Phosphofructokinase from Fasciola hepatica: activation by phosphorylation and other regulatory properties distinct from the mammalian enzyme.
Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for phosphorylation and Fru-2,6-P2 in regulation of fluke phosphofructokinase.
Kamemoto
E S
ES
Department of Pharmacology, Stanford University School of Medicine, California 94305.
Iltzsch
M H
MH
Lan
L
L
Mansour
T E
TE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005210:Fasciola hepatica / Q000201:enzymology*; D005634:Fructose-Bisphosphate Aldolase / Q000494:pharmacology; D005635:Fructosediphosphates / Q000378:metabolism / Q000494:pharmacology; D005636:Fructosephosphates / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives*; D005958:Glucosephosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
D002951:Citrates; D005635:Fructosediphosphates; D005636:Fructosephosphates; D005958:Glucosephosphates; D010710:Phosphates; D019343:Citric Acid; D019298:Glucose-6-Phosphate; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; C016473:glucose-1,6-bisphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D010732:Phosphofructokinase-1; D010749:Phosphoprotein Phosphatases; D005634:Fructose-Bisphosphate Aldolase