Modulation of muscle phosphofructokinase at physiological concentration of enzyme

Two approaches have been used to study the allosteric modulation of phosphofructokinase at physiological concentration of enzyme; a "slow motion" approach based on the use of a very low Mg2+/ATP ratio to conveniently lower Vmax, and the addition of polyethylene glycol as a "crowding" agent to favor aggregation of diluted enzyme. At 0.6 mg/ml muscle phosphofructokinase exhibited a drastic decrease in the ATP inhibition and the concomitant increase in the apparent affinity for fructose-6-P, as compared to a 100-fold diluted enzyme. Similar results were obtained with diluted enzyme in the presence of 10% polyethylene glycol (Mr = 6000). Results with these two approaches in vitro were essentially similar to those previously observed in situ (Aragón, J. J., Felíu, F. E., Frenkel, R., and Sols, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6324-6328), indicating that the enzyme is strongly dependent on homologous interactions at physiological concentrations. With polyethylene glycol it was observed that within the physiological range of concentration of substrates and the other positive effectors, fructose-2,6-P2 still activates the liver phosphofructokinase although it no longer significantly affects the muscle isozyme. In the presence of polyethylene glycol, muscle phosphofructokinase can approach its maximal rate even in the presence of physiologically high concentrations of ATP. Three minor activities of muscle phosphofructokinase have been studied at high enzyme concentration: the hydrolysis of MgATP (ATPase) and fructose-1,6-P2 (FBPase), produced in the absence of the other substrate, and the reverse reaction from MgADP and fructose-1,6-P2. The kinetic study of these activities has allowed a new insight into the mechanisms involved in the modulation of phosphofructokinase activity. The binding of (Mg)ATP at its regulatory site reduces the ability of the enzyme to cleave the bond of the terminal phosphate of MgATP at the substrate site. The positive effectors (Pi, cAMP, NH+4, fructose-1,6-P2, and fructose-2,6-P2) decrease the inhibitory effect of MgATP. Citrate and fructose-2,6-P2 both act as mechanistically "secondary" effectors in the sense that citrate does not inhibit and fructose-2,6-P2 does not activate the FBPase activity, requiring both the presence of ATP to affect the enzyme activity. In conclusion it appears that the regulatory behavior of mammalian phosphofructokinases is utterly dependent on the fact of their high concentrations in vivo.     

The effect of trypsin treatment on rabbit muscle phosphofructokinase

The consequences of trypsin treatment of rabbit muscle phosphofructokinase, in terms of the physical and kinetic properties of the enzyme, have been investigated. At 1% trypsin (w/w) and 25 °C, no activity is lost over a period of 60 min. The complex sedimentation behavior at pH 8 (three peaks) is unchanged by this treatment as is the extent of dissociation of the enzyme when the pH is lowered from 8 to 6 or reassociation when the pH is raised back to 8. However, the trypsin-treated enzyme shows a subunit molecular weight, determined in the guanidine HCl or 0.5 m acetic acid, of 35,000–40,000 compared to the subunit molecular weight of the untreated enzyme at 75,000–80,000. Similarly, SDS gels give only a single species of about 80,000 for the native enzyme but two species, 42,000 and 48,000, for the trypsin-treated enzyme. Kinetic studies showed no differences in the regulatory properties of the enzyme including fructose 6-phosphate cooperativity, ATP inhibition, NH_4⁺ activation, and cAMP activation. Small differences in stability and inhibition by citrate and creatine phosphate were observed.     

Studies on specific elution of rabbit muscle phosphofructokinase with allosteric ligands]

The specific elution of rabbit skeletal muscle phosphofructokinase (PFK) from DEAE-cellulose is studied in the linear gradient of different allosteric ligands. Citrate and fructose-6-phosphate elute PFK at concentrations of 1.0 and 2.5 mM respectively, i.e. without increasing the ionic strength of the starting buffer (similar to 0.12). The specificity of elution is confirmed by comparison of the ionic strength of these solutions with that of buffer eluting PFK in buffer gradient (mu=0.17) as well as by comparison with the eluting ability of other ligands. Fructose-1,6-diphosphate elutes PFK only at the concentration of 5.5 mM which corresponds to the ionic strength 0.17. MgATP and AMP are inefficient as specific eluents whereas ATP and ADP elute only a small part of PFK with concomitant substantial increase of the ionic strength (up to 0.17--0.18). These results are discussed in terms of a charge compensation mechanism as a result of the displacement of PFK conformers equilibrium under the influence of the allosteric ligands.     

On the quaternary structure of native rabbit muscle phosphofructokinase.

Small angle X-ray scattering measurements on solutions of native rabbit muscle phosphofructokinase (EC 2.7.1.11; ATP; D-fructose-6-phosphate 1 phosphotransferase) show that the dimer has a radius of gyration of 32.5 Å and a molecular weight of 160,000, and that the biologically active tetramer has a radius of gyration of 51.5 Å and a molecular weight of 320.000. A possible model was calculated from scattering curves of the dimer and tetramer suggesting two hollow cylinders with cell dimensions for the dimer of a height of 78.0 Å and a long half axis of 38.0 Å, and for the tetramer of a height of 155.0 Å and an outer radius of 35.0 Å. The tetramer is formed along the 78.0 Å axis of the dimer by means of an end-to-end aggregation. The overall particle dimensions of the protomer of molecular weight 80,000 is calculated to be 35.0 × 30.0 × 55.0 Å, assuming an elliptical molecule. The distance between the centers of the two dimeric units within the tetramer is 104.5 ± 1.5 Å.     

Factors affecting the activation of rabbit muscle phosphofructokinase by actin

The consistent application of phosphatase inhibitors and a novel final purification step using a connected series of DE-51, DE-52, and DE-53 anion-exchange chromatography columns facilitate the preparation of electrophoretically homogeneous subpopulations of rabbit muscle phosphofructokinase which differ in their catalytic properties and endogenous covalent phosphate content. A band of "high"-phosphate enzyme (fraction II) flanked by regions of "low"-phosphate enzyme (fractions I and III) is an unusual feature of the final purification profile. Fractions I (containing in this case 0.42 mol of P/82 000 g of enzyme) and II (containing 1.26 mol of P/82 000 g of enzyme) exhibit the most pronounced functional differences of the fractions. Following our original report [Liou, R.-S., & Anderson, S. R. (1980) Biochemistry 19, 2684], both are activated by the addition of rabbit skeletal muscle F-actin. Under the assay conditions, half-maximal stimulation of phosphofructokinase activity occurs at 15.4 nM actin (in terms of monomer) for fraction I and 9.7 nM for fraction II. The low-phosphate enzyme is synergistically activated in the presence of 0.12 microM actin plus 3.0 microM fructose 2,6-bisphosphate, with a marked increase in Vmax, while the high-phosphate enzyme is not. Neither fraction is activated appreciably by the addition of G-actin or the chymotrypsin-resistant actin "core". The covalently cross-linked trimer of actin stimulates the activity of both the low- and high-phosphate enzyme fractions. However, the previously mentioned synergistic activation characteristic of fraction I fails to occur in solutions containing the trimer plus fructose 2,6-bisphosphate. Phosphorylation of fraction I in an in vitro reaction catalyzed by the cAMP-dependent protein kinase causes its properties to become more like those of fraction II. The total amount of covalent phosphate present after in vitro phosphorylation approaches 2 mol of P/82 000 g of enzyme for both fractions.     

Self-association of rabbit muscle phosphofructokinase: effects of ligands

The effects of ligands on the self-association of rabbit muscle phosphofructokinase (PFK) were investigated by velocity sedimentation at pH 7.0 and 23 degrees C. The concentration dependence of the weight-average sedimentation coefficient was monitored in the presence of these ligands. The mode of association and equilibrium constants characterizing each association step were determined by theoretical fitting of the sedimentation data. The simplest mode of association for the PFK system is M in equilibrium M2 equilibrium M4 in equilibrium M16. Ligands and temperature would perturb the various equilibrium constants without altering the mode of association. The apparent equilibrium constants for the formation of tetramer, K4app, are increased in the presence of 0.1 mM ATP and 1.0 mM fructose 6-phosphate. The value of the sedimentation coefficient for the tetramer, S4 degrees, that would best fit the data is 12.4 S instead of 13.5 S determined in the absence of substrates, thus implying a structural change in the tetramer induced by substrates. Only an insignificant amount of dimer is present under the experimental conditions. The presence of activators, ADP or phosphate, enhances the formation of tetramers, and S4 degrees assumes a value of 13.5 S. Similar results are obtained with decreasing concentrations of proton. The presence of the inhibitor, citrate, however, favors the formation of dimers. The equilibrium constants determined as a function of ADP concentration were further analyzed by the linked-function theory derived by Wyman [Wyman, J. (1964) Adv. Protein Chem. 19, 224--285], leading to the conclusion that the formation of a tetramer involves the binding of two additional molecules of ADP per monomer. Similar analysis results in a conclusion that the formation of a dimer involves the binding of one additional molecule of citrate per phosphofructokinase subunit.     

A calorimetric study of the interaction of ATP with rabbit muscle phosphofructokinase.

The heat of interaction of ATP with phosphofructokinase from rabbit muscle was determined at 25 degrees C in 0.1 M potassium phosphate, pH 7.0 and 8.0. The limiting value of the enthalpy change at high ATP concentrations was found to be -11.5 kcal mol of enzyme polypeptide chains. Since phosphate and imidazole have very different heats of ionization (+0.8 and +7.5 kcal/mol, respectively), this suggests that the binding of at least two protons to the enzyme occurs concomitantly with the binding of ATP at the regulatory site.