Catabolism of d-fructose and d-ribose by Pseudomonas doudoroffii

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Abstract
1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudomonas doudoroffii were partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. The pH optima of these enzymes were 9.0 and 8.5, respectively. 2. When the concentrations of the substrates of the 1-PFK reaction were varied, Michaelis-Menten kinetics were observed. The Kms for d-fructose-1-P (F-1-P) and ATP were 3.03×10-4 M and 3.39×10-4 M, respectively. Variation of MgCl2 at fixed concentrations of F-1-P and ATP resulted in sigmoidal kinetics; about 10 mM MgCl2 was necessary for maximal activity. Activity of 1-PFK was inhibited when the ratio of ATP: Mg++ was higher than 0.5, suggesting that ATP: 2Mg++ was the substrate and that free ATP was inhibitory. Although an absolute requirement for K+ or NH + 4 could not be demonstrated, these cations stimulated the rate of the reaction. Activity of 1-PFK was not significantly affected by 3 mM AMP, cyclic-AMP, Pi, d-fructose-6-P (F-6-P), ADP, P-enolpyruvate (PEP), pyruvate, citrate, or l-glutamate. 3. Sigmoidal kinetics were observed for 6-PFK when the concentration of F-6-P was increased and the level of ATP was kept constant. Activity of 6-PFK was increased by ADP, inhibited by PEP, and unaffected by 3 mM AMP, cyclic-AMP, Pi, F-1-P, pyruvate, or citrate.