Phosphofructokinase-1 (PDB ID: 3O8O) from Saccharomyces cerevisiae
Created by: Mariam Yaktieen
Phosphofructokinase-1 (PFK1) (PDB ID: 3O8O) is an allosteric multisubunit enzyme that falls under the Transferases Class (1). The proteins under this class catalyze phosphorylation reactions in a cell (9). PFK1 catalyzes the phosphorylation of fructose-6-phospate with adenosine triphosphate (ATP) to fructose-1,6-bisphosphate in which it transfers the γ-phosphate from ATP to the fructose-6-phosphate (1). PFK1 is a cytosolic protein and a key reagent in glycolytic pathways (Image 1) for it is the committed step, the second priming reaction of glycolysis, and has a negative delta G, which makes it a highly regulated enzyme (4, 9). Glycolysis is necessary for metabolism of cells converting glucose into pyruvate and thus is a response for the cell’s needs for energy and building blocks for biosynthesis (1).
The Saccharomyces cerevisiae phosphofructokinase-1 (ScPFK1) is composed of two polymers in β2α4β2 subunits. These subunits are classified into chains A-H with chain D (beta subunit) and chain E (alpha subunit) being unique chains (10). Chains A, C, E and G make up the alpha subunit and B, D, F and H make up the beta subunit. Chains A, C and E all contain 44% alpha helices and 16% beta sheets. Chains B, F, and H contain 48% alpha helices and 15% beta sheets. Chain D contains 47% alpha helices and 15% beta sheets. Chain G contains 43% alpha helices and 16% beta sheets. The remaining percentages of all of the secondary structures consist of turns, beta bridges, 3/10-helixes, bends and empty spaces to make up all components of its secondary structures (13). These proteins form tetrameric or octameric structures such as this crystalized structure from yeast and consist of 6,212 amino acids with a molecular weight of 677,183.3 Da. It has a theoretical isoelectric point that is predicted to be 6.32 (6).
ScPFK1 contains four active sites (Pocket 1), which are located between the two subunits and are conserved between eukaryotes and prokaryotes near the N-terminal halves (Image 2) (1). The N-terminal half is capable of forming a functional PFK active site by itself. The C-terminus is not catalytic since it only holds the allosteric binding sites, but it is important for the stability of the enzyme by promoting formation of the eukaryotic tetrameric structure. The connecting peptide that joins the two domains is also necessary. Even though no participation of the C-terminal residues are necessary for the catalytic sites contained in the N-terminal half, the C-terminal is very important for the evolution of eukaryotic PFK from prokaryotic PFK (12). Important basic residues in ScPFK1 are Arg-391 in the alpha subunit and Arg-383 in the beta subunit for the Fructose-6-phosphate in the binding pocket. These residues are fully conserved in the bacterial PFK (1).
Because PFK is a regulated step in glycolysis, it is bound to inhibitors and activators. Both citrate and high levels of adenosine triphosphate (ATP) are allosteric inhibitors for the enzyme (9). Increases in citrate from the Citric Acid Cycle can inhibit the enzyme as a signal to stop the production of pyruvate since the two pathways are coupled via citrate. When ATP binds, there is a conformational change to the protein structure where it goes from 75 degrees when bound to its substrate, to 46 degrees (Image 4) going into its inactive form known as the T-state formation (1, 2). Fructose-2,6-bisphosphate (F-2,6-BP) and adenosine monophosphate (AMP) are both allosteric activators. F-2,6-BP binding increases the affinity of ScPFK1 for its substrate fructose-6-phosphate and it overrides the inhibition of ATP (1, 9). A pair of activator sites is located on the side opposite to the pair of active sites on each α and β dimers (1). There are many residues in the allosteric binding pocket near the C-terminal halves of ScPFK1 for this activator (4 pockets – Image 3). It must accommodate for the extra phosphate on carbon-2. Residues Arg952, Arg665, and Glu694 of the alpha subunit and Arg935, Arg658 and Glu688 of the beta form a motif for binding this activator (1). A certain alignment of subunits when ScPFK has fructose-6-phopshate and F-2,6-BP bound allows it to be active and this conformation has been seen to resemble the bacterial PFK tetramers of E. Coli (PDB: 4PFK) known as the active R-state. With this superimposition, it can be concluded that the reaction mechanisms for these two proteins are conserved as well.
ScPFK1 has around twenty effectors total as opposed to the two, an inhibitor (phosphoenyolpyruvate) and an activator (adenosine diphosphate), in bacterial PFK1 (2,3). As noted earlier, eukaryotic PFK1 exhibit more complicated regulatory mechanisms (1). Another distinguished difference is the doubled size of the eukaryotic PFK1 compared to the prokaryotic PFK. This has risen from a process of second gene duplication and fusion of the bacterial gene by the α and β subunits (3).
Although there are subsequent evolutionary modified structure and regulatory functions, prokaryotic PFK1 has been preserved to a certain degree. Eukaryotic PFK1 four αβ dimers resemble a bacterial PFK tetramer (1). Comparing tertiary structures of crystallized ScPFK1 with the crystal structure of PFK from bacillus stearothermophilus (PDB: 3U39) (Bacillus S.) using the DALI server, it is found that it has a Z-score of 30.0. The alpha subunit of ScPFK1 is also very similar in comparison to another eukaryotic PFK1 found in rabbit skeletal muscle (RMPFK) (PDB: 3O8N). It has a Z-score of 47.1 calculated by the server DALI (7). When comparing the beta subunit of ScPFK1 with other proteins using the same server, it is found that crystal structure of PFK from bacillus subtilis (PDB: 4A3S) is significant in comparison. It has a Z-score of 39.6 relative to the ScPFK1. RMPFK also showed a Z-score of 49.6 compared to ScPFK1 (7).
Having crystallized the RMPFK with ADP, research has shown a nucleotide binding cavity for this substrate. The ADP is buried inside the protein and its many interactions, such as its stacking interactions with Phe308 and Phe671, indicate a well-tailored binding site (1). This binding interaction is similar to bacterial PFK and is fully conserved with the interacting residues (1). The ADP binding site in the RMPFK could be the active site for what is in the bacterial PFK. With this study it was found that ATP would also bind to this same site, which is an inhibitor of PFK1 (1). One can make the assumption that this is where an ADP or ATP would bind for the ScPFK1 because the structure of ScPFK1 and RMPFK are so similar based on the Z-score produced from the DALI server (7). However, the research team (1) did not crystalize ScPFK1 with the nucleotide but only with the fructose-6-phosphate and F-2,6-BP ligands. By crystallizing both ScPFK and RMPFK it can be shown how the structure of this protein has evolved but more importantly has been conserved over prokaryotes to eukaryotes.
The primary structure of ScPFK1 can be compared to other primary structures of other organisms and proteins. Using NCBI BLAST server, the alpha subunit (Chain E) of ScPFK1 can be compared to 6-phosphofructokinase in Taphrina deformans, a fungus and plant pathogen, which has an overall E-value of 0.0 within the amino acid residues 200-952 of the subject protein (8). The beta subunit (Chain D) of ScPFK1 can be compared to 6-phosphofructokinase in Marssonina brunnea, a fungal pathogen found in plants, which has an overall E-value of 0.0 between amino acid residues 12-796 (8).
PFK1 is a protein of focus for much research. Understanding the detailed mechanism of the specific F-2,6-BP activation opens rational design of metabolic modulators with potential applications in industry, medicine, and disease (1). Research has shown that effector F-2,6-BP is a very specific compound that acts only on PFK as well as fructose-1,6-bisphophatase in gluconeogenesis and thus the binding site is highly selective and high selectivity is beneficial as a means to use to target certain functions of the enzyme (1). For example, a study done by the National Institute of Health (NIH) found that by blocking glycosylation of ScPFK1 at Ser529 reduced the proliferation of cancer cells and impaired tumor formation in vitro (5). Also in a different study done by a team of researchers in China, it was observed that breast cancer tissues as well as paracancer cancer tissues have an increased degree of glycolytic efficiency and that PFK1 levels are higher in human breast cancer tissues than in paracancer tissues (11). It is clear to see the degree of importance this enzyme plays in mammals and so the use of model organisms like Saccharomyces cerevisiae can help in the development of such observations. This complex protein has and continues to provide in-depth analysis and insight to metabolism.