Phosphofructokinase-1 (PFK-1) is a crucial regulatory enzyme in glycolysis, the foundation for both anaerobic and aerobic respiration. As a rate-controlling enzyme, its activity determines the speed of glycolysis. PFK-1 catalyzes the "committed" step of glycolysis, which is the conversion of fructose 6-phosphate and ATP to fructose 1,6-bisphosphate and ADP. This enzyme is allosteric, composed of four subunits, and is regulated by various activators and inhibitors.

Function and Regulation

PFK regulates glycolysis through allosteric inhibition, allowing the cell to adjust the rate of glycolysis according to its energy needs. For example, a high ATP to ADP ratio inhibits PFK and, consequently, glycolysis. In eukaryotes, PFK is activated by fructose 2,6-bisphosphate, which overrides ATP inhibition, providing eukaryotes with greater sensitivity to hormones like glucagon and insulin.

PFK-1 belongs to the phosphotransferase family and facilitates the transfer of γ-phosphate from ATP to fructose-6-phosphate. The active site of PFK-1 includes both the ATP-Mg²⁺ and the F6P binding sites. Allosteric activators like AMP and ADP bind to the allosteric site to promote the formation of the R state by inducing structural changes, while inhibitors like ATP and PEP bind to the same allosteric site to facilitate the formation of the T state, thus inhibiting enzyme activity.

Phosphofructokinase Assay Kits

Phosphofructokinase assay kits offer a simple and direct method for measuring PFK activity in different samples. These kits capitalize on the role of PFK in converting fructose-6-phosphate to fructose-1,6-diphosphate, which is the rate-limiting step of glycolysis. PFK activity is regulated by cofactors and post-translational modifications and can be used to measure glycolytic flux in tissues. Deficiencies in PFK activity can lead to glycogen storage diseases such as glycogenosis type VII (Tarui’s disease).

Assay Principle

The PFK activity is measured through a coupled enzyme assay. In this assay, PFK converts fructose-6-phosphate and ATP into fructose-1,6-diphosphate and ADP. Subsequently, the enzyme mix converts ADP to AMP and NADH. The resulting NADH reduces a colorless probe, producing a colorimetric product that is proportional to the PFK activity.