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Pyruvate kinase

About: Pyruvate kinase is a research topic. Over the lifetime, 5683 publications have been published within this topic receiving 180020 citations. The topic is also known as: ATP:pyruvate 2-O-phosphotransferase & phosphoenolpyruvate kinase.


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01 Sep 2010
TL;DR: In this paper, the authors demonstrate that phosphoenolpyruvate (PEP) can act as a phosphate donor in mammalian cells because PEP participates in the phosphorylation of the glycolytic enzyme phosphoglycerate mutase (PGAM1) in PKM2-expressing cells.
Abstract: Proliferating cells, including cancer cells, require altered metabolism to efficiently incorporate nutrients such as glucose into biomass. The M2 isoform of pyruvate kinase (PKM2) promotes the metabolism of glucose by aerobic glycolysis and contributes to anabolic metabolism. Paradoxically, decreased pyruvate kinase enzyme activity accompanies the expression of PKM2 in rapidly dividing cancer cells and tissues. We demonstrate that phosphoenolpyruvate (PEP), the substrate for pyruvate kinase in cells, can act as a phosphate donor in mammalian cells because PEP participates in the phosphorylation of the glycolytic enzyme phosphoglycerate mutase (PGAM1) in PKM2-expressing cells. We used mass spectrometry to show that the phosphate from PEP is transferred to the catalytic histidine (His11) on human PGAM1. This reaction occurred at physiological concentrations of PEP and produced pyruvate in the absence of PKM2 activity. The presence of histidine-phosphorylated PGAM1 correlated with the expression of PKM2 in cancer cell lines and tumor tissues. Thus, decreased pyruvate kinase activity in PKM2-expressing cells allows PEP-dependent histidine phosphorylation of PGAM1 and may provide an alternate glycolytic pathway that decouples adenosine triphosphate production from PEP-mediated phosphotransfer, allowing for the high rate of glycolysis to support the anabolic metabolism observed in many proliferating cells.

460 citations

Journal ArticleDOI
TL;DR: It is suggested that mixed-function oxidation system-catalyzed inactivation of enzymes is a regulatory step in enzyme turn-over and the implication of oxidative inactivation reactions in ageing is suggested by the fact that many of the enzymes inactivated by mixed- function oxidation systems are known to accumulate as inactive forms during ageing.
Abstract: Several mixed-function oxidation systems catalyze the inactivation of Escherichia coli glutamine synthetase. Inactivation involves modification of a single histidine residue in each enzyme subunit and makes the enzyme susceptible to proteolytic degradation. We show here that 10 key enzymes in metabolism are inactivated by a bacterial NADH oxidase and by an oxidase system comprised of NADPH, cytochrome P-450 reductase, and cytochrome P-450 isozyme 2 from rabbit liver microsomes. Most of the inactivatable enzymes require a divalent cation for activity and all but one (enolase) possess a nucleotide binding site. Glutamine synthetase, pyruvate kinase, and phosphoglycerate kinase are protected from inactivation by their substrates; substrate protection of other enzymes was not tested. We propose that inactivation involves mixed-function oxidization system-catalyzed synthesis of H2O2 and reduction of Fe(III) to Fe(II) followed by oxidation of enzyme-bound Fe(II) by H2O2 to generate oxygen radicals that attack a histidine (or other oxidizable amino acid) at the metal binding site of the enzyme. This is supported by the following: (i) most of the inactivation reactions are inhibited by EDTA and by catalase, (ii) both mixed-function oxidation systems reduce Fe(III), and (iii) H2O2 together with Fe(II) catalyzes nonenzymic inactivation of glutamine synthetase. In view of the fact that inactivation of glutamine synthetase makes it susceptible to proteolytic degradation, it is possible that mixed-function oxidation system-catalyzed inactivation of enzymes is a regulatory step in enzyme turn-over. In addition, the implication of oxidative inactivation reactions in ageing is suggested by the fact that many of the enzymes inactivated by mixed-function oxidation systems are known to accumulate as inactive forms during ageing.

460 citations

Book ChapterDOI
TL;DR: This chapter describes the assay, purification, and properties of glutamine synthetase from Escherichia coli, which is of cardinal importance in biosynthetic metabolism and is a strategic target for cellular regulation.
Abstract: Publisher Summary This chapter describes the assay, purification, and properties of glutamine synthetase. The synthesis of glutamine in microorganisms may be regarded as the first step in a highly branched pathway, which leads ultimately to the biosynthesis of a large number of different compounds, including tryptophan, adenylic acid, cytidylic acid, and glucosamine 6-phosphate. Glutamine synthetase from Escherichia coli is, therefore, of cardinal importance in biosynthetic metabolism and is a strategic target for cellular regulation. The purified enzyme may be assayed by measuring the production of inorganic phosphate in the biosynthetic reaction. Alternatively, a continuous recording of catalytic activity may be achieved by coupling the production of adenosine diphosphate (ADP) in the biosynthetic assay to the oxidation of diphosphopyridine nucleotide (DPNH) by the addition of phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase in excess. Preparations of purified glutamine synthetase may differ from one another in the amount of covalently bound adenosine monophosphate (AMP) residues that they contain. Mn 2+ stabilizes the native dodecameric structure of the enzyme thus, protecting the enzyme from reacting with sulfhydryl reagents and other mild denaturants.

453 citations

Journal ArticleDOI
10 Oct 2013-Cell
TL;DR: The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism as mentioned in this paper, but it is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors.

435 citations

Journal ArticleDOI
TL;DR: The results suggest that AMPK is involved in the inhibition of glucose-activated gene expression but not in the induction pathway, and demonstrates that the two mutants generated will provide valuable tools for studying the wider physiological role of AMPK.
Abstract: In the liver, glucose induces the expression of a number of genes involved in glucose and lipid metabolism, e.g., those encoding L-type pyruvate kinase and fatty acid synthase. Recent evidence has indicated a role for the AMP-activated protein kinase (AMPK) in the inhibition of glucose-activated gene expression in hepatocytes. It remains unclear, however, whether AMPK is involved in the glucose induction of these genes. In order to study further the role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (alpha1(312)) acts as a constitutively active kinase, while the other (alpha1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to express these mutants in primary rat hepatocytes in culture in order to determine their effect on AMPK activity and the transcription of glucose-activated genes. Expression of alpha1(312) increased AMPK activity in hepatocytes and blocked completely the induction of a number of glucose-activated genes in response to 25 mM glucose. This effect is similar to that observed following activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of alpha1DN markedly inhibited both basal and stimulated activity of endogenous AMPK but had no effect on the transcription of glucose-activated genes. Our results suggest that AMPK is involved in the inhibition of glucose-activated gene expression but not in the induction pathway. This study demonstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.

421 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023215
2022201
2021147
2020166
2019150
2018138