The pH dependence of the activity of the allosteric phosphofructokinase from Escherichia coli has been studied in the pH range from 6 to 9, in the absence or presence of allosteric effectors. The sigmoidal cooperative saturation of phosphofructokinase by fructose 6-phosphate has been analyzed according to the Hill equation, and the following results have been obtained: (i) the apparent affinity for Fru-6P, as measured by the half-saturating concentration, [Fru-6P]0.5, does not change with pH; (ii) the cooperativity, as measured empirically by the Hill coefficient, nH, increases markedly with pH and reaches a value of 5.5-6 at pH 9; (iii) the catalytic rate constant, kcat, is controlled by the ionization of a critical group which has a pK of 7 in the absence of effector and must be deprotonated for phosphofructokinase to be active. The observation that pH affects both the cooperativity and the maximum velocity suggests that the catalytic efficiency of a given active site could be modified by the binding of fructose 6-phosphate to other remote sites. Finding values of the cooperativity coefficient larger than the number of substrate binding sites indicates that slow conformational changes may occur in phosphofructokinase. The cooperative saturation of phosphofructokinase by fructose 6-phosphate appears more complex than described by the classical concerted model at steady state and could involve two slowly interconverting states which differ in both their turnover rate constants and their affinities for fructose 6-phosphate. The presence of GDP shifts the pK of the critical group which controls kcat from 7 to 6.6.(ABSTRACT TRUNCATED AT 250 WORDS)