The use of mass spectrometry to examine the formation and hydrolysis of the phosphorylated form of phosphoglycerate mutase

Nairn, Jacqueline; Krell, Tino; Coggins, John R.; Pitt, Andrew R.; Fothergill‐Gilmore, Linda A.; Walter, Rebecca A.; Price, Nicholas C.

doi:10.1016/0014-5793(95)00044-a

Cited by 24 publications

(19 citation statements)

References 17 publications

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“…4d). These hydrogen bonds also contribute to the stabilisation of the phosphorylated-histidine group captured in the crystal, which, otherwise, is generally unstable in the aqueous solution with a half-life around 30 minutes 20 .…”

Section: Resultsmentioning

confidence: 99%

Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing the active conformation

et al. 2013

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How oncogenic signalling coordinates glycolysis and anabolic biosynthesis in cancer cells remains unclear. We recently reported that the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) regulates anabolic biosynthesis by controlling intracellular levels of its substrate 3-phosphoglycerate (3-PG) and product 2-phosphoglycerate (2-PG). Here we report a novel mechanism in which Y26 phosphorylation enhances PGAM1 activation through release of inhibitory E19 that blocks the active site, stabilising cofactor 2,3-bisphosphoglycerate binding and H11 phosphorylation. We also report the crystal structure of H11-phosphorylated PGAM1 and find that phospho-H11 activates PGAM1 at least in part by promoting substrate 3-PG binding. Moreover, Y26-phosphorylation of PGAM1 is common in human cancer cells and contributes to regulation of 3-PG and 2-PG levels, promoting cancer cell proliferation and tumour growth. Since PGAM1 as a negative transcription target of TP53 is commonly upregulated in human cancers, these findings suggest that Y26 phosphorylation represents an additional acute mechanism underlying PGAM1 upregulation.

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Section: Resultsmentioning

confidence: 99%

Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing the active conformation

et al. 2013

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“…The resulting model has 0.28 occupancy phosphohistidine and 0.72 occupancy histidine plus three water molecules. We had no reason to expect histidine phosphorylation prior to crystallization, because the half-life of the phosphohistidine is expected to be of the order of 35 min, as observed with the S. cerevisiae enzyme (25).…”

Section: Methodsmentioning

confidence: 99%

High Resolution Structure of the Phosphohistidine-activated Form of Escherichia coli Cofactor-dependent Phosphoglycerate Mutase

Bond

White²,

Hunter

2001

Journal of Biological Chemistry

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The active conformation of the dimeric cofactor-dependent phosphoglycerate mutase (dPGM) from Escherichia coli has been elucidated by crystallographic methods to a resolution of 1.25 Å (R-factor 0.121; R-free 0.168). The active site residue His 10 , central in the catalytic mechanism of dPGM, is present as a phosphohistidine with occupancy of 0.28. The structural changes on histidine phosphorylation highlight various features that are significant in the catalytic mechanism. The Cterminal 10-residue tail, which is not observed in previous dPGM structures, is well ordered and interacts with residues implicated in substrate binding; the displacement of a loop adjacent to the active histidine brings previously overlooked residues into positions where they may directly influence catalysis. E. coli dPGM, like the mammalian dPGMs, is a dimer, whereas previous structural work has concentrated on monomeric and tetrameric yeast forms. We can now analyze the sequence differences that cause this variation of quaternary structure.Phosphoglycerate mutases (PGMs) 1 are enzymes involved in glycolysis and gluconeogenesis. They can be subdivided into two types: cofactor-dependent PGM (dPGM) and cofactor-independent PGM (iPGM). Whereas vertebrates, yeasts, and many bacteria have only dPGM, and higher plants, nematodes, archaea, and many other bacteria have only iPGM, a small number of bacteria including Escherichia coli have both (1).dPGMs have three catalytic activities. The main activity is that of a mutase (EC 5.4.2.1), catalyzing the interconversion between 2-phosphoglycerate and 3-phosphoglycerate. A second activity is as a phosphatase (EC 3.1.3.13), converting 2,3-bisphosphoglycerate and water to 3-phosphoglycerate or 2-phosphoglycerate and phosphate. The third activity is the synthase activity (EC 5.4.2.4), where 1,3-bisphosphoglycerate is converted to 2,3-bisphosphoglycerate. The label "cofactor-dependent" comes from the observation in vitro that to be active, the native protein must be phosphorylated by 2,3-bisphosphoglycerate.The crystal structure of Saccharomyces cerevisiae dPGM 2 was first published in 1974 (Protein Data bank code 3PGM (2, 3)), and structures of different crystal forms and inhibitor complexes at increasing resolution have followed (4PGM, 5PGM, 1BQ3, 1BQ4, 1QHF (4 -7)). Schizosaccharomyces pombe dPGM has been studied by NMR, and a backbone assignment has been published (8). In most organisms for which a dPGM has been characterized, including E. coli and mammals, the active enzyme exists as a dimer. S. cerevisiae dPGM, however, is tetrameric, and S. pombe dPGM is monomeric. Most recently, the crystal structure of the iPGM from Bacillus stearothermophilus has been solved (9, 10), highlighting the absence of any similarity to dPGM in all aspects except its main mutase activity.dPGM is the archetype of the "phosphoglycerate mutaselike" protein fold superfamily (SCOP (11)), which also contains the phosphatase domain of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family as well as prostatic aci...

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“…For ScPGM to be catalytically competent His8 must be phosphorylated, which requires trace amounts of the cofactor 2,3-bisphosphoglycerate (2,3-BPG) to be present in vivo (Nairn et al, 1995). Site-directed mutagenesis has shown that replacing either of the active-site histidines (His8 and His181) with alanine dramatically reduces ScPGM isomerase activity by preventing the formation of the phosphohistidine moiety (White & Fothergill-Gilmore, 1992).…”

Section: Introductionmentioning

confidence: 99%

Structure of a phosphoglycerate mutase:3-phosphoglyceric acid complex at 1.7 Å

Crowhurst

Dalby

Isupov

et al. 1999

Acta Crystallogr D Biol Cryst

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The use of mass spectrometry to examine the formation and hydrolysis of the phosphorylated form of phosphoglycerate mutase

Abstract: The results are discussed in terms of the kinetic properties of the enzymes. Mass spectrometry would appear to be a powerful method to study the formation and breakdown of phosphorylated proteins, processes which are of widespread significance in regulatory mechanisms.

Cited by 24 publications

References 17 publications

Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing the active conformation

Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing the active conformation

High Resolution Structure of the Phosphohistidine-activated Form of Escherichia coli Cofactor-dependent Phosphoglycerate Mutase

Structure of a phosphoglycerate mutase:3-phosphoglyceric acid complex at 1.7 Å

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