Evidence for phosphorylation of yeast phosphofructokinase

Radioactively labelled material from yeast cells grown in the presence of [32P]phosphate was specifically recognized by antibodies raised against yeast phosphofructokinase. Purified yeast phosphofructokinase was phosphorylated in a cyclic AMP-independent manner by a protein kinase enriched from yeast extracts. This phosphorylation occurred specifically on the beta-subunit, and 0.56 mol of phosphate/mol of subunit was incorporated. The results indicate the phosphorylation of yeast phosphofructokinase both in vivo and in vitro. Phosphofructokinase phosphorylated in vitro was more stable against proteolytic degradation compared to the non-phosphorylated enzyme.     

Multiple genes control particulate phosphofructokinase of yeast

Summary Mutants of Saccharomyces cerevisiae lacking the particulate phosphofructokinase define at least four unlinked genes, PFK2, PFK3, PFK4 and PFK5. A structural role of PFK2 is indicated. Mutations in the other three have pleiotropic effects.     

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].     

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.     

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.     

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.     

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.     

Identification and cloning of yeast phosphofructokinase 2.

Fructose-6-phosphate 2-kinase ('phosphofructokinase 2') was purified from a strain of Saccharomyces cerevisiae lacking fructose-6-phosphate 1-kinase. After chromatography on DEAE-Sephacel, Sephacryl blue, CM-Sephadex and rechromatography on CM-Sephadex with fructose-6-phosphate elution, the specific activity was 1.6 U/mg protein. Although the latter value is high for fructose-6-phosphate 2-kinase, as was the purification factor of 3 x 10(4), staining with Coomassie blue showed the fraction to still contain many proteins. Incubation with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase gave a further increase in specific activity and labeling of, only, 96-kDa and 93-kDa polypeptides. Antiserum raised against these polypeptides recognized them in an immunoblot and could be used to remove the enzyme activity from crude extracts. Tryptic peptide profiles were obtained from about 10 pmol of the 96-kDa and 93-kDa polypeptides. The profiles were similar and sequencing allowed construction of mixed probes and identification of a putative single structural gene. Returned to yeast on a multicopy plasmid, phosphofructokinase 2 activity was considerably above the wild-type level, as was polypeptide revealed by immunoblotting.