Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger.
Phosphofructokinase (EC 2.7.1.11) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran--Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis--Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.
D000227:Adenine Nucleotides / Q000494:pharmacology; D000645:Ammonium Sulfate / Q000494:pharmacology; D001234:Aspergillus niger / Q000201:enzymology*; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D004789:Enzyme Activation / Q000187:drug effects; D005636:Fructosephosphates / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D000227:Adenine Nucleotides; D002951:Citrates; D005636:Fructosephosphates; D019343:Citric Acid; C027618:fructose-6-phosphate; D006160:Guanosine Triphosphate; D010732:Phosphofructokinase-1; D000645:Ammonium Sulfate