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The paper describes a simple kinetic model of an open monosubstrate enzyme reaction with substrate inhibition and product activation. A comparison between the model and the phosphofructokinase reaction shows a close resemblance between their dynamical properties. This makes it possible to explain qualitatively most experimental data on single-frequency oscillations in glycolysis. A mathematical analysis of the model has shown the following. In the model, at a definite relationship between the parameters, self-oscillations arise. The condition of self-excitation is satisfied more readily with a lower source rate, larger product sink rate constants, lower product-enzyme affinity and higher enzyme activity. Self-oscillations exist only in a certain range of values of the parameter determining the degree of substrate inhibition. This range increases with decreasing source rate. Too strong or, conversely, too weak substrate inhibition leads to damped oscillations. The period of self-oscillations depends on the degree of substrate inhibition, the source rate, the sink rate constant, the enzyme activity, the affinity of the substrate and the product for the enzyme; it decreases with an increase in these values. With an increase in the relative sink rate constant the steady state amplitude of self-oscillations initially increases until a definite maximum is reached and then drops to zero. A self-oscillatory state in the phosphofructokinase reaction exists only when the maximum rate of this reaction is essentially higher than the source rate, and lower than the maximum rate of the reactions controlling the sink of the products. An experimental investigation of self-oscillations in the phosphofructokinase reaction may be considerably simplified by using a reconstituted system consisting of a small number of reactions with an irreversible sink of the products and artificial substrate supply. In this case the above relationship (section 6) should hold. [ABSTRACT FROM AUTHOR]

The phosphofructokinase of the red cells of rats is exquisitely sensitive to inhibition by ATP. The apparent Ki for ATP is decreased with lower pH values and fructose-6-phosphate concentrations. 2,3-Bisphophoglycerate, which is present in the red cells of man and many mammals, inhibits the phosphofructokinase in a synergistic manner with ATP. The inhibitory effect of phosphoenolpyruvate is biologically insignificant. It may be estimated that the enzyme would be practically completely inactive under cellular conditions without the action of positive effectors. There exist a multitude of positive effectors. Significant activations under cellular conditions are exercised by AMP, glucose 1,6-bisphosphate and inorganic phosphate. Fructose 1,6-bisphosphate activates to a limited extent and ADP only at alkaline pH values. All positive effectors produce a decreased inhibition by ATP and increased affinity of the enzyme for fructose 6-phosphate. AMP is the activator with the highest affinity, while glucose 1,6-bisphosphate may be considered as a baseline activator owing to its sluggish turnover. The data may be fitted in a qualitative manner to the model of Monod, Wyman and Changeux. It is possible that the negative anionic effectors act on the same site. [ABSTRACT FROM AUTHOR]

By applying analytical gel chromatography technique the association behaviour of human erythrocyte phosphofructokinase has been studied. The molecular weight distribution of the polymerized forms of this enzyme covers a range of 380000 to about six millions. The degree of polymerization has been found to depend on the protein concentration, on the presence of various substrates and effectors and on the pH-value. ATP favours the aggregation of the enzyme with increasing pH, whereas in presence of fructose 6-phosphate or fructose 1,6-bisphosphate respectively the degree of association appears to be independent of the pH value between pH 6 and 9. In the microgram range the specific activity of the enzyme decreases with an increase of the enzyme concentration. [ABSTRACT FROM AUTHOR]

A new and efficient method for preparation of pure yeast phosphofructokinase is described, leading rapidly to an enzyme with minimum proteolytic alterations. Essential is an affinity chromatography step on the reactive dye Cibacron blue F3G-A® which has been covalently bound to Sephadex G-100. The enzyme prepared by this method has a significantly higher sedimentation constant (20.5 S) than those forms of yeast phosphofructokinase purified by procedures previously described (17.8 S) and is composed of two different kinds of subunits having molecular weights of 130000 and 96000, respectively. By gel filtration various enzyme fractions have been obtained, containing either mainly subunits of the 130000 or of the 96000 type, respectively. Samples of phosphofructokinase which have been prepared by the original and by the new method were compared by means of analytical gel chromatography and dodecylsulphate elcetrophoresis. In the course of the preparation procedure described previously, the enzyme is evidently proteolytically converted to the 570000 molecule by degradation of the subunits having 130000 molecular weight to those of 96000. [ABSTRACT FROM AUTHOR]