Evidence for a phosphoenzyme intermediate in the reaction pathway of rat hepatic fructose-2,6-bisphosphatase.
We re-examined the kinetics of the bisphosphatase reaction of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase after depleting the enzyme of bound fructose 6-phosphate and found a hyperbolic dependence on fructose 2,6-bisphosphate at concentrations below 100 nM. The Michaelis constant was 4 nM, the Vmax was about 12 nmol X mg-1 X min-1 at 22 degrees C but the substrate inhibited at concentrations above 100 nM. Both phosphate and alpha-glycerol phosphate strongly inhibited phosphoenzyme formation and hydrolytic rate below 100 nM, but relieved the inhibition by substrate at higher concentrations probably by antagonizing substrate binding. A number of observations support the proposition that the phosphoenzyme is a necessary participant in catalysis. 1) The amount of phosphoenzyme measured during steady-state hydrolysis as a function of substrate concentration correlated with the velocity profile. 2) Rapid mixing experiments demonstrated that over a broad range of substrate concentrations phosphoenzyme formation was faster than the net rate of hydrolysis. 3) Both phosphate and alpha-glycerol phosphate inhibited the rate of phosphoenzyme formation and, at low substrate concentrations, reduced the steady-state phosphoenzyme levels. The latter correlated with inhibition of substrate hydrolysis. 4) Both phosphate and alpha-glycerol phosphate stimulate the rate of phosphoenzyme breakdown, consistent with their stimulation of substrate hydrolysis at high substrate concentrations. 5) The fractional rate of phosphoenzyme breakdown, which was pH and substrate dependent, multiplied by the amount of phosphoenzyme obtained in the steady state at that pH and substrate concentration approximated the observed rate of hydrolysis. We conclude that the phosphoenzyme is a reaction intermediate in the hepatic fructose-2,6-bisphosphatase reaction.
D000818:Animals; D001667:Binding, Competitive; D005635:Fructosediphosphates / Q000378:metabolism / Q000494:pharmacology; D005636:Fructosephosphates / Q000494:pharmacology; D005994:Glycerophosphates / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010710:Phosphates / Q000494:pharmacology; D025481:Phosphofructokinase-2; D010750:Phosphoproteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats
D005635:Fructosediphosphates; D005636:Fructosephosphates; D005994:Glycerophosphates; D010710:Phosphates; D010750:Phosphoproteins; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; C029620:alpha-glycerophosphoric acid; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases