Inactivation of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii by pyridoxal 5'-phosphate. Determination of the pH dependence of enzyme-reactant dissociation constants from protection against inactivation.
The pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii is rapidly inactivated by low concentrations of pyridoxal 5'-phosphate (PLP). The inactivation is first order with respect to PLP and the rate increases linearly with PLP concentrations suggesting that over the concentration range used no significant E-PLP complex accumulates during inactivation. The rate of inactivation decreases at high and low pH and this is discussed in terms of the mechanism of Schiff base formation. The presence of any reactants decreases the rate of inactivation to 0 at infinite concentration. This protection against inactivation has been used to obtain the pH dependence of the dissociation constants of all enzyme-reactant binary complexes. Reduction of the PLP-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 6-phosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphate probably directly coordinated to these phosphates.
Cho
Y K
YK
Department of Biochemistry, North Texas State University/Texas College of Osteopathic Medicine, Denton 76203.
Cook
P F
PF
D000465:Algorithms; D011756:Diphosphates / Q000494:pharmacology*; D005636:Fructosephosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors*; D011424:Propionibacterium / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*
D011756:Diphosphates; D005636:Fructosephosphates; D011732:Pyridoxal Phosphate; C027618:fructose-6-phosphate; D010732:Phosphofructokinase-1; D008239:Lysine