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 of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase fromPropionibacterium 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/sub i/ ↔ MgPP/sub i/, and Mg[32P]PP/sub i/ ↔ 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/sub i/ and MgPP/sub i/P/sub i/ substrate-product pairs and weakly inhibited at high concentrations of the MgPP/sub i/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:P/sub i/, E:MgPP/sub i/:P/sub i/, and E:FBP:MgPP/sub i/, in agreement with initial velocity studies. Neither back-exchange by [32P]P/sub i/ nor positional isotope exchange of 18O-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