Because the 32
P-phosphate (and 18
O-phosphate) from PEP is transferred to the catalytic histidine of PGAM1, we wondered whether we were observing a net increase in H11 phosphorylation of PGAM1 or merely observing an exchange of phosphate already present on PGAM1 with phosphate from PEP (as can occur during the interconversion of 2PG and 3PG). To address this issue, we added recombinant PGAM1 to a cell extract in the presence or absence of PEP. These extracts were then subjected to 2D gel electrophoresis and analyzed by Western blot with an antibody to PGAM. Consistent with PEP phosphorylation of H11, a new more acidic isoelectric species of both endogenous and recombinant PGAM1 was detected in the PEP-containing lysate (). No change in the isoelectric forms of PGAM1 was observed when lysates were incubated with ATP instead of PEP (Fig. S10A
). To confirm that this species did indeed represent the H11-phosphorylated form of PGAM1, we added PEP to a cell lysate to phosphorylate PGAM1 and then incubated the reaction either at neutral pH or at pH 2 to chemically disrupt H11 phosphorylation. Analysis of 2D Western blots with an antibody to PGAM1 showed that incubation at pH 2 resulted in loss of the most acidic PGAM1 species (Fig. S10B
). Therefore we concluded that 2D Western blots could be used to assess H11 phosphorylation status in cells. These data also demonstrate that PEP can cause a net increase in H11 phosphorylated PGAM1, and that the phosphorylation of PGAM we observe cannot be accounted for by exchange of the PEP phosphate with a previously phosphorylated H11.
Association of PGAM1 phosphorylation with conversion of PEP into pyruvate in the absence of pyruvate kinase
To determine if H11 phosphorylated PGAM1 is catalytically competent for enzymatic activity, we assayed the ability of PEP-phosphorylated PGAM1 to convert 3PG to 2,3-bisphosphoglycerate (2,3-BPG), the intermediate in 3PG to 2PG conversion (23
). Recombinant His-tagged PGAM1 was incubated with PEP and cell extract to allow phosphorylation on His11 and the protein was recovered through association with Ni-agarose beads. Addition of 3PG to the recovered PGAM1 resulted in 2,3-BPG production as determined by selected reaction monitoring (SRM) using hybrid quadrupole linear ion trap mass spectrometry (Fig. S11
). Thus, phosphorylation of PGAM1 by PEP leads to an enzyme species that is active to carry out the known enzymatic function of PGAM1.
We fractionated a cell lysate over a weak anion exchange column and isolated the PEP-dependent PGAM1 phosphorylating activity in a fraction that was separate from the enolase-containing fraction as determined by both enzyme activity assays and Western blot (; Fig. S12A
). The fraction containing the PGAM1 phosphorylating activity was also separated from pyruvate kinase as determined by both enzyme activity assay and Western blot. In support of this finding that pyruvate kinase is not involved in the transfer of phosphate to PGAM1, we found that shRNA knockdown of pyruvate kinase resulted in the enhanced ability of a cell lysate to transfer 32
P from 32
P-labeled PEP to PGAM1 with no change in the level of PGAM1 protein (Fig. S12, B–D
). It has been reported that a complex containing the nucleoside diphosphate kinase nm23 and GAPDH can phosphorylate PGAM1 (24
). However, neither GAPDH nor nm23 co-purify in significant quantities with the PEP-dependent PGAM1-phosphorylating activity (Fig. S12E
), suggesting that these proteins are not involved in the activity we observe.
We further investigated the consequences of metabolizing PEP through phosphotransfer to PGAM1. To test whether PEP is converted to pyruvate during the phosphotransfer reaction, we incubated the anion exchange fraction containing the PGAM1 phosphorylating activity (D500 fraction) with 13
C-labeled PEP and recombinant PGAM1. Similar reactions with a whole cell lysate served as a positive control and a 13
C-labeled PEP sample that contained no cellular material served as a negative control. We then extracted metabolites from the resulting reactions to study the products derived from the labeled PEP by [1
C] HSQC NMR (25
). We detected 13
C-labeled pyruvate in the whole cell lysate as determined by an isolated peak corresponding to a 13
C-labeled methyl group of pyruvate (26
). No pyruvate was observed in the mock-treated control, indicating that PEP did not undergo spontaneous dephosphorylation and tautomerization to pyruvate under the reaction conditions. Incubation with the anion exchange fraction containing the PGAM1 phosphorylating activity also caused generation of pyruvate. The amount of 13
C-labeled pyruvate produced by the D500 fraction was approximately 50% of the amount produced by a whole cell lysate (). Thus, one or more factors in the partially purified fraction from cell lysates lacking pyruvate kinase mediates PEP-dependent phosphorylation of PGAM1 and conversion of PEP to pyruvate.
C-labeled pyruvate was produced from PEP in the D500 fraction at a rate of approximately 30–60 μM/minute. Given that the number of PGAM1 molecules in this fraction is small relative to the number of PEP molecules consumed, this fraction must also contain the ability to release inorganic phosphate (Pi
). To determine whether Pi
production from PEP also occurred in this fraction, we incubated the D500 fraction with 32
P-labeled PEP and recombinant PGAM1 and assessed the release of 32
over time (Fig. S13A
). We also tested whether the rate of Pi
production was enhanced by PGAM1. Addition of PGAM1 should have no impact on (or decrease) the rate of Pi
production from PEP if this reaction is independent of PEP-mediated PGAM1 phosphorylation. However, PGAM1 addition stimulated Pi
production in the fraction lacking pyruvate kinase (Fig. S13B
) suggesting a link between PEP-dependent PGAM1 phosphorylation and PEP to pyruvate conversion with Pi
release. These results suggest that release of Pi
from either phosphorylated PGAM1, PEP, or both occurs in this fraction; and accounts for how PEP to pyruvate conversion can occur at a rate that is super-stoichiometric to the amount of PGAM1 present.