pH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli

Abstract

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)

Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of Activity

“…Two notable examples are the feed-forward activation of phosphofructokinase-1 (PFK-1) by fructose 6-phosphate and the activation of pyruvate dehydrogenase by pyruvate. Hill coefficient studies have shown that the catalytic efficiency of a given active site in PFK-1 is increased by the binding of fructose 6-phosphate to remote binding sites, thus constituting a homoallosteric phenomenon (31). Heteroallosterically, fructose 6-phosphate can also increase PFK-1 catalytic efficiency by first being phosphorylated by phosphofructokinase-2 to yield fructose 2,6,-bisphosphate, a metabolite that is then able to serve as a potent allosteric activator of PFK-1.…”

Interaction between the carboxyl groups of Asp127 and Asp129 in the active site of Escherichia coli phosphofructokinase

“…All activity measurements were performed in buffer A (0.1 M Mes, 51 niM N-ethylmorpholine, 51 mM diethanolamine, 10 mM magnesium acetate) which maintains a constant ionic strengh over a wide pH range (Ellis and Morrison, 1982). Both coupled assays are only valid in the range pH 5.8-9.2 (Deville- Bonne et al, 1991a;Auzat and Garel, 1992). Measurements of activity for the forward reaction were made in the presence of the allosteric activator GDP, so that PFK did not show any cooperative behavior (Blangy et al, 1968).…”

Cytosolic Ca2+ and H+ buffers in green algae: A reply

“…However, we feel that our approach is superior to the Hill equation for several reasons. Firstly, the Hill equation is a completely phenomenological approach and not related to any mechanism (Deville-Bonne et al 1991, Hill 1910. Monod et al (1965) emphasised the fact that the exponent n must not be interpreted in terms of interacting sites.…”