Activation by phosphate of yeast phosphofructokinase.

The activity of yeast phosphofructokinase assayed in vitro at physiological concentrations of known substrates and effectors is 100-fold lower than the glycolytic flux observed in vivo. Phosphate synergistically with AMP activates the enzyme to a level within the range of the physiological needs. The activation by phosphate is pH-dependent: the activation is 100-fold at pH 6.4 while no effect is observed at pH 7.5. The activation by AMP, phosphate, or both together is primarily due to changes in the affinity of the enzyme for fructose-6-P. Under conditions similar to those prevailing in glycolysing yeast (pH 6.4, 1 mM ATP, 10 mM NH4+) the apparent affinity constant for fructose-6-P (S0.5) decreases from 3 to 1.4 mM upon addition of 1 mM AMP or 10 mM phosphate; if both activators are present together, S0.5 is further decreased to 0.2 mM. In all cases the cooperativity toward fructose-6-P remains unchanged. These results are consistent with a model for phosphofructokinase where two conformations, with different affinities for fructose-6-P and ATP, will present the same affinity for AMP and phosphate. AMP would diminish the affinity for ATP at the regulatory site and phosphate would increase the affinity for fructose-6-P. The results obtained indicate that the activity of phosphofructokinase in the shift glycolysis-gluconeogenesis is mainly regulated by changes in the concentration of fructose-6-P.     

Binding of MgATP to yeast phosphofructokinase.

Binding of MgATP to yeast phosphofructokinase was investigated by the gel filtration equilibrium dialysis technique. Per subunit of yeast phosphofructokinase two molecules of MgATP are bound in the absence of fructose-6-phosphate, one to a high-affinity and one to a low-affinity site. The experimental data were compared with a kinetic model of yeast phosphofructokinase as described by Freyer et al. [3].     

Binding of manganese to phosphofructokinase from yeast.

The binding of manganese to yeast phosphofructokinase has been studied using the equilibrium dialysis technique. Three independent binding sites per enzyme subunit have been found with identical affinities. The dissociation constant for Mn²⁺ binding is 2,26 mM.     

Reaction of yeast phosphofructokinase with succinic and maleic anhydride.

Modification of yeast phosphofructokinase by succinic and maleic anhydride influences the catalytic activity and the allosteric behaviour of the enzyme. Depending on the degree of succinylation and maleinylation a decrease of maximum activity, an increase of the apparent affinity for fructose-6-phosphate, a decrease of the Hill-coefficient and a diminution of ATP-inhibition are observed. Up to about 40% of the lysyl residues could be succinylated without dissociation of the hexameric protein, however with a decrease of the enzyme activity. More extensive succinylation or maleinylation causes a dissociation into subunits. The sedimentation coefficient is lowered from 20 S to about 3 S. The molecular weight of the smallest dissociation product was determined to 50 000 (+/- 10 000) by the sedimentation equilibrium method. The number of bound succinyl groups, as determined from radioactivity incorporation, exceeds the content of lysyl groups of the enzyme, indicating that the modifying reagent is also reacting with other amino acid residues.     

Rapid purification and crystallization of yeast phosphofructokinase

A model is described for the ion equilibria between the main salt constituents of bovine milk diffusate. A mass balance equation is constructed for each of the strongly interacting components, consisting of the sum of the concentrations of free and complexed forms of that substance, and an efficient algorithm is given for solving the set of mass balance equations. The model gives calculated free calcium ion concentrations which lie between experimental values determined by ion-selective electrode and murexide methods. Calculated free calcium and magnesium ion concentrations are in general agreement with values determined by a resin equilibrium procedure. The calculated concentrations of ions and complexes in a typical milk diffusate are tabulated.     

Complementation of soluble phosphofructokinase activity in yeast mutants.

We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae: those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele-testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutations in the catalytic subunit but also in the regulatory subunit. Also, a mutation in the catalytic subunit can give rise to an enzyme altered in its kinetic properties in a manner phenomenologically similar to that caused by a mutation in the regulatory subunit. The results of the complementation studies in diploids suggest that, in spite of their distinct functions, both the subunits are essential for activity to be detected in vitro. This is confirmed by the reconstitution of an active PFK I enzyme by mixing cell-free extracts of two complementing parents, each of which lacks the enzyme activity. PFK activity appears in the mixture, reaching a maximum value of 60-100% of that of the diploid in 15-30 min at 24 degrees C. Unlike the catalytic subunit which exists in various multimeric states in cell-free extracts of the mutant bearing only this subunit, the regulatory subunit exists largely as a monomer in a mutant devoid of the catalytic subunit. The reconstituted enzyme, however, is indistinguishable from that of the wild type, as analysed by sedimentation studies and Western blot analysis, demonstrating that only the heteromeric complex of the two subunits is active, while neither of the individual subunits displays activity in vitro.     

Differences in the degradation of yeast phosphofructokinase by proteinases A and B from yeast.

Proteinase A from yeast causes a stepwise degradation of yeast phosphofructokinase, resembling the action of subtilisin on this enzyme. Proteinase B, however, exhibits a limited proteolysis similar to the action of alpha-chymotrypsin.     

Binding of fructose-6-phosphate to phosphofructokinase from yeast.

Yeast phosphofructokinase binds one molecule of fructose-6-phosphate per subunit. The binding curve exhibits sigmoidality and yields a good fit to an equation derived from the kinetic model as developed previously for this enzyme. The results show that the allosteric kinetic response of the enzyme to fructose-6-phosphate is due to cooperativity of the binding process.     

Some kinetic and molecular properties of yeast phosphofructokinase

Yeast PFK had a sedimentation coefficient of 16.7 S both in the absence and in the presence of ATP, and did not dissociate even at very low protein concentrations. Sodium dodecyl-sulphate caused dissociation of the protein to sub-units of 3.2 S.The effects of pH on substrate affinities are described. In the presence of UTP, acting as non-inhibiting phosphate donor, the behaviour of the enzyme towards F-6-P was co-operative, with a Hill coefficient of 2.2.     

Effects of yeast proteinase A, proteinase B and carboxypeptidase Y on yeast phosphofructokinase.

Incubation of a crude yeast extract containing phosphofructokinase with proteinase A, proteinase B or carboxypeptidase Y gave the following results: Proteinase B and carboxypeptidase Y did not change the activity of phosphofructokinase during incubation. On the other hand, incubation with proteinase A resulted in a 40-100% activation; continued incubation, however, led to an inactivation of the enzyme. Addition of allosteric effectors did not change the activation or inactivation process. The activated phosphofructokinase was not changed with respect to pH optimum and ATP inhibition. Molecular weight determination of phosphofructokinase in crude extracts in the presence of inhibitors of proteinase A indicated a molecular weight of 700000. Without inhibitors of proteinase A, the molecular weight was determined to be 600 000, while after 40-100% activation by proteinase A, a molecular weight of 500 000 was obtained. The activity profile of proteinase A in density gradients indicated that this enzyme is bound to variety of cellular proteins.     

Allosteric activation and competitive inhibition of yeast phosphofructokinase by d-fructose.

Purified phosphofructokinase from bakers yeast is activated by D-fructose in low concentrations (up to 1 mM) and inhibited by high concentrations. The stimulatory effect of D-fructose is similar, but smaller than that of AMP. In the presence of AMP (0.4 mM or higher) D-fructose does no longer stimulate, but its inhibitory effect persists (KI = 8 mM). Its dualistic action on phosphofructokinase activity indicates that D-fructose might induce low frequency in glycolytic oscillations by direct interaction with the enzyme.     

Studies on the structure of yeast phosphofructokinase

In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.