Phosphoglycerates and Protein Phosphorylation: Identification of a Protein Substrate as Glucose‐1,6‐Bisphosphate Synthetase

Abstract

We have previously reported the occurrence of two endogenous protein phosphorylation systems in mammalian brain that are enhanced in the presence of 3-phosphoglycerate (3PG) and ATP. We present here a study of one of these systems, the phosphorylation of the 72-kDa protein (3PG-PP72). This system was separated into the substrate, 3PG-PP72, and a kinase by ammonium sulfate fractionation, hydroxyapatite chromatography, and hydrophobic interaction HPLC. The substrate protein was shown to be directly phosphorylated with [1-32P]1,3-bisphosphoglycerate [( 1-32P]1,3BPG) with an apparent Km of 1.1 nM. Nonradioactive 1,3BPG inhibited 32P incorporation in the presence of [gamma-32P]ATP and 3PG. Phosphopeptide mapping and phosphoamino acid analyses indicated that the site of phosphorylation of 3PG-PP72 observed in the presence of 3PG and ATP is a serine residue identical to that observed with [1-32P]1,3BPG. Moreover, [32P]phosphate incorporated into 3PG-PP72 in the presence of 3PG and ATP was removed by subsequent incubation with glucose-1-phosphate or glucose-6-phosphate. Finally, 3PG-PP72 showed chromatographic behaviors identical to those of glucose-1,6-bisphosphate (G1,6P2) synthetase. Based upon these observations, we conclude that 3PG-PP72 is G1,6P2 synthetase and that it is phosphorylated directly by 1,3BPG, which is formed from 3PG and ATP by 3PG kinase present in a crude 3PG-PP72 preparation.

Molecular Identification of Mammalian Phosphopentomutase and Glucose-1,6-bisphosphate Synthase, Two Members of the α-D-Phosphohexomutase Family

“…However, this enzyme proved to have a very low phosphomutase activity on the three tested substrates (glucose 1-phosphate, ribose 1-phosphate, and deoxyribose 1-phosphate). The finding that PGM2L1 is expressed at a high level in brain, its molecular mass of 72 kDa and its (low) phosphomutase activity are all properties that PGM2L1 shares with glucose-1,6-bisphosphate synthase (17,18,26), an enzyme whose molecular identity had not yet been established.…”

“…As previously shown, this reaction is the first step of aldose-bisphosphate synthesis. The second step is the transfer of the phosphate group from the phosphorylated serine onto a suitable sugar-phosphate acceptor (18,26).…”

Glycolysis and Glutamate Accumulation into Synaptic Vesicles

“…The column was equilibrated with 0.4 M sodium phosphate buffer (pH 3.2), and glycolytic intermediates and nucleotides were eluted isocratically, as described previously (14). Retention times for glycolytic intermediates were 4.1 min for DHAP and GAP, 5.3 min for P i , 5.9 min for 2-phosphoglycerate and 3-PG, 6.0 min for ADP, 7.4 min for phosphoenolpyruvate, 15.4 min for 1,3-BPG, and 30.0 min for ATP.…”

“…Fleck et al (7) have shown that substantial reduction of extracellular glucose results in a decrease in stimulus-evoked Glu release, with no changes in ATP levels. These studies together suggest that glycolysis or glycolytic intermediate(s) are necessary for normal synaptic transmission independent of global cellular ATP levels.In an attempt to reveal the underlying mechanism of hypoglycemia-induced aberrant synaptic transmission, we previously explored the possibility that glycolytic intermediates could modify proteins localized in the nerve ending (13,14). 3-Phosphoglycerate (3-PG) 1 was demonstrated to stimulate phosphorylation of 155-and 72-kDa proteins.…”

“…In an attempt to reveal the underlying mechanism of hypoglycemia-induced aberrant synaptic transmission, we previously explored the possibility that glycolytic intermediates could modify proteins localized in the nerve ending (13,14). 3-Phosphoglycerate (3-PG) 1 was demonstrated to stimulate phosphorylation of 155-and 72-kDa proteins.…”