3-Phosphoglycerate Mutase
Created by: Kyle Miller
3-Phosphoglycerate mutase (PDB = 3PGM) is an enzyme belonging to the cofactor-dependent phosphoglycerate mutase family (1). The 3-phosphoglycerate mutase enzyme catalyzes the interconversion of 2- and 3-phosphoglycerate in the organism Saccharomyces cerevisae (2). The usual direction of interest is from 2-phosphoglycerate to 3-phosphoglycerate, as this conversion is involved in the second phase of glycolysis – more specifically, the 8th step of the glycolytic pathway (3). The 3-phosphoglycerate product is then dehydrated by enolase. This is followed by dephosphorylation by pyruvate kinase, ultimately resulting in the transfer of a phosphoryl group to ADP (3). Thus, this enzyme has important biological implications, in that the lack of this enzyme would inhibit the production of ATP and ultimately impede the metabolic pathway. The molecular weight of 3-phosphoglycerate mutase is 54,241.9 Da, and has an isoelectric point of 9.11 (4).
The quaternary structure of 3-phosphoglycerate mutase coincides with its function as a phosphoryl transferase. At a physiological level, 3-phosphoglycerate mutase exists as a tetramer composed of four identical subunits. These units display nearly perfect 222 symmetry (2). This degree of symmetry is further reflected by His-8 and His-179, the two critical residues, which are nearly parallel to each other and only 0.4 nm apart while they are quite separated in the primary structure (2). This degree of symmetry hastens the encasing of a substrate by lessening the necessity of enzyme orientation, thus increasing the rate of association between enzyme and substrate.
The secondary structure of 3-phosphoglycerate mutase is predominately random coils, and alpha-helices dominate over beta-sheets (5). 3-phopshoglycerate is composed of 28% alpha-helices and 7% beta-sheets, broken into 8 segments of each (6). The residues of one of the subunits folds into a tunnel-shape, which ultimately leads to the active site of 3-phosphoglycerate mutase (2). Each of the subunits of 3-phosphoglycerate mutase are composed of both alpha-helices and beta-sheets. These secondary structures are arranged so that when the subunits are associated, alpha helices surround the beta-sheet core (2).
The association of 3-phosphoglycerate mutase’s subunits is driven by its non-polar residue placement and composition. 3-Phosphoglycerate mutase is composed of approximately 41% polar residues, and the highest concentrations of these residues are found on the exterior surfaces of the alpha helices surrounding the beta-sheet core (7). This leaves the other 59% of 3-phosphoglycerate mutase to be non-polar residues, which are primarily situated in the interior of the protein. The alpha helices that line the exterior of 3-phosphoglycerate mutase are considered amphiphilic, as the exterior surfaces of the helices are highly polar and the interior surfaces highly non-polar. The predominately non-polar interior of 3-phosphoglycerate mutase provides significant hydrophobic interactions, which drives the folding and association of 3-phosphoglycerate mutase’s subunits. Furthermore, the non-polar interior of 3-phosphoglycerate mutase shields the negatively charged 3-phosphoglycerate substrate from solvation by water molecules.
The core of the active site of 3-phosphoglycerate mutase is composed of two critical histidine residues, His-8 and His-179 (2). The positively charged imidazole groups of the histidine residues enable them to ionically bond with a negatively charged phosphate group. The phopshophate group bound to the histidine is ultimately transferred to the C-2 position of the 3-phosphoglycerate substrate, and the C-3 phosphoryl group is abstracted afterwards (2). A histidine bound to a phosphate group forms a “phosphohistidine” intermediate, which is a key step in the reaction mechanism of 3-phosphoglycerate mutase (2). A 3-phosphoglycerate mutase with an unphosphorylated histidine residue is considered inactive. One experiment showed that replacement of one of the critical histidine residues with alanine lowered the reaction rate by a factor of 1.6 x 104 in phosphoglycerate mutases. (8). Additional stabilization of the phosphohistidine intermediate comes from Glu-15 and Thr-20 (6). Both of these residues form hydrogen bonds with one of the phosphoryl oxygen atoms to negate the anionic character. The substrate requirements for phosphoglycerate mutase include the following: C-1 must have a negative charge, C-2 and C-3 must by hydroxyl groups, and either of the C-2 or C-3 hydroxyl groups must be phosphorylated to bind to 3-phosphoglycerate mutase (2).
Another key feature of the 3-phosphoglycerate mutase active site is the positively charged residues closely associated with His-8 and His-179 (2). These include Lys-172 and Arg-184 for His-179, and Arg-7 and Lys-16 for His-8. These residues help stabilize the 3-phophoglycerate substrate’s negative charge, overcoming electrostatic repulsion in the enzyme-substrate interaction.
The 3-phosphoglycerate mutase enzyme contains two different ligands per active site, with four active sites per molecule at the physiological level. These ligands include two sulfate ions, SO42-, and one 3-phosphoglycerate per active site. Thus, there are a total of eight sulfate ions and four 3-phosphoglycerates per 3-phosphoglycerate mutase enzyme (9). These sulfate ions behave in a similar fashion to Glu-15 and Glu-86, whose carboxyl groups only require a minor repositioning to interact with the substrate-histidine complex and produce a proton withdrawing effect (2). This effect is produced through interaction with the 2’ and 3’ hydroxyl groups of the 3-phosphoglycerate substrate. The proton-withdrawing effect enhances the leaving-group ability of the C-3 phosphate on 3-phosphoglycerate, thereby enhancing the reaction rate.
The 3-phosphoglycerate ligand is the substrate itself, in complex with the critical histidine residues His-8 and His-179. The carbonyl oxygen of His-179 acts as a hydrogen bond acceptor for the 2’ hydroxyl of 3-phosphoglycerate, and the hydrogen bound to the nitrogen in the imidazole ring acts as a hydrogen bond donor to the 3’ oxygen (2). The unhydrogenated nitrogen of His-8 then donates 2 electrons to the phosphate hydroxyl, creating a third hydrogen bond (6). Lastly, Glu-15 and Thr-20 act as hydrogen bond donors to the anionic oxygen atoms of 3-phosphoglycerate’s phosphate group (6).
Biphosphoglycerate mutase, found in Homo sapiens, is a well-suited comparison protein for 3-phosphoglycerate mutase. While biphosphoglycerate mutase (PDB ID= 2A9J) did not show up under an initial PSI-BLAST, a sequence-to-sequence comparison resulted in an E score of 6e-77 (10). The E value is a result of comparing the primary structures of both proteins and identifying sequence overlaps (10). An E score lower than .05 indicates a significant amount of sequence homology between biphosphoglycerate and 3-phosphoglycerate mutase (10). Furthermore, in a DALI search, biphosphoglycerate mutase produced a Z score of 27.0 in comparison with 3-phosphoglycerate mutase (5). DALI searches are based on tertiary structure homology founded on a “sum-of-pairs” method. Anything above a Z=2 indicates a high degree of tertiary structure similarity, so the value of 27.0 for biphosphoglycerate mutase is significant (5).
At a physiological level, the structures of biphosphoglycerate mutase and 3-phosphoglycerate mutase are slightly dissimilar. This is expected as biphosphoglycerate mutase acts on a different substrate and in a different environment. However, the quarternary structures of biphosphoglycerate mutase and 3-phosphoglycerate mutase are both highly symmetrical. Biphosphoglycerate has a slightly different physiological structure to enable a different primary function, which is the synthesis of 2,3-biphosphoglycerate (11). 2,3-Biphosphoglycerate is the allosteric effector of hemoglobin, and therefore is vital in oxygen transport (11). This is in contrast to the 3-to-2-phosphoglycerate interconversion that 3-phosphoglycerate mutase catalyzes (2). Additionally, biphosphoglycerate mutase exists as a heterodimer, while 3-phosphoglycerate mutase is a homeotetramer.
Despite the slight structural differences at the physiological level, the secondary structures of biphosphoglycerate mutase and 3-phosphoglycerate mutase elucidate their similarities. Both enzymes are composed primarily of loops, and alpha helices dominate over the beta-sheet content. However, biphosphoglycerate mutase is 43% alpha helices and 11% beta-sheets, while 3-phosphoglycerate mutase is 28% and 7% respectively (6,12). Biphosphoglycerate mutase also contains slightly more variety in its secondary structure – with 15 alpha-helices and 11 beta-sheets – in contrast to 3-phosphoglycerate mutase’s 8 alpha-helices and 8 beta-sheets (6,12).
On the level of primary structure, both biphosphoglycerate mutase and 3-phosphoglycerate operate via a highly conserved mechanism. Both biphosphoglycerate mutase and 3-phosphoglycerate mutase require a critical histidine residue to be phosphorylated in order to be catalytically active. In biphosphoglycerate mutase it can be either His-11 or His-188, and His-8 or His-179 in 3-phosphoglycerate mutase (2,11). This phosphohistidine is considered a critical intermediate in the reaction pathways of both 3-phosphoglycerate and biphosphoglycerate mutase (2,11). The critical histidines are a conserved component of phosphoryl transferases and illustrate an evolutionary connection. Furthermore, in both enzymes the phosphoglycerate substrate is stabilized by a slight rearrangement of nearby Glu residues – Glu-15 and Glu-89 in 3-phosphoglycerate mutase, and Glu-89 in biphosphoglycerate mutase. (2,11). Finally, the degree of structural similarity is represented by the fact that biphosphoglycerate mutase can complex with and function on 3-phosphoglycerate, the substrate of 3-phosphoglycerate mutase (11).