Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase

• Published in: Biochemistry (Easton) Vol. 27; no. 9; pp. 3320 - 3325
• Main Authors: Cho, Yong Kweon, Matsunaga, Terry O, Kenyon, George L, Bertagnolli, Byron L, Cook, Paul F
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 03.05.1988
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbon Radioisotopes; Diphosphates - pharmacology; Enzymes and enzyme inhibitors; Fabaceae - enzymology; Fundamental and applied biological sciences. Psychology; Isotope Labeling - methods; isotopes; Kinetics; Oxygen Isotopes; Phosphofructokinase-1 - metabolism; Phosphorus Radioisotopes; Plants - enzymology; Plants, Medicinal; Propionibacterium - enzymology; Propionibaterium freudenreichii; Radioisotope Dilution Technique; Transferases; Pyrophosphate―fructose-6-phosphate «1-»phosphotransferase; Enzyme; Propionibacteriaceae; Reaction mechanism; Bacteria; Actinomycetes; Kinetics; Propionibacterium freudenreichii
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00409a030

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.