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.     

Antagonistic effect of cAMP and cGMP on the kinetic behaviour of phosphofructokinase from rat erythrocytes and reticulocytes

Fructose-6-phosphate (F6P)-saturation curves (up to 5 mM F6P) for phosphofructokinase (PFK) have been studied at physiological pH (7.1) and inhibitory (1.5 mM) or non-inhibitory (0.25 mM) ATP levels, in rat erythrocytes and reticulocytes. The addition of 300 microM cAMP to control samples activates the enzyme and displaces F6P-saturation curve towards the left, while the addition of cGMP inhibits the enzyme and shifts the curve to the right. The cAMP positive allosteric effect is more evident at inhibitory ATP levels, while the inhibitory effect of cGMP is very similar at both ATP levels. This antagonistic effect is exerted at the same regulatory site, since cAMP also activates the enzyme when cGMP is previously present in the reaction mixture. The physiological significance of this antagonism is not yet clear.     

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.     

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].     

Influence of phosphate on the allosteric behavior of yeast phosphofructokinase.

Initial rate data obtained with purified yeast phosphofructokinase (PFK) show an ATP dependent kinetic cooperativity with respect to fructose-6-phosphate. In the presence of 25 mM phosphate, the cooperativity index (Hill number) is related to the half saturation concentration of fructose-6-phosphate as predicted by the concerted allosteric model in the case of a “K-system”. In the absence of phosphate, however, the kinetic behavior of yeast PFK is more complex and the cooperativity index is invariant with respect to the half saturation concentration of fructose-6-phosphate which is increased by ATP. In both cases, 5′AMP behaves as a strong activator of the enzyme. These kinetic data suggest that the two distinct functions of ATP as phosphate donnor and as allosteric inhibitor, respectively, are supported by different binding sites. These regulatory properties of yeast PFK are discussed in relation to glycolytic oscillations.     

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.     

The effect of repeated temperature shock on baker's yeast

Summary In a recycle system in which evaporation is used for ethanol recovery during fermentation, temperature changes of the broth in the loop will occur. These repeated temperature shocks may have an effect on the microbial ethanol production rate. In this study such repeated temperature changes were simulated in a recycle system with ethanol production by baker's yeast. The magnitude of the temperature change, as well as the time of exposure to this change were found to have an effect on the ethanol production rate. A temperature increase from 30°C in the fermentor to 35°C or more in the recycle loop led to a significantly lower ethanol concentration in the broth. This effect became negligible at a short exposure time of 18 s of the yeast to the higher temperature. Correspondence to: R. G. J. M. van der Lans