Catabolism of D-fructose and D-ribose by Pseudomonas doudoroffii. II. Properties of 1-phosphofructokinase and 6-phosphofructokinase.
1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudmonas 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 X 10(-4) M and 3.39 X 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 NH4+ 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-gluamate. 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.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D004789:Enzyme Activation; D005636:Fructosephosphates; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000302:isolation & purification / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011549:Pseudomonas / Q000187:drug effects / Q000201:enzymology*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology
D005636:Fructosephosphates; D000644:Quaternary Ammonium Compounds; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D008274:Magnesium; D011188:Potassium