Phosphoglycerate Mutase
Created by Sana Hafeez
Phosphoglycerate mutase (PGM) (PDB ID = 1E58), studied in Escherichia coli (E. coli), has an isoelectric point of 5.86 and a molecular weight of 28425.21 Daltons (4). PGMs are transferase enzymes that exist in two evolutionarily unrelated forms: cofactor-dependent phosphoglycerate mutase (dPGM) and cofactor-independent phosphoglycerate mutase (iPGM) (8). Cofactor-dependent phosphoglycerate mutase exists as a homodimer composed of alpha/beta subunits with a
two-fold 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 two-fold axis of the dimer contains a
chloride ion.
The dPGM enzyme catalyzes the reversible interconversion of 3-phosphoglycerate (3-PGA) and 2-phosphoglycerate (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-bisphosphoglycerate (1,3-BPG) to 2,3-BPG. 1,3-BPG phosphorylates the
active site histidine, His10, making dPGM catalytically active (3). In the inactive conformation, His10 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 0.28Å 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 graphics due to 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 bisphosphoglycerates, 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 bisphosphoglycerates (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 beta-pleated sheet, denoted C-B-D-A-E-F, in which all are parallel with the exception of E. Furthermore, the six-stranded beta-sheet is surrounded by
six alpha-helices (7). The active site is located on the edge of the C-terminus of the beta-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 towards the middle and extends to the C-terminus of the
alpha carbon backbone of the monomer.
The
active site of dPGM is 16Å deep and 10 by 8 Å wide and is in the shape of a cup. The volume of this active site is ~1200 Å3 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 His10. The
12-residue alpha-helix that makes up the active site is noticeable in the ribbon model because the helix also lies more perpendicular to the beta-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 beta-hairpin that is based around a
beta-turn from residues 243-246. The beta-hairpin motif extends across the active site opening, forming hydrogen bonds with the residues of the rim and substrate-binding region (2).
There are several drugs that deactivate dPGM catalytic activity. The deactivating reaction is stimulated by the presence of an activator molecule such as vanadate, VO3-, a potent inhibitor of the dPGM mutase activity. VO3- is useful because the structure of vanadate-dPGM complex represents the dephosphorylated, inactive conformation of dPGM, which can be used to compare to 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).
Phosphoglycerate mutase (PGM) in E. coli is similar to
PGM in Saccharomyces cerevisiae (S. cerevisiae) (PDB ID = 5PGM-E). PGM in S. cerevisiae has a similar primary structure to PGM in E. coli (E = 3*10-65) (4). The two proteins also have similar tertiary structures (Z = 34.7) (4). Furthermore, the average distance between the backbones of superimposed proteins, the root mean squared deviation (rmsd), is 1.3Å (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 kDa (larger than E. coli PGM) (5). The S. cerevisiae PGM active site also contains two histidine residues,
His-8 and His-181 while the E. coli PGM active site contains two different histidine residues, His-10 and His-183. In E. coli dPGM, His-10 is phosphorylated while
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. Phosphogycerate mutase deficiency in humans causes muscular dystrophy. This deficiency in PGM function can easily be controlled by monitoring lifestyle and eating habits.