosteric with 3-phosphoglycerate. Thus, a decrease in the substrate size and the accompanying stereochemical changes cannot be tolerated by the catalytic mechanism. Instead, both analogues acted as relatively poor competitive inhibitors with respect to both 3-phosphoglycerate and MgATP. AT pH 8.5 and 20 degrees C, the inhibitory constants (Ki) of phosphonolactate and arsnolactate against both substrates are 17 +/- 5 mM and 30 +/- 7 mM, respectively. Surprisingly, however, both analogues proved to be more effective than either 3-phosphoglycerate or its isosteric analogues in protecting the enzyme against modification of its fast-reacting thiols. This comparison suggests that the shorter analogues bind differently, and that the catalytic mechanism demands a precise fitting of the -CH2-O-PO32- segment of the substrate.
Topics: Animals; Arsenicals; Binding Sites; Glyceric Acids; Kinetics; Muscles; Phosphoglycerate Kinase; Sulfhydryl Compounds; Swine
PubMed: 2269289
DOI: 10.1111/j.1432-1033.1990.tb15663.x -
Enzyme 1976Human phosphoglycerate kinase has been totally purified from leukemic granulocytes by double 'affinity elution' with ATP and 3-phosphoglycerate. This purification...
Human phosphoglycerate kinase has been totally purified from leukemic granulocytes by double 'affinity elution' with ATP and 3-phosphoglycerate. This purification procedure allowed to obtain 19 mg of protein, specific activity of which was 400 IU/mg i.e. a 105-fold purification and an overall yield of 47%. Purified enzyme was homogenous when tested by acrylamide sodium dodecyl sulfate electrophoresis and immunodiffusion. Specific antibodies raised in rabbits totally inactivated phosphoglycerate kinase of crude extracts as well as of the purified preparation. The molecular specific activity (i.e. the ratio enzyme activity/immunological reactivity) was identical in leukocytes, platelets, erythrocytes and was identical in these cells to the value found for the totally purified phosphoglycerate kinase.
Topics: Antigen-Antibody Reactions; Blood Platelets; Chromatography, Affinity; Erythrocytes; Granulocytes; Humans; Leukemia, Myeloid; Leukocytes; Phosphoglycerate Kinase; Pyruvate Kinase
PubMed: 1066269
DOI: 10.1159/000458891 -
Autophagy Jul 2017Macroautophagy/autophagy is a cellular defense response to stress conditions and is crucial for cell homeostasis maintenance. However, the precise mechanism underlying...
Macroautophagy/autophagy is a cellular defense response to stress conditions and is crucial for cell homeostasis maintenance. However, the precise mechanism underlying autophagy initiation, especially in response to glutamine deprivation and hypoxia, is yet to be explored. We recently discovered that PGK1 (phosphoglycerate kinase 1), a glycolytic enzyme, functions as a protein kinase, phosphorylating BECN1/Beclin 1 to initiate autophagy. Under glutamine deprivation or hypoxia stimulation, PGK1 is acetylated at K388 by NAA10/ARD1 in an MTOR-inhibition-dependent manner, leading to the interaction between PGK1 and BECN1 and the subsequent phosphorylation of BECN1 at S30 by PGK1. This phosphorylation enhances ATG14-associated PIK3C3/VPS34-BECN1-PIK3R4/VPS15 complex activity, thereby increasing phosphatidylinositol-3-phosphate (PtdIns3P) generation in the initiation stage of autophagy. Furthermore, NAA10-dependent PGK1 acetylation and PGK1-dependent BECN1 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumor formation. Our work reveals the important dual roles of PGK1 as a glycolytic enzyme and a protein kinase in the mutual regulation of cell metabolism and autophagy in maintaining cell homeostasis.
Topics: Autophagy; Carcinogenesis; Cell Hypoxia; Glycolysis; Neoplasms; Phosphoglycerate Kinase
PubMed: 28486006
DOI: 10.1080/15548627.2017.1313945 -
Methods in Enzymology 1982
Topics: Amino Acids; Geobacillus stearothermophilus; Kinetics; Molecular Weight; NAD; Phosphoglycerate Kinase; Spectrophotometry, Ultraviolet; Substrate Specificity
PubMed: 7154941
DOI: 10.1016/s0076-6879(82)90118-5 -
Methods in Enzymology 1982
Topics: Animals; Erythrocytes; Humans; Isoenzymes; Kinetics; Male; Mice; Molecular Weight; Muscles; Phosphoglycerate Kinase; Rabbits; Spectrophotometry, Ultraviolet; Substrate Specificity; Testis; Trout
PubMed: 7154939
DOI: 10.1016/s0076-6879(82)90114-8 -
Biochemistry Mar 2008L-Nucleosides comprise a new class of antiviral and anticancer agents that are converted in vivo by a cascade of kinases to pharmacologically active nucleoside... (Comparative Study)
Comparative Study
Differences in the transient kinetics of the binding of D-ADP and its mirror image L-ADP to human 3-phosphoglycerate kinase revealed by the presence of 3-phosphoglycerate.
L-Nucleosides comprise a new class of antiviral and anticancer agents that are converted in vivo by a cascade of kinases to pharmacologically active nucleoside triphosphates. The last step of the cascade may be catalyzed by 3-phosphoglycerate kinase (PGK), an enzyme that has low specificity for nucleoside diphosphate (NDP): NDP + 1,3-bisphosphoglycerate <--> NTP + 3-phosphoglycerate. Here we compared the kinetics of the formation of the complexes of human PGK with d- and its mirror image l-ADP and the effect of 3-phosphoglycerate (PG) on these by exploiting the fluorescence signal of PGK that occurs upon its interaction with nucleotide substrate. Two types of experiment were carried out: equilibrium (estimation of dissociation constants) and stopped-flow (transient kinetics of the interactions). We show that under our experimental conditions (buffer containing 30% methanol, 4 degrees C) PGK binds d- and l-ADP with similar kinetics. However, whereas PG increased the dissociation rate constant for d-ADP by a factor of 8-which is a kinetic explanation for "substrate antagonism"-PG had little effect on this constant for l-ADP. We explain this difference by a molecular modeling study that showed that the beta-phosphates of d- and l-ADP have different orientations when bound to the active site of human PGK. The difference is unexpected because l-ADP is almost as catalytically competent as d-ADP [ Varga, A. et al. (2008) Biochem. Biophys. Res. Commun. 366, 994-1000].
Topics: Adenosine Diphosphate; Binding Sites; Buffers; Catalysis; Cold Temperature; Glyceric Acids; Humans; Kinetics; Methanol; Models, Molecular; Phosphoglycerate Kinase; Stereoisomerism
PubMed: 18288812
DOI: 10.1021/bi7023145 -
Pediatric Hematology and Oncology Aug 2019The human phosphoglycerate kinase-1 enzyme is the first of two energy generating steps in the glycolysis. Since its discovery in 1968, many pathologically mutated forms...
The human phosphoglycerate kinase-1 enzyme is the first of two energy generating steps in the glycolysis. Since its discovery in 1968, many pathologically mutated forms of PGK1 have been described. PGK1 is expressed in all tissues. The clinical manifestations of PGK1 deficiency are some combination of anemia, central nervous system and/or musculoskeletal manifestations. We describe a case of PGK1 in an African-American child, which to our knowledge, has never been described to date. The manifestations of PGK1-Detroit (c.1105A > C (p.Thr369Pro)) include hematologic and central nervous manifestations.
Topics: Black or African American; Amino Acid Substitution; Child, Preschool; Genetic Diseases, X-Linked; Humans; Male; Metabolism, Inborn Errors; Mutation, Missense; Phosphoglycerate Kinase
PubMed: 31424298
DOI: 10.1080/08880018.2019.1639863 -
Biophysical Journal Jul 2018Phosphoglycerate kinase (PGK) is a highly conserved enzyme that is crucial for glycolysis. PGK is a monomeric protein composed of two similar domains and has been the...
Phosphoglycerate kinase (PGK) is a highly conserved enzyme that is crucial for glycolysis. PGK is a monomeric protein composed of two similar domains and has been the focus of many studies for investigating interdomain interactions within the native state and during folding. Previous studies used traditional biophysical methods (such as circular dichroism, tryptophan fluorescence, and NMR) to measure signals over a large ensemble of molecules, which made it difficult to observe transient changes in stability or structure during unfolding and refolding of single molecules. Here, we unfold single molecules of PGK using atomic force spectroscopy and steered molecular dynamic computer simulations to examine the conformational dynamics of PGK during its unfolding process. Our results show that after the initial forced separation of its domains, yeast PGK (yPGK) does not follow a single mechanical unfolding pathway; instead, it stochastically follows two distinct pathways: unfolding from the N-terminal domain or unfolding from the C-terminal domain. The truncated yPGK N-terminal domain unfolds via a transient intermediate, whereas the structurally similar isolated C-terminal domain has no detectable intermediates throughout its mechanical unfolding process. The N-terminal domain in the full-length yPGK displays a strong unfolding intermediate 13% of the time, whereas the truncated domain (yPGKNT) transitions through the intermediate 81% of the time. This effect indicates that the mechanical properties of yPGK cannot be simply deduced from the mechanical properties of its constituents. We also find that Escherichia coli PGK is significantly less mechanically stable as compared to yPGK, contrary to bulk unfolding measurements. Our results support the growing body of observations that the folding behavior of multidomain proteins is difficult to predict based solely on the studies of isolated domains.
Topics: Biomechanical Phenomena; Enzyme Stability; Escherichia coli; Mechanical Phenomena; Molecular Dynamics Simulation; Phosphoglycerate Kinase; Protein Domains; Protein Unfolding; Saccharomyces cerevisiae
PubMed: 29972811
DOI: 10.1016/j.bpj.2018.05.028