Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate ⇌fructose 6-phosphate, [32P]Pi⇌ MgPPi, and Mg[32P]PPj⇌ fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate—product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Piand MgPPj/Pisubstrate—product pairs and weakly inhibited at high concentrations of the MgPPj/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi,in agreement with initial velocity studies [Bertagnolli, B. L., & Cook, P. F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pinor positional isotope exchange ofl8O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.
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