2. Phosphoglycerate Mutase 1E58 References Images
Created by Sana Hafeez
Phosphoglycerate mutase (PGM; PDB ID: 1E58), studied in Escherichia coli, has an isoelectric
point of 5.86, and a molecular weight of 28,425.21 daltons (4). PGMs are transferase enzymes that
exist in two evolutionarily unrelated forms: cofactor-dependent PGM (dPGM) and cofactor-
independent PGM (iPGM) (8). dPGM exists as a homodimer composed of α/β-subunits with a
twofold symmetry about the central core. All dPGMs, whether monomeric, dimeric, or tetrameric,
have the same essential activity; however, they differ in their quaternary assemblies (2). The dPGM
homodimer in E. coli is formed when the C-strands of two monomers are aligned in an antiparallel
fashion. Residues 58–78 and 136–139 form dimer interactions that join the two monomers
together. Residues 58–63 in the monomer form hydrophobic contacts that also participate in
dimer formation. Each monomer is asymmetric and contains an active-site histidine residue. The
twofold axis of the dimer contains a chloride ion.
The dPGM enzyme catalyzes the reversible interconversion of 3-phosphoglycerate (3-PGA) and
2-PGA in step eight of glycolysis and gluconeogenesis (see Image 1). For this reaction to be
catalyzed, the cofactor 2,3-bisphosphoglycerate (2,3-BPG) must be present (5). The synthase
activity of dPGM converts 1,3-BPG to 2,3-BPG. 1,3-BPG phosphorylates the active-site histidine,
His-10, making dPGM catalytically active (3). In the inactive conformation, His-10 is
dephosphorylated (see Image 2). The phosphatase activity deactivates dPGM, in which 2,3-BPG is
hydrolyzed to 2- or 3-PGA and phosphate. The following slide depicts the closed cavities found in
PGM.
The active-site residue, His-10, is the nucleophilic histidine that participates in dPGM's catalytic
mechanism and is phosphorylated to phosphohistidine, occupying 0.28 � in its active
conformation. Although there are two histidines, His-10 and His-183, that play a role in catalysis,
the active-site histidines refer specifically to His-10, shown as X10 in the images because of
histidine phosphorylation of each subunit. Histidine phosphorylation causes key structural changes
that are important for the catalytic mechanism of dPGM. With the exception of the final two
residues, the C-terminal tail of the protein is ordered when dPGM is phosphorylated. This ordered
conformation inhibits solvent access, which prevents phosphoenzyme hydrolysis. When the
enzyme is dephosphorylated, it attracts the BPGs, 1,3-BPG and 2,3-BPG, that ultimately
phosphorylate His-10. However, when dPGM is phosphorylated, the C-terminal tail can be
accessed by 2-PGA and 3-PGA. Once the tail is in place, the shielding of positive charge lowers
the affinity for the larger and more negatively charged BPGs (1).
The polypeptide chain folds to form each subunit that consists of a central core. The central core
is made up of a six-stranded β-pleated sheet, denoted C-B-D-A-E-F, in which all are parallel with
the exception of E. Furthermore, the six-stranded β-sheet is surrounded by six α-helices (7). The
active site is located on the edge of the C terminus of the β-sheet and is made up of stretches of
sequence dispersed throughout the amino acid sequence (Arg-9, His-10, Gly-11, Asn-16, Arg-61,
Glu-88, His-183, and Gly-184). These sequences are part of the catalytic machinery and interact to
form hydrogen bonds. The catalytic machinery is located toward the middle and extends to the C
terminus of the α-carbon backbone of the monomer.
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3. The active site of dPGM is 16� deep and 10 by 8 � wide and is in the shape of a cup. The
volume of this active site is ~1200 �3 and contains two sulfate ions that form hydrogen bonds
with His-10 and Arg-61. The active site catalytic machinery is made up of the residues that are
closest to His-10: Arg-9, Gly-11, Asn-16, Arg-61, Glu-88, His-183, and Gly-184. Residues
involved in the substrate binding region include Ser-13, Thr-22, Gly-23, Arg-89, Tyr-91, Lys-99,
Arg-115, and Arg-116. Arg-115 and Arg-116 also form part of the active site opening, along with
Asn-19 and Asp-1085. The active site cavity is lined by atoms of 43 residues (9–23, 36, 61, 88–91,
99, 111–116, 183–188, 203–209, and 239–247) that are involved in three major functions: they are
part of the catalytic machinery, responsible for substrate binding, and are the site of access where
the substrates enter and the products leave. The His-10 side chain is stabilized in the
dephosphorylated and phosphorylated forms through hydrogen bonding between the N delta 1 of
the imidazole ring and the adjacent Gly-11 amide oxygen. In the phosphorylated form, the
hydrogen bond length decreases, allowing residues 9–22 to move. The residue adjacent to His-10,
Asn-16, alters its side chain conformation in order for the N delta 2 of the His-10 imidazole ring to
hydrogen bond with the phosphate oxygen and the O delta 1 of Asn-16 to participate in a CH—O
hydrogen bond with C epsilon 1 of His-10. The 12-residue α-helix that makes up the active site is
noticeable in the ribbon model because the helix also lies more perpendicular to the β-sheet than
other surrounding helices. The aliphatic segments of Arg-9 and Arg-61 side chains contribute to
hydrophobic contacts that orient and stabilize the imidazole ring of His-10 (1).
Arg-115 and Arg-116 provide a shift that causes the substrate at the active site to induce a
change between active and inactive forms by making the interactions with the tail residues more or
less favorable. In its active form, the tail interacts with Asp-108, allowing the conformation to be
more stable. The conformation of Asn-16 preserves phosphohistidine by forming two hydrogen
bonds with the phosphohistidine. Asn-16 lies on the loop that is formed by residues 9–21 and is
constrained to its active form by the interaction of Asn-19 with the C-terminal tail residues
238–247. The secondary structure of the C-terminal tail is a β-hairpin that is based around a β-turn
from residues 243–246. The β-hairpin motif extends across the active-site opening, forming
hydrogen bonds with the residues of the rim and substrate-binding region (2).
Several drugs deactivate dPGM catalytic activity. The deactivating reaction is stimulated by the
presence of an activator molecule such as vanadate, VO
3−
, a potent inhibitor of the dPGM mutase
activity. VO
3−
is useful because the structure of vanadate–dPGM complex represents the
dephosphorylated, inactive conformation of dPGM, which can be used to compare with the
phosphorylated, active form. Such a comparison helps elucidate the specific roles of key amino
acid residues and can be used to study the different oligomerization states of dPGMs (6).
PGM in E. coli is similar to PGM in Saccharomyces cerevisiae (PDB ID: 5PGM-E). PGM in S.
cerevisiae has a similar primary structure to PGM in E. coli (E value = 3 � 10
−65
) (4). The two
proteins also have similar tertiary structures (Z score = 34.7) (4). Furthermore, the average
distance between the backbones of superimposed proteins, the root mean squared deviation
(rmsd), is 1.3 � (4). PGM in S. cerevisiae acts as a catalyst in the transfer of phosphate groups
within the carbon atoms of phosphoglycerates, a key function in glycolysis. S. cerevisiae PGM's
role in glycolysis and gluconeogenesis is similar to that of PGM in E. coli. The two proteins have
similar primary and tertiary structures, which explain their similar function. Despite the similarities in
function, there are a few notable differences. Unlike the E. coli dimeric PGM, S. cerevisiae PGM is
a homotetramer composed of four subunits that are identical, having a total molecular weight of 112
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4. kD (larger than E. coli PGM) (5). The S. cerevisiae PGM active site also contains two histidine
residues, His-8 and His-181, whereas the E. coli PGM active site contains two different histidine
residues, His-10 and His-183. In E. coli dPGM, His-10 is phosphorylated, whereas His-8 is
phosphorylated in S. cerevisiae (7). The slight differences in the primary structures of E. coli and S.
cerevisiae PGMs account for the differences in function of the two proteins.
Most PGMs have similar structures and have particular catalytic functions in glycolysis and
gluconeogenesis. This pathway is vital in both lower and higher organisms, and is conserved
among most living things. Phosphoglycerate mutase deficiency in humans causes muscular
dystrophy. This deficiency in PGM function can easily be controlled by monitoring lifestyle and
eating habits.
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5. Phosphoglycerate Mutase 1E58 References Images
References
1. Johnsen, U., and Sch�nheit, P., 2007. Characterization of cofactor-dependent and cofactor-independent
phosphoglycerate mutases from Archaea. Biomedical and Life Sciences 11:647–657.
2. White, M. F., Fothergill-Gilmore, L. A., Kelly, S. M., and Price, N. C., 1993. Dissociation of the tetrameric
phosphoglycerate mutase from yeast by a mutation in the subunit contact region. Biochemistry Journal
295:743–748.
3. Bond, C. S., White, M. F., and Hunter, W. N., 2001. High resolution structure of the phosphohistidine-
activated form of Escherichia coli cofactor-dependent phosphoglycerate mutase. Journal of Biological
Chemistry 276:3247–3253.
4. Holm, L., and Rosenstr�m, P., 2010. Dali server: Conservation mapping in 3D. Nucleic Acids Research
38:W545–W549.
5. Hajigeorgiou, G. M., Kawashima, N., Bruno, A., et al., 1999. Manifesting heterozygotes in a Japanese
family with a novel mutation in the muscle-specific phosphoglycerate mutase (PGAM-M) gene.
Neuromuscular Disorders 9:399–402.
6. Bond, C. S., White, M. F., and Hunter, W. N., 2002. Mechanistic implications for Escherichia coli cofactor-
dependent phosphoglycerate mutase based on the high resolution crystal structure of a vanadate complex.
Journal of Molecular Biology 316:1071–1081.
7. Winn, S. I., Watson, H. C., Harkins, R. N., and Fothergill, L. A., 1981. Structure and activity of
phosphoglycerate mutase. Philosophical Transactions of the Royal Society of London. Series B, Biological
Sciences 293:121–130.
8. Fraser, H. I., Kvaratskhelia, M., and White, M. F., 1999. The two analogous phosphoglycerate mutases of
Escherichia coli. FEBS Letters 455:344–348.
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