An enzymatic method for [32P]phosphoenolpyruvate synthesis

Mattoo, Roshan L.; Waygood, E. B.

doi:10.1016/0003-2697(83)90372-x

Cited by 47 publications

(26 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Indeed, [ 32 P]-PEP is synthesized from commercial [ 32 P]-ATP using pyruvate kinase (Mattoo and Waygood, 1983; Vander Heiden et al, 2010), and despite a step to enrich for [ 32 P]-PEP, these preparations are contaminated by small quantities of [γ- 32 P]-ATP (Figure 2A). Preparative HPLC of [ 32 P]-PEP allowed for quantitative separation from the contaminating [γ- 32 P]-ATP.…”

Section: Resultsmentioning

confidence: 99%

Lack of Evidence for PKM2 Protein Kinase Activity

et al. 2015

View full text Add to dashboard Cite

Summary The role of pyruvate kinase M2 (PKM2) in cell proliferation is controversial. A unique function of PKM2 proposed to be important for the proliferation of some cancer cells involves the direct activity of this enzyme as a protein kinase; however, a detailed biochemical characterization of this activity is lacking. Using [32P]-phosphoenolpyruvate (PEP) we examine the direct substrates of PKM2 using recombinant enzyme and in vitro systems where PKM2 is genetically deleted. Labeling of some protein species from [32P]-PEP can be observed, however most were dependent on the presence of ADP, and none were dependent on the presence of PKM2. In addition, we also failed to observe PKM2-dependent transfer of phosphate from ATP directly to protein. These findings argue against a role for PKM2 as a protein kinase.

show abstract

Section: Resultsmentioning

confidence: 99%

Lack of Evidence for PKM2 Protein Kinase Activity

et al. 2015

View full text Add to dashboard Cite

show abstract

“…The Quik-Change site-directed mutagenesis kit was obtained from Stratagene. [ 32 P]Phosphoenolpyruvate (PEP) was produced as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

The Aspartyl Replacement of the Active Site Histidine in Histidine-containing Protein, HPr, of the Escherichia coliPhosphoenolpyruvate:Sugar Phosphotransferase System Can Accept and Donate a Phosphoryl Group

Napper¹,

Delbaere

Waygood

1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

The active site residue, His 15 , in histidine-containing protein, HPr, can be replaced by aspartate and still act as a phosphoacceptor and phosphodonor with enzyme I and enzyme IIA glucose , respectively. Other substitutions, including cysteine, glutamate, serine, threonine, and tyrosine, failed to show any activity. Enzyme I K m for His 15 3 Asp HPr is increased 10-fold and V max is decreased 1000-fold compared with wild type HPr. The phosphorylation of Asp 15 led to a spontaneous internal rearrangement involving the loss of the phosphoryl group and a water molecule, which was confirmed by mass spectrometry. The protein species formed had a higher pI than His 15 3 Asp HPr, which could arise from the formation of a succinimide or an isoimide. Hydrolysis of the isolated high pI form gave only aspartic acid at residue 15, and no isoaspartic acid was detected. This indicates that an isoimide rather than a succinimide is formed. In the absence of phosphorylation, no formation of the high pI form could be found, indicating that phosphorylation catalyzed the formation of the cyclization.

show abstract

“…Hydrophobic interaction high performance liquid chromatography (Synchropak) replaced or was added to the final step in the purification of CheW, CheA, CheY, and Enzyme I. Phosphoenolpyruvate carboxykinase was partially purified (free of PEP carboxylase, NADH oxidase and pyruvate kinase) by gel filtration (Sephadex G-100) of a 40-60% ammonium sulfate cut from the crude extract. [32P]PEP was synthesized with phosphoenolpyruvate carboxykinase and purified as described by Mattoo and Waygood [17]. Overexpression vectors pDV4 (cheA cheW; [18]), pRL22 (che Y; [13]), pDS20 (ptsH.…”

Section: Protein Purificationmentioning

confidence: 99%