3-Phosphoglycerate Kinase
Created by Courtney Killough
3-Phosphoglycerate kinase (3-PGK) (PDB ID = 1PHP) serves a critical role in cellular metabolism. The enzyme is specifically a glycolytic enzyme (1). Thus, it is a key player in glycolysis; the formation of high energy molecules, ATP and NADH, from the breakdown of glucose in all living organisms. 3-PGK is directly involved in the 7th step of glycolysis, when 1,3-bisphosphoglycerate reacts with MgADP to produce 3-phosphoglycerate and MgATP. The 3-PGK actually binds both the MgADP and the 1,3-bisphosphoglycerate simultaneously and facilitates the transfer of one phosphate group over to the MgADP which creates MgATP. 3-PGK is very effective at performing its enzymatic activity thanks to its hinge region that allows for it to bend and conceal the phosphate transfer within a protected hydrophobic area, as seen in Image 1 (2).
3-Phosphoglycerate kinase has 394 amino acids that make up its one chain. The chain’s weight is approximately 43kD. Its secondary structure is made of 23 a-helices and 20 ß-strands (1). In addition, some of the residues remain in their primary structures and connect the neighboring helices and strands. Those are specifically called "3/10 helices" and "random coils". The single amino acid chain of 3-phosphoglycerate kinase, however, is separated into 2 domains. Each domain moves as an individual unit within the larger motif. The N-domain in particular binds 1,3-bisphosphoglycerate or 3-phosphoglycerate at the catalytic site on residue 46, while the C-domain binds a nucleotide, either MgADP or MgATP (3). The particular bound substrate on each domain depends upon whether or not the enzyme has reached catalysis, signified by the transfer of a phosphoryl group. Therefore, catalysis requires domain closure.
The MgADP complex that binds to the C-domain is the nucleotide that is able to interact with 1,3-bisphosphoglycerate on the N-domain during the 7th step of glycolysis. ATP is produced via the transfer of a phosphoryl group within the hydrophobic, shielded core of the closed enzyme 3-PGK. There are multiple binding sites for ADP, while magnesium only binds at residue 352. This demonstrates both the complex shape of ADP (as compared to the simple, elemental shape of magnesium) and the way in which 3-PGK creates a perfectly shaped pocket that clings onto the MgADP nucleotide complex (1).
The specific residues of 3-PGK reveal a lot about its function and its motion. Looking further into the ADP binding site, for example, it is clear that the following amino acid residues are conserved among all organisms: 250, 324, 348, 350, 352, 353, 354, 355, 386, 387, and 388. Although they are not positioned right next to each other in the open conformation, protein folding always brings them together to constitute the ADP binding site (4). There are 4 a-helices in particular that are part of the interdomain region. The interdomain a-helices connect the N-domain and the C-domain to successfully bring together the two substrates during glycolysis via a hinge motion. These a-helices are formed from residues 169-185 and residues 351-394 (5). Thus, the binding site residues are moved into their prime locations as the interdomain region closes.
The protein 3-PGK is classified more broadly as a phosphoglycerate kinase. All phosphoglycerate kinases are involved in glycolysis and work to transfer phosphate groups over to ADP, so that the higher energy version, ATP, can be utilized in the cell. These phosphoglycerate kinases are cytosolic enzymes that perform substrate-level phosphorylation (6). It is medically important to study this general group of molecules, because a few serious disorders are directly caused by genetic defects within phosphoglycerate kinases. One disease in particular, Hereditary Hemolytic Anemia, creates a form of anemia due to non-functioning phosphoglycerate kinases. The lack of PGKs leads to the breakdown of erythrocytes and leukocytes, which obviously hinders the transport of oxygen throughout the body. These detrimental effects all stem from a single mutation that causes an amino acid shift at residue 46, known as the PGK-Barcelona mutation (7). Phosphoglycerate kinases are integral enzymes that must function correctly for an organism to reach its maximum energy potential.
The particular PGK in this document, 3-phosphoglycerate kinase, has been isolated from a bacterial cell found in Bacillus stearothermophilus. However, among all organisms, there has been notable conservation of the genetic code for phosphoglycerate kinases. “Over 32 PGK sequences are known, with about 12% of the residues conserved among all species” (1). Among the homologous structures to 3-PGK, the proteins all share four main conserved sites: the substrate binding site, the ADP binding site, the catalytic site and the hinge region. Because the two binding sites, catalytic site, and hinge region are utilized in every glycolysis reaction, the enzymatic activity of 3-PGK depends heavily upon these conserved regions. 3-PGK found in pig muscles is one such homolog of 3-PGK from Bacillus stearothermophilus. It too performs glycolytic enzyme activities through a “phospho-transfer reaction” (8).
Utilizing highly conserved regions, a metal cofactor, and hinge movements, 3-PGK demonstrates the connection between form and function in enzymes. Overall, the structural characteristics of 3-phosphoglycerate kinase in Bacillus stearothermophilus directly contribute to its ability to facilitate the 7th step of glycolysis and to produce the first coveted molecules of ATP.