Isotope Exchange as a Probe of the Kinetic Mechanism of Pyrophosphate-Dependent Phosphofructokinase

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]P_i⇌ MgPPi, and Mg[³²P]PP_j⇌ 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)/P_iand MgPP_j/P_isubstrate—product pairs and weakly inhibited at high concentrations of the MgPP_j/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:P_i, E:MgPP_i:P_i, and E:FBP:MgPP_i,in agreement with initial velocity studies [Bertagnolli, B. L., & Cook, P. F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [³²P]P_inor positional isotope exchange of^l8O-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.