Data on the phosphorylation state of the catalytic serine of enzymes in the α-D-phosphohexomutase superfamily

Most enzymes in the α-D-phosphohexomutase superfamily catalyze the reversible conversion of 1- to 6-phosphosugars. They play important roles in carbohydrate and sugar nucleotide metabolism, and participate in the biosynthesis of polysaccharides, glycolipids, and other exoproducts. Mutations in genes encoding these enzymes are associated with inherited metabolic diseases in humans, including glycogen storage disease and congenital disorders of glycosylation. Enzymes in the superfamily share a highly conserved active site serine that participates in the multi-step phosphoryl transfer reaction. Here we provide data on the effects of various phosphosugar ligands on the phosphorylation of this serine, as monitored by electrospray ionization mass spectrometry (ESI-MS) data on the intact proteins. We also show data on the longevity of the phospho-enzyme under various solution conditions in one member of the superfamily from Pseudomonas aeruginosa, and present inhibition data for several ligands. These data should be useful for the production of homogeneous samples of phosphorylated or unphosphorylated proteins, which are essential for biophysical characterization of these enzymes.


a b s t r a c t
Most enzymes in the α-D-phosphohexomutase superfamily catalyze the reversible conversion of 1-to 6-phosphosugars. They play important roles in carbohydrate and sugar nucleotide metabolism, and participate in the biosynthesis of polysaccharides, glycolipids, and other exoproducts. Mutations in genes encoding these enzymes are associated with inherited metabolic diseases in humans, including glycogen storage disease and congenital disorders of glycosylation. Enzymes in the superfamily share a highly conserved active site serine that participates in the multi-step phosphoryl transfer reaction. Here we provide data on the effects of various phosphosugar ligands on the phosphorylation of this serine, as monitored by electrospray ionization mass spectrometry (ESI-MS) data on the intact proteins. We also show data on the longevity of the phospho-enzyme under various solution conditions in one member of the superfamily from Pseudomonas aeruginosa, and present inhibition data for several ligands. These data should be useful for the production of homogeneous samples of phosphorylated or unphosphorylated proteins, which are essential for biophysical characterization of these enzymes. &

Value of the data
The catalytic phosphoserine of enzymes in the α-D-phosphohexomutase superfamily plays an integral role in enzyme mechanism.
We present data on the phosphorylation state of the serine under varying conditions. These data will be useful information for preparation of homogeneous samples of phospho-or dephospho-enzyme for future biophysical and kinetic studies.

Data
The data in this article show the effects of phosphosugar ligands and other variables on the phosphorylation state of the catalytic serine in several enzymes in the α-D-phosphohexomutase superfamily. Data were obtained by ESI-MS. Data on enzyme inhibition by several ligands is also presented.

Incubation with phosphosugar ligands
Ligands tested for effects on phosphorylation included two bisphospho-sugars, and various monophosphosugars (e.g., substrate or product in the enzyme reaction), which have been reported, in different instances, to either phosphorylate or dephosphorylate these enzymes [5,6]. The compounds were prepared as aqueous stock solutions at 1-200 mM, and mixed with protein to determine their effect on the phosphorylation level of the active site serine. For mass spectrometry, enzymes at 40-120 M were incubated with a 6.25 M excess of compound for 18 h at 4°C. Samples were flash frozen and stored at À 80°C until analysis.

ESI-MS data collection and analysis
Analysis of intact proteins by mass spectrometry was done as described previously [7] with a NanoLC-Nanospray QTOF (Agilent 6520) and C8 column chromatography. Expected and observed molecular masses of the proteins are found on Table 1. Duplicate spectra of two identical samples showed phosphorylation levels within 2% of each other, indicating good reproducibility. No degradation of the protein samples was observed during any of the conditions tested.
The percentage phosphorylation was calculated by normalizing the sum of the dephosphorylated and phosphorylated peak heights to 1.0. As the proteins characterized herein are known to be phosphorylated on the conserved active site serine, and ESI-MS data confirmed a single phosphorylation site, no additional attempts were made to localize the site of phosphorylation. The exception to this was StPGM, which showed two phosphorylation sites via ESI-MS (see Supplementary Methods and Fig. S1).

Enzyme inhibition assays
Enzymatic activity of PaPMM was quantified by measuring the formation of glucose 6-phosphate (G6P) in a coupled assay with G6PDH. The conversion of NAD to NADH was monitored by UV-vis spectrophotometry on a CARY 100 spectrophotometer at 25°C, as previously described [3]. Time courses of enzyme activity in the presence of glucosamine 1-phosphate (GlcN1P) and glucosamine 6-phosphate (GlcN6P) were conducted using 0.14 mM enzyme with 0.5 mM of glucose 1,6-bisphosphate (G16P), and 135 mM of substrate, glucose 1-phosphate (G1P).
Ki values for GlcN1P and the substrate analog X1P were determined as follows.For X1P, assays were performed with 0.1 mM enzyme, 1 mM G16P, and 10-500 mM G1P (substrate). For GlcN1P, assays were performed with 0.3 mM enzyme, 0.5 mM G16P, and 6.8-272 mM G1P. Data were fitted to the Michaelis-Menten equation. Apparent K m (for X1 P studies) or apparent k cat (for GlcN1P) values obtained at each inhibitor concentration were fitted using Eq. (1) or (2), respectively, to calculate K I ,

Data on phosphorylation by bisphosphosugars
A broad assessment of the effects of phosphosugar ligands on the phosphorylation of the catalytic serine was conducted on PaPMM, one of the best-characterized members of the superfamily [8][9][10][11]. Phospho-and dephospho-enzyme are easily distinguished in spectra (Fig. 1). Data on the effects of ligands are shown in Table 2. The phosphorylation level can be increased to $ 95% through incubation with G16P, a known activator for the superfamily. An alternate bisphosphosugar, fructose 1,6-bisphosphate (F16P), also increases the phosphorylation level of PaPMM to $ 85%.
Several other sequence-diverse proteins in the superfamily were examined for changes in phosphorylation: BaPNGM, FtPNGM, StPGM, and hPGM1. For all proteins, both G16P and F16P increase phosphorylation level of the active serine (Table 3). For two of the proteins, FtPNGM and StPGM, F16P appears to be somewhat more effective than G16P under the conditions tested, with phosphorylation levels after incubation ranging from 90-100%.
As noted in Section 2.4, ESI-MS of StPGM indicated two phosphorylation sites. To determine if one of these was the catalytic phosphoserine, the protein was subjected to proteolysis and phosphopeptides identified (Supplemental methods). A single phosphopeptide was identified by these studies (Fig. S1), corresponding to residues 134-156, which includes the active site serine (residue 146). Only one region of the protein was not covered by this analysis (residues 42-53), which could contain the second site of phosphorylation as it includes two serines and one threonine.

Data on dephosphorylation of enzymes
Incubations of PaPMM with G1P and G6P and the related sugars GlcN1P and GlcN6P were performed (Table 1). Under the conditions tested, most of the monophosphosugars were associated with a reduction in the level of phosphorylation, by up to 50%. Incubation with GlcN1P, however, substantially reduces the phosphorylation level of PMM/PGM, by $ 90%, consistent with previous observations [12]. GlcN1P was also found to reduce phosphorylation of hPGM1, but had no effect on StPGM or PNGM proteins (Table 3).
Several concentrations of G1cN1P were tested at various time points in incubations with PaPMM. A time course of dephosphorylation (i.e., loss of phosphoryl group due to hydrolysis) in the presence of increasing molar equivalents of GlcN1P is shown in (Fig. 2A). The effect of temperature on the longevity of phosphorylation of PMM/PGM was also assessed. Samples were collected after incubation of the protein at 4°and 37°C for various length of times, and after prolonged storage at À 80°C. The phosphorylation level was unchanged at 4°C for at least two days and at À 80°C for 21 months. At 37°C, a reduction in % phosphorylation was observed within 8 h, with complete loss over three days (Fig. 2B).

Data on the effects of ligands on enzyme activity
To assess interactions between PaPMM and G1cN1P or GlcN6P, these two phosphosugars were tested as inhibitors for the PGM activity (Fig. 3). GlcN6P shows no effect on time courses of enzyme activity at the concentrations tested (Fig. 3A), while GlcN1P shows increasing reductions in activity at higher concentrations (Fig. 3B). As a control, the substrate analog X1P, which has the same stereochemistry as glucose but lacks the O6 hydroxyl necessary for phosphoryl transfer, was also assessed as an inhibitor. The effects of X1P are consistent with those of a competitive inhibitor (Fig.3C), with a K i of 8.6 70.3 μM. GlcN1P shows evidence of noncompetitive inhibition with a K i of 307729 μM (Fig. 3D).
These data provide information on ligands and solution conditions that affect the phosphorylation state and lifetime of the active site serine of enzymes in the α-D-phosphohexomutase superfamily, and may assist with preparation of homogeneous protein samples for further studies.