Limited proteolysis of yeast phosphofructokinase by subtilisin. Alterations in enzyme activity, subunit composition, and hydrodynamic properties.

Yeast phosphofructokinase having a molecular weight of 750000--800000 (20 S) has been subjected to limited proteolysis by subtilisin and yeast proteases. Two steps of proteolytic degradation could be distinguished: in the first step, which is accompanied by an increase in molecular activity, the subunits alpha and beta (Mr 120000) are converted to alpha' and beta' (Mr approximately 900000), and in the second step, accompanied by a decrease in enzyme activity, alpha' is converted to alpha' (Mr 80000) and two further fragments having Mr 45000 and 35000 become detectable. In the course of the conversion the sedimentation value of the undissociated enzyme drops from 20 S to about 17 S. The two substrates fructose 6-phosphate and ATP exhibit characteristic protective effects on enzyme activity and on subunit degradation. Whereas the first step is not strongly influenced by the substrates, fructose, 6-phosphate inhibits significantly the degradation of alpha' and beta', whereas ATP prevents only degradation of beta'. When in presence of ATP alpha' is degraded to alpha', the quaternary structure of the 17-S enzyme is no longer stable and a dissociation of the molecule occurs to a 12-S form which is enzymically active and ATP-sensitive and in which the ratio of alpha' to beta' is one-to-one.     

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.     

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.     

Yeast phosphofructokinase. II. Dependence of the ATP desensitization on effectors

The conversion of ATP-sensitive PFKs to ATP-desensitized PFKd is dependent on the following effectors: ADP, Fru-6-P, NH₄⁺, MgF⁺ (or CaF⁺). For half-maximal rate of desensitization, the necessary concentrations of effectors are: 0.01 mm ADP, 0.006 mm Fru-6-P, 0.5 mm NH₄⁺, and 0.011 mm MgF⁺. Except for the unphysiological MgF⁺, these concentrations generally lie below those encountered in intact cells. On this basis the biological significance of the effector-controlled ATP desensitization of phosphofructokinase is discussed.     

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.     

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.     

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].     

Modification of native sheep liver fructose-1,6-bisphosphatase by subtilisin.

Digestion of native sheep liver fructose-1,6-bisphosphatase by subtilisin resulted in a parallel decrease in activity and sensitivity to AMP inhibition at neutral pH and an increase in specific activity at alkaline pH. During the course of digestion the 35,500 subunit was progressively replaced by two peptides of approximately 29,000 and 6,000 molecular weight, respectively.A comparison of native and digested fructosebisphosphatase showed no significant changes in molecular weight or tryptophan content; however, their catalytic and regulatory properties were markedly different.     

Adenylyl cyclase in yeast. Hydrodynamic properties and activation by trypsin

The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have determined hydrodynamic properties of yeast adenylyl cyclase in taurocholate extracts of wild type and RAS-deficient membranes. In taurocholate extracts of both kinds of membranes, the enzyme is insensitive to guanine nucleotide stimulation; in the presence of 0.5 M NaCl, the taurocholate-solubilized enzyme has a sedimentation coefficient of 12.5 S and a Stokes radius of 11 nm, consistent with a molecular weight of 594,000 for the protein-detergent complex. Treatment of particulate fractions with trypsin (less than 10 micrograms/ml) markedly activates membrane-bound adenylyl cyclase activity, abolishes stimulation by guanine nucleotides, and reduces the sedimentation coefficient of the detergent-solubilized enzyme; higher concentrations of trypsin release a still smaller water-soluble enzyme complex (7.5 S, 6.1 nm Stokes radius, calculated Mr = 190,000) from the membrane. In combination with genetic evidence (Kataoka, T., Broek, D., and Wigler M., (1985) Cell 43, 493-505), our data are consistent with a structural and functional model of yeast adenylyl cyclase in which GTP-activated RAS proteins stimulate cAMP synthesis by relieving an inhibitory constraint on the activity of the CYR1 gene product. This constraint may be mediated by the amino-terminal portion of the CYR1 polypeptide.     

A kinetic model for phosphofructokinase based on the paradoxical action of effectors.

Effectors of muscle phosphofructokinase show opposing action on the activity of the enzyme depending upon the concentration of phosphoryl donor employed in the assay. Established inhibitors, such as citrate, activate at low ATP or ITP concentrations while known activators, such as AMP, ADP, and cyclic AMP inhibit at low ATP or ITP concentrations. Inorganic phosphate, on the other hand, activates at all substrate concentrations. The paradoxical effects at low substrate concentrations are dependent upon the order of addition of reaction components. A model is proposed to explain these and other regulatory phenomena of phosphofructokinase.     

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.     

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.     

Tetramer-dimer conversion of phosphofructokinase from Thermus thermophilus induced by its allosteric effectors

Phosphofructokinase (PFKase) was purified from an extreme thermophile. Thermus thermophilus. Allosteric natures of T. thermophilus PFKase is similar to those of Bacillus stearothermophilus PFKase, that is, hyperbolic plots of the activity versus concentration of fructose 6-phosphate (F6P) were changed into a sigmoidal shape by the addition of phosphoenolpyruvate (PEP), while further addition of ADP caused it to revert to a hyperbolic shape. The native T. thermophilus PFKase has an Mr of 148,000 consisting of four 36,500 subunits. However, it exists as a two-subunit form of Mr 74,000 in the presence of PEP. The two-subunit form was catalytically inactive. The four-subunit enzyme was regenerated by addition of either F6P or Mg.ADP, or by removal of PEP from the solution. This reversible dissociation was observed within a wide range of pH (6.5 to 8.4) and temperature (4 degrees C to 65 degrees C). Thus, unlike PFKase from other sources, the allosteric kinetics of T. thermophilus PFKase can be explained well, at least qualitatively, by the dynamic equilibrium between the active four-subunit form and inactive two-subunit form that is modulated by PEP, F6P and Mg.ADP. Parallel suppression of the PEP-induced conversion in molecular form and kinetics by high concentrations of sulfate and phosphate supports the above explanation. Also, the observation that the degree of PEP inhibition was dependent on the protein concentration of the PFKase in the assay solution is consistent with the presence of this equilibrium.