Phosphofructokinase-2 (6-phosphofructo-2-kinase, PFK-2) or fructose bisphosphatase-2 (FBPase-2), is an enzyme indirectly responsible for regulating the rates of glycolysis and gluconeogenesis in cells. It catalyzes formation and degradation of a significant allosteric regulator, fructose-2,6-bisphosphate (Fru-2,6-P2) from substrate fructose-6-phosphate. Fru-2,6-P2 contributes to the rate-determining step of glycolysis as it activates enzyme phosphofructokinase 1 in the glycolysis pathway, and inhibits fructose-1,6-bisphosphatase 1 in gluconeogenesis. Since Fru-2,6-P2 differentially regulates glycolysis and gluconeogenesis, it can act as a key signal to switch between the opposing pathways. Because PFK-2 produces Fru-2,6-P2 in response to hormonal signaling, metabolism can be more sensitively and efficiently controlled to align with the organism's glycolytic needs. This enzyme participates in fructose and mannose metabolism. The enzyme is important in the regulation of hepatic carbohydrate metabolism and is found in greatest quantities in the liver, kidney and heart. In mammals, several genes often encode different isoforms, each of which differs in its tissue distribution and enzymatic activity. The family described here bears a resemblance to the ATP-driven phospho-fructokinases; however, they share little sequence similarity, although a few residues seem key to their interaction with fructose 6-phosphate. PFK-2 is known as the "bifunctional enzyme" because of its notable structure: though both are located on one protein homodimer, its two domains act as independently functioning enzymes. One terminus serves as a kinase domain (for PFK-2) while the other terminus acts as a phosphatase domain (FBPase-2). In mammals, genetic mechanisms encode different PFK-2 isoforms to accommodate tissue specific needs. While general function remains the same, isoforms feature slight differences in enzymatic properties and are controlled by different methods of regulation; these differences are discussed below.
Structure The monomers of the bifunctional protein are clearly divided into two functional domains. The kinase domain is located on the N-terminal. It consists of a central six-stranded β sheet, with five parallel strands and an antiparallel edge strand, surrounded by seven α helices. The domain contains nucleotide-binding fold (nbf) at the C-terminal end of the first β-strand. The PFK-2 domain appears to be closely related to the superfamily of mononucleotide binding proteins including adenylate cyclase. On the other hand, the phosphatase domain is located on the C-terminal. It resembles the family of proteins that include phosphoglycerate mutases and acid phosphatases. The domain has a mixed α/ β structure, with a six-stranded central β sheet, plus an additional α-helical subdomain that covers the presumed active site of the molecule. Finally, the N-terminal region modulates PFK-2 and FBPase2 activities, and stabilizes the dimer form of the enzyme. While this central catalytic core remains conserved in all forms of PFK-2, slight structural variations exist in isoforms as a result of different amino acid sequences or alternative splicing. With some minor exceptions, the size of PFK-2 enzymes is typically around 55 kDa. Researchers hypothesize that the unique bifunctional structure of this enzyme arose from a gene fusion event between a primordial bacterial PFK-1 and a primordial mutase/phosphatase.
Function This enzyme's main function is to synthesize or degrade allosteric regulator Fru-2,6-P2 in response to glycolytic needs of the cell or organism, as depicted in the accompanying diagram. In enzymology, a 6-phosphofructo-2-kinase (EC 2.7.1.105) is an enzyme that catalyzes the chemical reaction:
ATP + beta-D-fructose 6-phosphate ⇌ {\displaystyle \rightleftharpoons } ADP + beta-D-fructose 2,6-bisphosphate Thus, the kinase domain hydrolyzes ATP to phosphorylate the carbon-2 of fructose-6-phosphate, producing Fru-2,6-P2 and ADP. A phosphohistidine intermediate is formed within the reaction.
At the other terminal, the fructose-2,6-bisphosphate 2-phosphatase (EC 3.1.3.46) domain dephosphorylates Fru-2,6-P2 with the addition of water. This opposing chemical reaction is: beta-D-fructose 2,6-bisphosphate + H2O ⇌ {\displaystyle \rightleftharpoons } D-fructose 6-phosphate + phosphate
Because of the enzyme's dual functions, it can be categorized into multiple families. Through categorization by the kinase reaction, this enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. On the other hand, the phosphatase reaction is characteristic of the family of hydrolases, specifically those acting on phosphoric monoester bonds.
Regulation In almost all isoforms, PFK-2 undergoes covalent modification through phosphorylation/dephosphorylation based on the cell's hormonal signaling. Phosphorylation of a specific residue may prompt a shift that stabilizes either kinase or phosphatase domain function. This regulation signal thus controls whether F-2,6-P2 will be synthesized or degraded. Furthermore, the allosteric regulation of PFK2 is very similar to the regulation of PFK1. High levels of AMP or phosphate group signifies a low energy charge state and thus stimulates PFK2. On the other hand, a high concentration of phosphoenolpyruvate (PEP) and citrate signifies that there is a high level of biosynthetic precursor and hence inhibits PFK2. Unlike PFK1, PFK2 is not affected by ATP concentration.
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