A Kinetic Study of Pyrophosphate: Fructose‐6‐Phosphate Phosphotransferase from Potato Tubers

TLDR

The study describes a method to quantify fructose 2,6‑bisphosphate at sub‑picomole levels based on the enzyme’s kinetic properties. The purified PPi‑PFK from potato tubers exhibits sigmoidal saturation for fructose‑6‑phosphate and fructose‑1,6‑bisphosphate, hyperbolic for PPi and Pi, and its affinity for fructose‑2,6‑bisphosphate is dramatically increased (Ka 5 nM forward, 150 nM reverse), with the effect amplified by fructose‑6‑phosphate and reduced by Pi.

Abstract

Pyrophosphate: fructose‐6‐phosphate phosphotransferase (PP i ‐PFK) has been purified 150‐fold from potato tubers and the kinetic properties of the purified enzyme have been investigated both in the forward and the reverse direction. Saturation curves for fructose 6‐phosphate and also for fructose 1,6‐bisphosphate were sigmoidal whereas those for PP i and P i were hyperbolic. In the presence of fructose 2,6‐bisphosphate, the affinity for fructose 6‐phosphate and for fructose 1,6‐bisphosphate were greatly increased and the kinetics became Michaëlian. The effect of fructose 2,6‐bisphosphate was increased by the presence of fructose 6‐phosphate and decreased by the presence of P i . Consequently, the K a for fructose 2,6‐bisphosphate was as low as 5 nM for the forward reaction and reached 150 nM for the reverse reaction. On the basis of these properties, a procedure allowing one to measure fructose 2,6‐bisphosphate in amounts lower than a picomole, is described.