Mechanism of activation of fructose-2,6-bisphosphatase by cAMP-dependent protein kinase.
The bisphosphatase reaction sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase involves a phosphoenzyme intermediate. Catalysis is activated in vitro by cAMP-dependent protein kinase-catalyzed phosphorylation. We investigated the mechanism of this activation by studying the effect of protein kinase-catalyzed phosphorylation on the formation and breakdown of the phosphoenzyme intermediate. The significant findings were as follows. 1) Phosphorylation decreased the rate of phosphoenzyme formation. 2) More importantly, phosphorylation increased the much slower rate of phosphoenzyme breakdown both in the absence and presence of the regulatory ligands, inorganic phosphate and alpha-glycerol phosphate. The increase in the rate of phosphoenzyme breakdown correlated with the degree of activation of the bisphosphatase; both were increased about 2-fold. 3) The potent inhibition of phosphoenzyme breakdown by fructose 6-phosphate indicates that, in the catalytic sequence, the release of nascent fructose 6-phosphate from the active site precedes phosphoenzyme breakdown and Pi release. 4) Phosphorylation reduced the fructose 6-phosphate inhibition of phosphoenzyme breakdown both in the absence and presence of phosphate and alpha-glycerol phosphate. 5) Phosphorylation decreased the potent substrate inhibition which occurs at physiological substrate concentrations. It appears that protein kinase-catalyzed phosphorylation activates fructose-2,6-bisphosphatase by promoting the dissociation of fructose 6-phosphate and fructose 2,6-bisphosphate from the same phosphoenzyme intermediate, hastening its exposure to water and thereby relieving both product and substrate inhibitions.
D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D000242:Cyclic AMP; D011494:Protein Kinases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases