Kinetic mechanism of Ascaris suum phosphofructokinase desensitized to allosteric modulation by diethylpyrocarbonate modification

The kinetic mechanism of phosphofructokinase has been determined at pH 8 for native enzyme and pH 6.8 for an enzyme desensitized to allosteric modulation by diethylpyrocarbonate modification. In both cases, the mechanism is predominantly steady state ordered with MgATP binding first in the direction of fructose 6-phosphate (F6P) phosphorylation and rapid equilibrium random in the direction of MgADP phosphorylation. This is a unique kinetic mechanism for a phosphofructokinase. Product inhibition by MgADP is competitive versus MgATP and noncompetitive versus F6P while fructose 1,6-bisphosphate (FBP) is competitive versus fructose 6-phosphate and uncompetitive versus MgATP. The uncompetitive pattern obtained versus F6P is indicative of a dead-end E.MgATP.FBP complex. Fructose 6-phosphate is noncompetitive versus either FBP or MgADP. Dead-end inhibition by arabinose 5-phosphate or 2,5-anhydro-D-mannitol 6-phosphate is uncompetitive versus MgATP corroborating the ordered addition of MgATP prior to F6P. In the direction of MgADP phosphorylation, inhibition by anhydromannitol 1,6-bisphosphate is noncompetitive versus MgADP, while Mg-adenosine 5'(beta, gamma-methylene)triphosphate is noncompetitive versus FBP. Anhydromannitol 6-phosphate is a slow substrate, while anhydroglucitol 6-phosphate is not. This suggests that the enzyme exhibits beta-anomeric specificity.