Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds |
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Authors: | Botha Frederik C; Small JG Chris; de Vries Christa |
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Institution: | Department of Botany, University of the O.F.S. Bloemfontein 9300, South Africa |
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Abstract: | Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min1.mg protein1 in the presenceof 1 µM fru-2,6-P2. The pH optima is similar for boththe forward and reverse reactions (pH 7.57.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P2. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P2 stimulates activity by increasing Vmax and increasingthe affinity for fru-6-P, fru-1,6-P2 and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986) |
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