Phosphofructokinase
created by Brenda Ann Peterson
Phosphofructokinase (4PFK) from Geobacillus stearothermophilus is an enzyme that catalyses the key control step of glycolysis. Glycolysis is the metabolic pathway by which energy is released from the sugar and captured in the form of ATP over the course of five steps (Garrett, 551). In the reaction, fructose-6-phosphate is phosphorylated by ATP to form fructose-1,6-biphosphate (F1,6P2) and ADP (Evans, 53). Fructose-6-phosphate couples with PFK to make the reaction exergonic, releasing energy. This reaction forces the cell to metabolize the sugar in an irreversible reaction instead of converting it to another sugar or storing it, thus generating energy necessary for cell life. For this reason, the phosphorylation of fructose-6-phosphate is the most important step in the glycolytic pathway (Garrett, 580-588).
The fructose-6-phosphate and ATP ligands are attached to two separate sites on PFK. When ATP levels are high, the cell no longer needs energy produced from the metabolic pathway. The ATP binds to PFK on two sites, as opposed to one, and lowers the affinity of PFK to fructose-6-phosphate. This incidentally slows the glycolysis, making PFK the perfect regulatory enzyme (Phosphofructokinase, 1). As the concentration of ATP decreases, the reaction rate is able to increase.
PFK is a monomer consisting of one subunit. The tertiary structure of PFK in mammals and bacteria is a homotetramer, meaning there are four identical subunits; however, the bacterial enzyme is much smaller. This is because bacteria do not have rigid, compartmental controls found in eukaryotic cells by presence of mitochondria and other organelles (Sanwal, 20). The molecular weight of PFK in Geobacillus stearothermophilus is 34117.85 Da, and its isoelectric point (pI) is 6.64 according to Expasy, the Expert Protein Analysis System, (Expasy, 1).
PFK’s secondary structure consists of 319 residues. It is 44% helical and 19% beta sheet. There are 13 helices from 143 residues and 13 strands from 61 residues. It folds to a globular shape. Each subunit has two domains of a β-sheet between α-helices. There are three binding sites for ligands per subunit (Evans, 57).
The structure of PFK directly brings about its function. The catalytic site lies between the two domains in the subunit. The ATP molecule is almost entirely bound by domain one. Domain two binds to fructose-6-phosphate. Arg-162 and Arg-243 are an exception to this rule. They are in the other subunit and bind to fructose-6-phosphate. If these residues are removed, the binding affinity for fructose-6-phosphate is greatly reduced and therefore it will no longer react with ATP to catalyze the glycolysis pathway (Evans, 61).
In the catalytic site fructose-6-phosphate undergoes nucleophilic attack on the γ-phosphate. Even though Asp-127 is on a different subunit, it is close to the fructose-6-phosphate and acts as a base catalyst for the reaction by increasing the nucleophilicity of the hydroxyl. Arg-252 is on the same subunit as fructose-6-phosphate, and it is central to a network of hydrogen bonds linking the fructose-6-phosphate to the other subunit, to His-160, and by way of Asn-12 to Thr-156. These residues form a major part of the interaction between these two subunits (Evans, 59).
The effector site is between the two subunits. ADP, the activator in bacteria, is bound with its diphosphate group and the Mg2+ ion buried deep in the pocket of the two subunits. The adenine group is on the surface. Mg2+ bridges the alpha and beta phosphates of ADP. It is bound to PFK by Gly-185 through the main carbonyl chain, Glu-187, and His-215. The phosphates are tightly bound by groups from both subunits through hydrogen bonding (Evans, 61).
The weak forces in the sequence form the two subunits. ATP and fructose-6-phosphate are on separate subunits. The subunits are held closely by Arg-162 and Arg-243. There is a pocket between the two subunits. This pocket is where the fructose-6-phosphate and ATP react, generating energy to catalyze the glycolysis pathway.
PFK is extremely important because it provides a means for the phosphorylation of fructose-6-phosphate in glycolysis. It is a fundamental protein required for cellular respiration. For this reason, it is in multiple species. PFK from Lactobacillus delbrueckii (LbPFK) is very similar to the Geobacillus stearothermophilus enzyme. LbPFK has a Protein Data Bank ID of 1ZXX.
BLAST is a program used to find proteins with a similar primary structure to the protein in question. The similarities of the PFK in the different species can be seen through the E value. A low E value, below one, corresponds to a similar amino acid sequence. 1.79e-132 is the value for LbPFK and PFK, so they are very similar (Altschul). The Dali Server is a method for finding proteins with tertiary, three-dimensional, structure similarities to the protein in question. The Z-score shows the similarities of the tertiary structures. A high Z-score means the tertiary structures are similar (Holm). The two proteins have a Z-score of 48.4.
LbPFK is different than PFK, because it is a nonallosteric analogue of PFK. The effector binding site is very different from PFK. MgADP binds to the allosteric site much less readily. Two inorganic sulfate ions are found on LbPFK. This is similar to the inorganic phosphate found on PFK. The residues involved in binding sites with the phosphate groups of ADP are also similar (Paricharttanakul, 1).
LbPFK has an aspartic residue at 187, as opposed to glutamic acid found in PFK. Glu-187 in PFK adopts a folded conformation and is able to coordinate to Mg2+. The octahedral coordination between the carboxyl of Asp-187 in LbPFK and Mg2+ is very weak or non-existent. This explains why MgADP has very weak interactions with the LbPFK (Paricharttanakul, 1). Greatly increasing the MgADP concentration would allow for more interactions.