Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Abstract

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase was purified over 700-fold from germinating cucumber (Cucumis sativus cv. Fletcher) seeds. The purified enzyme has a specific activity of 5.2 μmol.min−1.mg protein−1 in the presence of 1 μM fru-2,6-P2. The pH optima is similar for both the forward and reverse reactions (pH 7.5–7.8). Magnesium, manganese and cobalt activate the enzyme, with the highest affinity being for magnesium. The enzyme exhibits normal Michaelis-Menten kinetics in both the presence and absence of fru-2,6-P2. Half-maximum activation of the enzyme was obtained with 35 nM fru-2,6-P2. Fru-2,6-P2 stimulates activity by increasing Vmax and increasing the affinity for fru-6-P, fru-1,6-P2 and PPi. Phosphate causes noncompetitive inhibition with respect to both fru-6-P and PPi. On the basis of the steadystate substrate interaction and Pi inhibition data a sequential ternary complex mechanism is proposed.