Analysis of the cold lability behavior of rabbit muscle phosphofructokinase.

Rabbit skeletal muscle phosphofructokinase has been previously shown to exhibit the characteristic of cold lability in phosphate buffers at pH values below pH 7 [Bock, P.E. and Frieden, C. (1974) Biochemistry $\text{13}$, 4191–4196]. Studies of the residual activity as a function of pH, reflecting the equilibrium between active and inactive forms of the enzyme, have been performed. These experiments show that the cold lability can be ascribed to a shift of the apparent pK describing the pH-dependent inactivation to lower pH values at higher temperatures. The apparent pK, Hill interaction coefficient and heat of ionization for this process indicate that the equilibrium between the inactive and active forms of the enzyme may be controlled by the ionization of one or more histidine residues per enzyme subunit. In addition the apparent pK for the pH dependence of the residual activity at constant temperature is influenced by the presence of ligands which are substrates or effectors of the phosphofructokinase reaction.     

Structural properties of an active form of rabbit muscle phosphofructokinase

The quaternary structure of an active form of rabbit muscle phosphofructokinase was studied by sedimentation and electron microscopy. Active enzyme centrifugation studies at pH 7.0 and 23 +/- 1 degrees C showed that phosphofructokinase sediments as a single component with a sedimentation coefficient of 12.2 +/- 0.5 S. Identical results were obtained in two assay and three solvent systems. Boundary sedimentation studies of phosphofructokinase in the presence of 1.0 mM fructose 6-phosphate, 0.1 mM adenylyl imidodiphosphate at pH 7.0 and 23 +/- 1 degrees C were performed. The results showed that the sedimentation coefficient of phosphofructokinase remains constant within the range of protein concentration studied and assumes a value of 12.4 S. The molecular weights of the subunit and the 12.4 S component were measured by sedimentation equilibrium yielding values of 83,000 and 330,000 for the monomeric and polymeric species, respectively. It is, therefore, concluded that the active form of phosphofructokinase is indeed the tetrameric species. The structure of the phosphofructokinase tetramer was also studied by electron microscopy of negatively stained specimens. Particles identified as tetramers measured approximately 9 nm in diameter by 14 nm in length. The observed size and shape are consistent with the hydrodynamic measurements. Structural features within the tetramer were interpreted as due to the four individual subunits, each one approximately 4 X 6 X 6 nm in size, arranged with D2 symmetry.     

Identification of highly reactive cysteinyl and methionyl residues of rabbit muscle phosphofructokinase

The reactivity of the 16 thiol groups of rabbit skeletal muscle phosphofructokinase has been studied extensively over the past 20 years. Several of these thiols show high reactivity with a variety of reagents, display differential reactivity in the presence of allosteric ligands and substrates, and appear to be important to function because their modification changes activity and regulatory properties. In the present study, the location in the primary structure of several highly reactive thiol groups has been established by reaction with [14C]iodoacetate. In the course of these studies, 2 methionyl residues that are located at or near proposed ligand-binding sites are readily carboxymethylated by iodoacetate. In addition to confirming the presence of the most reactive thiol group at sequence position 88, a thiol protected from reaction by the presence of fructose-6-P and cyclic AMP has been found at position 169. Cysteine 169 is close to a residue important to the binding of fructose-6-P in the homologous structure from Bacillus stearothermophilis phosphofructokinase. The modification of Cys-169 brings about extensive, but not total, loss of activity. Another cysteine, at position 232, was found to be highly reactive also. Substrate provided partial protection against carboxymethylation at this position. Carboxymethylation of enzyme restricted to methionines 74 and 173 brought about no changes in the total activity or in the ATP inhibition profile of the enzyme. This is significant since position 74 was projected on the basis of the homologous procaryotic structure to be important in the binding of nucleotide to the allosteric site.     

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.     

Structure of cross-linked rabbit muscle phosphofructokinase in solution.

Cross-linked rabbit muscle phosphofructokinase in the active tetrameric and octameric state was studied in solution by hydrodynamic methods and small angle x-ray scattering techniques. The translational diffusion coefficients were determined by means of inelastic light scattering and were found to be 3.60 (+/- 0.02) x 10(-7) cm2 . s-1 for the tetramer and 2.54 (+/- 0.15) x 10(-7) cm2 . s-1 for the octamer. From small angle x-ray scattering measurements the radius of gyration, the specific inner surface area, and the volume were determined for both enzyme forms, revealing that the octameric cross-linked form is approximately spherical, with a diameter of 120.0 A, whereas the tetrameric form is asymmetric having an axial ratio of 2. By comparison of the scattering curves with triaxial geometric bodies which are equivalent in scattering, the tetrameric enzyme is described as a rectangular prism, with overall dimensions of A = 131.0 A, B = 131.0 A, and C = 65.0 A, and the octameric form as that of a cube with A = B = C = 120.0 A. The shape of the protomer, having a radius of gyration of 24.8 A, in the tetramer and octamer is similar to that for the native tetramer at pH 10 in the presence of 5 mM fructose 6-phosphate or 15 mM fructose 1,6-bis-phosphate. From the different shapes of the scattering curves of the native phosphofructokinase at pH 7.5 in the presence of 15 mM ATP and of the cross-linked tetramer or octamer, it can be inferred that the shapes of the protomers are different: in the presence of ATP the protomers are elongated, having an axial ratio of 1.8 to 2.0; the cross-linked state reveals a spherical protomer of radius 33.0 A, similar to that of the native enzyme at pH 7.5 in the presence of fructose 6-phosphate or fructose 1,6-bisphosphate.     

Protein-induced inactivation and phosphorylation of rabbit muscle phosphofructokinase.

Several previously untested proteins promote the reversible inactivation of rabbit skeletal muscle phosphofructokinase. Grouped in decreasing order of effectiveness, they include the following: skeletal muscle troponin C greater than troponin, the two smooth muscle myosin light chains, alpha-actinin, and S-100 much greater than parvalbumin and soybean trypsin inhibitor. The efficiency of troponin C in this process may even exceed that previously reported for calmodulin. Sequences near calcium binding site III are apparently involved in the troponin C-phosphofructokinase interaction. Troponin C and calmodulin exert calcium-dependent effects on the physical and chemical properties of muscle phosphofructokinase. When calcium is present, comigration with either protein allows the enzyme to enter the stacking gel during urea-polyacrylamide gel electrophoresis. Both enhance the phosphorylation of phosphofructokinase catalyzed by the cAMP-dependent protein kinase, with phosphate incorporations approaching 2 mol of P/mol of protomer. Reaction occurs at Ser774 and at Ser376--a novel site whose phosphorylation is highly sensitive to troponin C and less so to calmodulin. Maximum phosphorylation has slight effect on the catalytic activity of the enzyme under standard assay conditions. The troponin C induced or calmodulin-induced phosphorylation of phosphofructokinase requires calcium and is strongly inhibited by either fructose 2,6-bisphosphate or fructose 1,6-bisphosphate. Inactivation occurs in the presence or absence of calcium, with generally higher concentrations of effectors required for protection in the latter case. Liver and yeast phosphofructokinases shows little activity loss in the presence of either calmodulin or troponin C. We have developed and tested a general mathematical model for the protein-induced inactivation of phosphofructokinase which may find application to other systems.     

Sedimentation study of a catalytically active form of rabbit muscle phosphofructokinase at pH 8.55

The enzymatic active form of rabbit muscle phosphofructokinase (PFK) was observed directly by using the method of reacting or active enzyme centrifugation (AEC). These studies were performed in two assay systems: a coupled enzyme and a pH-dependent dye-linked system in glycylglycine buffer at pH 8.55 and 23 +/- 1 degree C. The sedimenting band of PFK was stabilized by three solvent systems: 50% (v/v) D2O, 10% (w/v) sucrose, and 4% (v/v) or 10% (v/v) glycerol. The active PFK species sediments as a single component with a sedimentation coefficient of 12.4 +/- 0.5 S, after correcting for protein--solvent interactions. Although PFK may undergo association--dissociation, there is no observable change in the value of s20,w over a 57-fold range of protein concentration. Throughout this range only a single active species of PFK was observed, and within an experimental uncertainty of +/- 10%, the enzymatic activity observed in the sedimentation studies accounts for the total enzymatic activity observed in the steady-state kinetics. Partially purified PFK was subjected to AEC analysis. Results reveal the presence of again a single active form sedimenting at the same rate as the purified enzyme. Results from sedimentation velocity studies indicate that the stabilizing solvents employed in AEC enhance the self-association of PFK. However, such an enhancement alone cannot account for the observation of a single active species with a sedimentation coefficient of 12.4 S. The interactions between solvent additives and PFK were studied by density measurements and by the application of multicomponent theory. Results from such a preferential solvent interaction study indicate that PFK is preferentially hydrated in the presence of sucrose or glycerol. The enhancement of PFK self-association is most likely due to a nonspecific solvent--protein interaction.     

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.     

Stabilization of active form of rabbit liver phosphofructokinase

(1) The active form of rabbit liver phosphofructokinase when preincubated in presence of F^? and effectors of the enzyme is stabilized against its conversion to less active form as a result of dilution. (2) The stabilized active form of enzyme has a Km value of 0.01 mM for fructose 6-phosphate, the same as measured in presence of all the positive effectors, and is lower, by 13 times, than the Km value of the non-stabilized control enzyme, and exhibits normal Michaelis-Menten kinetics, in contrast to the non-stabilized control enzyme which shows sigmoidal kinetics. (3) The stabilized active form of enzyme is neither inhibited by excess concentration of ATP nor activated by activators of phosphofructokinase. (4) The data thus support the proposition that the enzyme does indeed exist in two interconvertible forms with enormous difference in their affinities for fructose 6-phosphate and effectors.     

The mechanism of rabbit muscle phosphofructokinase at pH8.

The mechanism of rabbit muscle phosphofructokinase was investigated by measurement of fluxes, isotope trapping and steady-state velocities at pH8 in triethanolamine/HCl buffer with 4 mM free Mg2+. Most observations were made at I0.2. The ratio Flux of fructose 1,6-bisphosphate----fructose 6-phosphate/Flux of fructose 1,6-bisphosphate----ATP at zero ATP concentration increased hyperbolically from unity to about 3.2 as the concentration of fructose 6-phosphate was increased. Similarly, the ratio Flux of fructose 1,6-bisphosphate----ATP/Flux of fructose 1,6-bisphosphate----fructose 6-phosphate at zero fructose 6-phosphate concentration increased from unity to about 1.4 as the concentration of ATP was increased. The addition of substrates must therefore be random, whatever the other aspects of the reaction. Further, from the plateau values of the ratios, it follows that the substrates dissociate very infrequently from the ternary complex and that at a low substrate concentration 72% of the reaction follows the pathway in which ATP adds first to the enzyme. Isotope-trapping studies with [32P]ATP confirmed that ATP can bind first to the enzyme in rate-limiting step and that dissociation of ATP from the ternary complex is slow in relation to the forward reaction. No isotope trapping of [U-14C]-fructose 6-phosphate could be demonstrated. The ratios Flux of ATP----fructose 1,6-bisphosphate/Flux of ATP----ADP measured at zero ADP concentration and the reciprocal of the ratio measured at zero fructose 1,6-bisphosphate concentration did not differ significantly from unity. Calculated values for these ratios based on the kinetics of the reverse reaction and assuming ordered dissociations of products or a ping-pong mechanism gave values very significantly greater than unity. These findings exclude an ordered dissociation or a substantial contribution from a ping-pong mechanism, and it is concluded that the reaction is sequential and that dissociation of products is random. Rate constants were calculated for the steps in the enzyme reaction. The results indicate a considerable degree of co-operativity in the binding between the two substrates. The observations on phosphofructokinase are discussed in relation to methods of measurement and interpretation of flux ratios and in relation to the mechanism of other kinase enzymes.     

Analysis of the allosteric properties of rabbit muscle phosphofructokinase by means of affinity labeling with a reactive ATP analog.

A reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP-PCP, reacted specifically with the ATP inhibitory site of rabbit skeletal muscle phosphofructokinase without affecting the active site. Modification resulted in the incorporation of 1.01 mol of the reagent per mol of enzyme subunit. The modified enzyme was insensitive to allosteric inhibition by ATP and to activation by AMP at pH 7.2, where the native enzyme exhibits allosteric kinetic behavior. These observations demonstrate that we had succeeded in obtaining PFK fixed in the T state. Using the kinetic parameters of this modified enzyme, the kinetic properties of native enzyme can be quantitatively accounted for by the allosteric model of Monod-Wyman-Changeux. Further, the reagent was shown to have reacted with a specific cysteine residue near or at the ATP inhibitory site, and the sequence around the cysteine was determined as Cys-Lys-Asp-Phe-Arg.