Pyruvate kinase from Aspergillus niger: a regulatory enzyme in glycolysis?
Pyruvate kinase from the filamentous, citric acid producing fungus Aspergillus niger was purified about 100-fold by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration. The addition of fructose-1,6-diphosphate was necessary to prevent loss of activity during purification. The enzyme purified in the presence of fructose-1,6-diphosphate (FDP) exhibits hyperbolic kinetics with respect to phosphoenolpyruvate (PEP) and ADP. Monovalent cations activated the enzyme (K+, NH4+). FDP neither activated nor inhibited the enzymatic activity from extracts freshly prepared in the absence of exogenous FDP; ATP showed a weak activation. In contrast the enzyme from crude extracts which had been stored in the presence of glycerol for 3 days showed activation by FDP or a metabolite thereof and inhibition by ATP. In the absence of FDP sigmoidal kinetics were obtained with respect to PEP, which became hyperbolic kinetics after addition of FDP. ATP inhibition turned into slight ATP activation in the presence of FDP. However, it was possible to reactivate inactivated pyruvate kinase (after dialysis in the absence of FDP) by adding FDP to the enzyme assay. From these results and because of the very high affinity of pyruvate kinase for FDP (Ka less than 0.1 microM), it is concluded that the enzyme probably has FDP bound to the protein in vivo. The significance of this hypothesis to the regulation of glycolysis in A. niger, with special reference to the mechanism of citric acid accumulation, is discussed.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001234:Aspergillus niger / Q000201:enzymology*; D005635:Fructosediphosphates / Q000378:metabolism; D006019:Glycolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000032:analysis* / Q000302:isolation & purification
D005635:Fructosediphosphates; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D008274:Magnesium; C029063:fructose-1,6-diphosphate; D011188:Potassium