Self-Association of Human Erythrocyte Phosphofructokinase. Kinetic Behaviour in Dependence on Enzyme Concentration and Mode of Association

Abstract

The kinetic behaviour of human erythrocyte phosphofructokinase has been analyzed over a relative wide range of enzyme concentration (0.02 -1.7 pg/ml). The kinetic cooperativity which becomes apparent when the enzymic reaction rate is plotted versus the fructose 6-phosphate concentration decreases with increasing enzyme concentration. Simultaneously, a decrease of the half-saturation concentration for fructose 6-phosphate [S],., is observed. Maximum velocity passes through a maximum at increasing enzyme concentrations.Sets of curves representing specific enzymic activity of phosphofructokinase versus enzyme concentration obtained at various fixed concentrations of fructose 6-phosphate and ATP are analyzed. The shapes of these curves are interpreted in terms of an association model of human erythrocyte phosphofructokinase, in which an inactive dimer ( M , 190000) and active multimers of the dimeric form are involved.The conclusion is drawn that the sigmoidal shape of the plots of the enzymic reaction rate versus fructose 6-phosphate concentration is partially caused by a displacement of the equilibrium between different states of association of phosphofructokinase to multimers by this substrate. On the other hand, the inhibition of the enzyme by high concentrations of ATP may be partially caused by a shift of this equilibrium to the state of the inactive dimer.

A Mathematical Model for the Influence of Anionic Effectors on the Phosphofructokinase from Rat Erythrocytes

“…Furthermore, a shift of the Fru-6-P dependence to the left is observed. The results are in qualitative agreement with those reported for human erythrocyte phosphofructokinase [16]. However, the effects described here are less expressed than those found by Wenzel et al [16].…”

“…The results are in qualitative agreement with those reported for human erythrocyte phosphofructokinase [16]. However, the effects described here are less expressed than those found by Wenzel et al [16]. The data shown in Fig.6 were used to compare the experimentally varied changes in the enzyme concentration with the changes predicted by the associationdissociation model.…”

“…b) The dependence of the specific activity on the enzyme concentration which is either hyperbolic (muscle enzyme) or displays a maximum (erythrocyte enzyme) [12,16] (and this work).…”

The Enzyme Activity Allosteric Regulation Model Based on the Composite Nature of Catalytic and Regulatory Sites Concept

“…The steady state kinetics of the phosphofructokinase reaction catalyzed by PFK from human erythrocytes has been studied in detail by Wenzel and co-authors (44). They presented the data in a form of 66 kinetic curves describing the PFK specific activity as a function of concentration of F6P, ATP, Mg and enzyme.…”

“…The kinetics of the phosphofructokinase reaction F6P + ATP = Fl,6P + ADP, catalyzed by phosphofructokinase (EC 2.7.1.11) (PFK) has been well studied by numerous authors (43)(44)(45)(46)(47). The initial reaction rate is a function of the concentration of substrates and allosteric effectors as well as of the concentration of the enzyme.…”