pH dependence of the reverse reaction catalyzed by phosphofructokinase I from Escherichia coli: implications for the role of Asp 127

Protein Sci. 1992 Feb;1(2):254-8. doi: 10.1002/pro.5560010207.


The kinetics of the reverse reaction catalyzed by Escherichia coli phosphofructokinase, i.e., the synthesis of ATP and fructose-6-phosphate from ADP and fructose-1,6-bisphosphate, have been studied at different pH values, from pH 6 to pH 9.2. Hyperbolic saturations of the enzyme are observed for both substrates. The affinity for fructose-1,6-bisphosphate decreases with pH following the ionization of a group with a pK of 6.6, whereas the catalytic rate constant and perhaps the affinity for ADP are controlled by the ionization of a group with a pK of 6. Several arguments show that the pK of 6.6 is probably that of the carboxyl group of Asp 127, whereas the pK of 6 is tentatively attributed to the carboxyl group of Asp 103. The pK of 6.6 is assigned to the carboxyl group of Asp 127 in the free enzyme, and a simple model suggests that the same group would have an abnormally high pK, above 9.6, in the complex between phosphofructokinase and fructose-1,6-bisphosphate. It is proposed that the large pK shift of more than 3 pH units upon binding of fructose-1,6-bisphosphate is due to an electrostatic repulsion that could exist between the 1-phosphate group and the carboxyl group of Asp 127, which are close to each other in the crystal structure of phosphofructokinase (Shirakihara, Y. & Evans, P.R., 1988, J. Mol. Biol. 204, 973-994). The same interpretation would also explain the much higher affinity of the enzyme for fructose-1,6-bisphosphate when Asp 127 is protonated.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphate / metabolism
  • Aspartic Acid / chemistry*
  • Catalysis
  • Escherichia coli / enzymology*
  • Fructosediphosphates / metabolism
  • Fructosephosphates / metabolism
  • Hydrogen-Ion Concentration
  • Models, Biological
  • Models, Chemical
  • Phosphofructokinase-1 / chemistry
  • Phosphofructokinase-1 / metabolism*


  • Fructosediphosphates
  • Fructosephosphates
  • Aspartic Acid
  • Adenosine Diphosphate
  • fructose-6-phosphate
  • Adenosine Triphosphate
  • Phosphofructokinase-1
  • fructose-1,6-diphosphate