Kinetic Properties of Pyrophosphate:Fructose-6-Phosphate Phosphotransferase from Germinating Castor Bean Endosperm

Abstract

Pyrophosphate:fructose-6-phosphotransferase (PFP) was purifeid over 500-cold from endosperm of germinating castor bean. The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3-7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nm. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e., fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e., fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate and phosphate in the control of PFP are discussed.