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.     

A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.     

Influence of NaCl on the kinetic behaviour of mammalian muscle acetylcholinesterase

Acetylcholinesterase was solubilized from rabbit white muscle by means of dilute buffer and Triton X-100 (0.5%). About 50% of total activity was brought into solution with buffer, the rest being solubilized by extracting the tissue with buffer and Triton X-100. The enzyme activity recovered in the supernatants was 170% of that found in the homogenate in the absence of Triton X-100 indicating that, to some extent, the enzyme could be found in an occluded form in muscle. At suboptimum substrate concentration the Triton-solubilized acetylcholinesterase displayed a negative cooperativity, this phenomenon being greatly modified in the presence of NaCl. As the salt concentration increased (0-400 mM) the enzyme activity decreased, the Km values being linearly-dependent on the NaCl concentration in the assay medium. We propose a kinetic pattern to explain both the negative cooperativity produced by the substrate and the effect of NaCl on the kinetic behaviour on this enzyme. Our data are consistent with the hypothesis of binding of substrate to both the catalytic anionic site and a peripheral anionic site, the salt showing the capacity to compete with the substrate for these two binding sites.     

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.     

Rabbit muscle phosphofructokinase: the effect of the state of the enzyme and assay procedure on the kinetic properties.

The specific activity and/or the allosteric behavior of rabbit muscle phosphofructokinase is dependent on several factors including (1) the method of assay, and (2) the concentration, pH, and temperature of the enzyme solution from which dilution is made into the assay mixture. These observations suggest that quantitative interpretation of the allosteric characteristics of this enzyme may be in error because of lack of control of any or all of these factors. In some buffer systems, such as imidazole at pH 7, instability of the enzyme in the assay leads to further complications in the interpretation of previous studies.The results of the present paper show that under specific conditions it is possible to obtain allosteric kinetic data from which quantitative interpretations can be made. This is best accomplished by performing experiments in phosphate buffer and coupling the reaction through pyruvate kinase and lactic dehydrogenase. The experiments must be carried out either in the presence of excess fructose 1,6-bisphosphate or fructose diphosphatase in order to control the level of the activator fructose 1,6-bisphosphate. Under such conditions, the allosteric kinetic behavior observed at pH 6.5 does not appear to be a consequence of polymerization between an active (four subunit) and inactive (two subunit) form of the enzyme, but is inherent in the active form.     

The effect of temperature change on the kinetic behaviour of yeast phosphofructokinase.

Yeast phosphofructokinase does not exhibit any cold sensitivity. The kinetic properties of the enzyme have been investigated in the range between 10 degrees C and 30 degrees C in dependence on fructose 6-phosphate and ATP. Although a significant increase in the enzyme activity with rising temperature does not occur, the shape of the ATP velocity curves is not markedly altered. With increasing concentrations of fructose-6-phosphate the efficiency of temperature on the catalytic process increases, indicating a small temperature effect on the shape of the fructose-6-phosphate velocity curves. The results are interpreted in terms of an adequate kinetic model.     

Self-association of human erythrocyte phosphofructokinase. Kinetic behaviour in dependence on enzyme concentration and mode of association.

The kinetic behaviour of human erythrocyte phosphofructokinase has been analyzed over a relative wide range of enzyme concentration (0.01 -- 1.7 mug/ml). The kinetic cooperativity which becomes apparent when the enzymic reaction rate is plotted versus the fructose 6-phosphate concentration decreases with increasing enzyme concentration. Simultaneously, a decrease of the half-saturation concentration for fructose 6-phosphate [S]0.5 is observed. Maximum velocity passes through a maximum at increasing enzyme concentrations. Sets of curves representing specific enzymic activity of phosphofructokinase versus enzyme concentration obtained at various fixed concentrations of fructose 6-phosphate and ATP are analyzed. The shapes of these curves are interpreted in terms of an association model of human erythrocyte phosphofructokinase, in which an inactive dimer (Mr 190000) and active multimers of the dimeric form are involved. The conclusion is drawn that the sigmoidal shape of the plots of the enzymic reaction rate versus fructose 6-phosphate concentration is partially caused by a displacement of the equilibrium between different states of association of phosphofructokinase to multimers by this substrate. On the other hand, the inhibition of the enzyme by high concentrations of ATP may be partially caused by a shift of this equilibrium to the state of the inactive dimer.     

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.