title: pH dependence of the kinetic parameters for the pyrophosphate-dependent phosphofructokinase reaction supports a proton-shuttle mechanism.
abstract: The pH dependence of kinetic parameters for the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii suggests that the enzyme catalyzes its reaction via general acid-base catalysis with the use of a proton shuttle. The base is required unprotonated in both reaction directions. In the direction of fructose 6-phosphate phosphorylation the base accepts a proton from the hydroxyl at C-1 of F6P and then donates it to protonate the leaving phosphate. Whether this occurs in one or two steps cannot be deduced from the present data. The maximum velocity of the reaction is pH independent in both reaction directions while V/K profiles exhibit pKs for binding groups (including enzyme and reactant functional groups) as well as pKs for enzyme catalytic groups. These data suggest that reactants bind only when correctly protonated and only to the correctly protonated form of the enzyme. Specifically, the requirement for two enzyme epsilon-amino groups in the protonated form for reactant binding was detected as was the requirement for the ionized phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, MgPPi and HPO4(2-). The protonation state of enzyme and reactant binding groups is in agreement with data obtained previously [Cho, Y.-K., & Cook, P. F. (1988) J. Biol. Chem. 263, 5135].
authors.affiliation: Department of Biochemistry, Texas College of Osteopathic Medicine/University of North Texas, Forth Worth 76107.
authors.forename: Y K
authors.initials: YK
authors.lastname: Cho
authors.forename: P F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D001665:Binding Sites; D011756:Diphosphates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011424:Propionibacterium / Q000378:metabolism; D011522:Protons
chemical_list: D011756:Diphosphates; D011522:Protons; D010732:Phosphofructokinase-1