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 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 in equilibrium fructose 6-phosphate, [32P]Pi in equilibrium MgPPi, and Mg[32P]PPi in equilibrium 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)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/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]Pi 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.
Cho
Y K
YK
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Matsunaga
T O
TO
Kenyon
G L
GL
Bertagnolli
B L
BL
Cook
P F
PF
D002250:Carbon Radioisotopes; D011756:Diphosphates / Q000494:pharmacology*; D007887:Fabaceae / Q000201:enzymology; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D010103:Oxygen Isotopes; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010944:Plants / Q000201:enzymology; D010946:Plants, Medicinal; D011424:Propionibacterium / Q000201:enzymology; D011865:Radioisotope Dilution Technique
D002250:Carbon Radioisotopes; D011756:Diphosphates; D010103:Oxygen Isotopes; D010761:Phosphorus Radioisotopes; D010732:Phosphofructokinase-1