Rat Liver Phosphofructokinase PURIFICATION AND CHARACTERIZATION OF ITS REACTION MECHANISM

Abstract Rat liver phosphofructokinase was purified to homogeneity with a specific activity of 84 units per mg of protein. The sodium dodecyl sulfate-treated purified enzyme migrated as a single band in 5% sodium dodecyl sulfate polyacrylamide gel with an apparent molecular weight of 82,000 for the protomer. Polyacrylamide gradient gel electrophoresis demonstrated that the molecular weight of the purified enzyme was 325,000 to 365,000 indicating that the monomer consists of four protomers. The initial velocity kinetics in the forward and the reverse direction revealed clearly intersecting patterns. The product inhibition kinetics were as follows. Fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at nonsaturating and saturating concentrations of MgATP. In the reverse reaction, fructose-6-P was a competitive inhibitor versus fructose-1,6-P2 at saturating concentrations of MgADP. The noncompetitive inhibition of fructose-1,6-P2 versus MgATP was completely overcome by saturating concentrations of fructose-6-P. MgADP was a noncompetitive inhibitor versus MgATP both at nonsaturating and saturating concentrations of fructose-6-P. MgADP exhibited also noncompetitive inhibition versus fructose-6-P at nonsaturating concentrations of MgATP but the inhibition became uncompetitive at saturating concentrations of MgATP. The results are in accordance with an ordered Bi Bi reaction mechanism where fructose-6-P is the first, MgATP the second substrate to bind, and MgADP the first and fructose-1,6-P2 the second product to be released.