A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate.
Pyrophosphate : fructose-6-phosphate phosphotransferase (PPi-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 PPi and Pi 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 Pi. Consequently, the Ka 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.
D005635:Fructosediphosphates / Q000032:analysis*; D006598:Hexosediphosphates / Q000032:analysis*; D007700:Kinetics; D008832:Microchemistry; D010770:Phosphotransferases / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*
D005635:Fructosediphosphates; D006598:Hexosediphosphates; C027652:fructose 2,6-diphosphate; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase