The synthesis of Fru-2,6-P2 is performed through a bifunctional enzyme containing both PFK-2 and FBPase-2, which is dephosphorylated, allowing the PFK-2 portion to phosphorylate fructose 6-phosphate using ATP.

At physiological concentration, PFK-1 is almost completely inactive, but interaction with Fru-2,6-P2 activates the enzyme to stimulate glycolysis and enhance breakdown of glucose.

[4] Cellular stress as a result of oncogenesis or DNA damage among others, activates certain genes by the tumor suppressor p53.

TIGAR removes the allosteric effector, Fru-2,6-P2., therefore the activator does not enhance the affinity of the enzyme (PFK1) for its substrate (fructose 6-phosphate).

Insulin triggers the opposite response by activating protein phosphatases that dephosphorylate PFK-2, thereby inhibiting the FBPase-2 domain.

Fru-2,6-P2 plays an important role in the regulation of triose phosphates, the end products of the Calvin Cycle.