Interaction of calmodulin with muscle phosphofructokinase. Changes of the aggregation state, conformation and catalytic activity of the enzyme

Phosphofructokinase from muscle has been shown to be a calmodulin-binding protein [Mayr, G.W. and Heilmeyer, L.M.G., Jr (1983) FEBS Lett. 159, 51-57]. Details of the influence of calmodulin on the aggregation state, the conformation and the catalytic properties of phosphofructokinase have been studied by enzymatic and light-scattering analyses. Calmodulin acts as a Ca2+-dependent hysteretic inhibitor of the highly active enzyme. At least one mole of calmodulin binds to each protomer of the enzyme, induces a shift from the highly active tetrameric towards an inactive dimeric state and slowly changes the conformation of the dimers. Dissociation of calmodulin from conformationally changed dimers by removal of Ca2+ stops the inactivation. Without a significant regain of catalytic activity large polymers are rapidly formed. For a reactivation of the inactivated enzyme, calmodulin has to remain associated and the incubation conditions must be changed in a way to allow for a back isomerization and reassociation of dimers. The isomerization reaction is promoted by Mg . ATP, the reassociation reaction most effectively by fructose bisphosphate. A model for the calmodulin-phosphofructokinase interaction is proposed.     

Interaction of calmodulin with the phosphofructokinase target sequence

Ca4.calmodulin (Ca4.CaM) inhibits the glycolytic enzyme phosphofructokinase, by preventing formation of its active tetramer. Fluorescence titrations show that the affinity of complex formation of Ca4.CaM with the key 21-residue target peptide increases 1000-fold from pH 9.0 to 4.8, suggesting the involvement of histidine and carboxylic acid residues. 1H NMR pH titration indicates a marked increase in pKa of the peptide histidine on complex formation and HSQC spectra show related pH-dependent changes in the conformation of the complex. This unusually strong sensitivity of a CaM-target complex to pH suggests a potential functional role for Ca4.CaM in regulation of the glycolytic pathway.     

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.     

Interaction of inhibitors with muscle phosphofructokinase.

The interaction of several inhibitors with muscle phosphofructokinase has been studied by both equilibrium binding measurements and kinetic analysis. At low concentrations of citrate a maximum of 1 mol is bound per mol of enzyme protomer. Tight binding requires MgATP and very weak binding is observed in the absence of either magnesium ion or ATP. ITP at low concentrations cannot replace ATP. In the presence of MgATP and at pH 7.0, the dissociation constant for the enzyme-citrate complex is 20 muM. At 50 muM citrate and excess magnesium ion, the concentration of ATP required to give half-maximal binding of citrate is approximately 3 muM . Both P-enolpyruvate and 3-P-glycerate compete for the binding of citrate and the estimated Ki values are 480 and 52 muM, respectively. Creatine-P, another inhibitor of muscle phosphofructokinase, does not compete with the binding of citrate. Measurement of the equilibrium binding of ATP shows that citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P all increase the affinity of enzyme for MgATP with the concentration required to give an effect increasing in the order given. In kinetic studies, citrate, 3-P-glycerate and P-enolpyruvate each act synergistically with ATP to inhibit the phosphofructokinase reaction. This is indicated by the observation that the three metabolites do not inhibit the enzyme with ITP as the phosphoryl donor and that they inhibit at ATP concentrations that are not themselves inhibitory. Furthermore, the sensitivity to the inhibitors increases with increasing ATP concentrations. Striking differences in the extent of inhibition can be seen by varying the order of addition of assay components. Preincubation of the enzyme with ATP and citrate, 3-P-glycerate, or P-enolpyruvate results in greater inhibition than when the inhibitor is added after the reaction is started with fructose-6-P. Furthermore, the inhibition is reversed partially 10 to 15 min after the addition of fructose-6-P. This phenomenon is particularly striking with creatine-P as the inhibitor. Very high concentrations of this inhibitor are required to show any effect if the inhibitor is added after fructose-6-P. These effects are interpreted as reflecting slow conformational changes between an active form with high affinity for fructose-6-P and an inactive, or less active, conformation that binds the inhibitors. Citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P increase the rate of the phosphofructokinase at subsaturating concentrations of MgITP. The results indicate a common binding site on the enzyme for citrate, 3-P-glycerate, and P-enolpyruvate that is distinct from the ATP inhibitory site. An additional site (or sites) for creatine-P is indicated. All four inhibitors act synergistically with ATP by increasing the affinity of the enzyme for MgATP at an inhibitory site. The inhibitors appear also to increase the affinity of the catalytic nucleoside triphosphate site for substrate.     

Interaction of immobilized phosphofructokinase with soluble muscle proteins

Selected glycolytic enzymes (including phosphoglucose isomerase, aldolase, glyceraldehyde phosphate dehydrogenase, enolase, pyruvate kinase and lactate dehydrogenase), as well as glycogen phosphorylase, creatine kinase, and adenylate kinase, bound to phosphofructokinase immobilized on an agarose gel. The affinity of phosphofructokinase to these various proteins differed, with phosphorylase exhibiting the strongest binding. Binding was reversed either by: (1) elution with high-ionic-strength buffer (0.4 M KCl); (2) the addition of a 5-10 mM concentration of ATP; or (3) high concentrations of fructose 6-phosphate (5 mM).     

Specificity of rabbit muscle phosphofructokinase for nucleotides as substrates or as allosteric effectors.

Various ATP and AMP analogs with modifications in the base moiety or in the polyphosphate chain were tested as substrates and/or as allosteric effectors of rabbit muscle phosphofructokinase. The significance of different structural elements for the nucleotide-enzyme interaction is discussed. While all investigated triphosphate analogs with a modified purine base are substrates for phosphofructokinase, those with a modified polyphosphate chain are competitive inhibitors. 5′-Adenylyl-(β,γ-methylene) diphosphonate, which is a weak competitive inhibitor, is shown to have a high affinity for the allosteric site of phosphofructokinase. Among the investigated monophosphate analogs only adenosine-N¹-oxide 5′-monophosphate can reverse the inhibitory effect of excessive ATP. A qualitative correlation is found between the quenching of the phospho-fructokinase-8-anilino-1-naphthalene-sulfonate fluorescence and the ability of the nucleotide analogs to act as substrates or as allosteric effectors of phosphofructokinase. It is concluded that the interaction of ATP with the allosteric site is more complex than that with the substrate site and requires both an intact adenine moiety and an intact terminal phosphate group for full activity.     

Interaction of calmodulin with the particulate fraction of cardiac muscle

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.     

Interaction of calmodulin with lactoferrin.

Calmodulin, as a major intracellular calcium-binding protein, regulates many Ca(2+)-dependent enzymes and plays an important role in a wide spectrum of cellular functions of the eukaryotes. Interaction between calmodulin and human lactoferrin, a 78 kDa protein with antibacterial properties, was found in the presence of Ca2+ using (i) a method for the detection of calmodulin binding proteins with biotinylated calmodulin, (ii) affinity chromatography on an agarose-calmodulin column with subsequent detection by an enzyme-linked immunosorbent assay (ELISA). The binding of calmodulin to lactoferrin blocked the ability of lactoferrin to agglutinate Micrococcus lysodeikticus.     

Interaction of phosphofructokinase with antibodies. Kinetic properties of phosphofructokinase in complexes with antibodies.

The allosteric properties of phosphofructokinase (EC 2.7.1.11) from rabbit muscle are influenced by enzyme concentration, most probably due to changes in the association state of the enzyme. In this study, the behaviour of dispersed pre-cipitates of phosphofructolinase as produced by treatment with antibodies has been investigated. The enzyme is not capable of rapid dissociation in the precipitated state as is confirmed by the lack of inactivation upon dilution and by the absence of shifts in substrate saturation curves as measured in the presence of different concentrations of the enzyme. The Hill coefficient of phosphofructokinase is decreased from 1.96 to 1.04 by antibody treatment. The V at neutral pH is increased 3-fold while the K0.5 for fructose 6-phosphate is reduced significantly. On the other hand, antibody-treated phosphofructokinase retains its sensitivity to allosteric activation by glucose 1,6-bisphosphate in the rpesence of high ATP concentrations.     

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.     

Regulation of glycolysis in muscle. The role of ammonium and synergism among the positive effectors of phosphofructokinase

It is observed that the decrease in the energy charge, increase in P_i, NH₄⁺, and fructose-6-phosphate observed in stimulated frog muscle act synergistically in increasing the activity of rabbit muscle phosphofructokinase 300-fold over its activity observed at the concentrations of above effector substrates found in the muscle at rest.The activity of phosphofructokinase at various concentrations of Mg²⁺ and various fixed concentrations of NH₄⁺, at levels of energy charge and P_i corresponding to the resting and stimulated muscle were also studied.These results suggest that variations in the concentrations of effectors of phosphofructokinase resulting from contraction of muscle are responsible for the increase in the activity of enzyme in stimulated muscle and that this activation may not necessarily be geared to the contractile process itself as postulated by Karpatkin el al.     

Conformational adaptability of the active site of beta-galactosidase. Interaction of the enzyme with some substrate analogous effectors.

The action of different effectors, glycosides, and alcohols on the reactions catalyzed by beta-galactosidase is analyzed in this paper. Effectors as large as tri- and tetrasaccharides have no effect on the enzyme activity, suggesting that the binding site has rather small size. Most of the beta-galactosides produce a competitive inhibition. The other compounds assayed behave either as noncompetitive inhibitors, and they are deadened inhibitors, or as uncompetitive inhibitors which exhibit a better affinity for the chemical intermediate than for free enzyme; nearly all of them give transfer products. The analysis of the data indicates that the active center of beta-galactosidase is made up of two subsites: a galactose and a glucose subsite. This latter site is in a more favorable conformation in the galactosylenzyme than in free enzyme; possibly it might even by generated by the galactose binding. Conformational rearrangements of the active center deduced from the inhibition data have been directly observed by differential spectroscopy. The conformational adaptability of the enzyme and its consequence for the functional properties of beta-galactosidase are discussed.     

Rat thyroid phosphofructokinase. Comparison of the regulatory and molecular properties with those of rat muscle enzyme.

The kinetic and molecular properties of rat thyroid phosphofructokinase (specific activity 134 units/mg) were compared with those of rat muscle phosphofructokinase (specific activity 135 units/mg). Thyroid and muscle phosphofructokinase showed similar sedimentation patterns in sucrose density gradients; their affinity for DEAE-cellulose was similar but not identical. A comparison of the kinetic properties revealed differences in the pH optima. Striking differences in the kinetic properties were shown below pH 7.4; the thyroid enzyme was less inhibited by ATP or citrate and more sensitive to activation by cyclic 3':5'-AMP than the muscle enzyme. A study of the effects of some cyclic as well as linear mononucleotides, such as cyclic AMP, cyclic IMP, cyclic GMP, cyclic CMP, cyclic UMP, 5'-AMP, and 3'-AMP on thyroid phosphofructokinase showed that at concentrations as low as 1 micrometer only cyclic AMP and cyclic IMP were able to activate thyroid enzyme in the presence of low fructose-6-P and high ATP concentrations.     

Interaction of a phosphorylated site of muscle phosphofructokinase with the activation of the enzyme by NH4+ and K+.1

The crystal structure of the cyclic peptide disulfide

has been determined by X-ray diffraction. The peptide crystallizes in the space group P2₁2₁2₁, with a = 8.646(1), b = 18.462(2), c = 19.678(3)Å and Z = 4. The molecules adopt a highly folded compact conformation, stabilized by two intramolecular 4→ 1 hydrogen bonds between the Cys (1) and Pro (2) CO groups and the Cys (4) and methylamide NH groups, respectively. The backbone conformational angles for the peptide lie very close to those expected for a 3₁₀ helix. The S-S bridge adopts a right handed twist with a dihedral angle of 82°. The structure illustrates the role of stereochemically constrained residues, in generating novel peptide conformations.     

Interaction of group A streptococci with human plasmin(ogen) under physiological conditions

A series of methods for analyzing the interaction of group A streptococci with the human plasminogen system are described. Examples of group A streptococcal isolates capable of assembling surface plasminogen activator activity when grown in human plasma are presented and the key requirements for this process are evaluated. The stabilities of cell-associated plasmin and plasminogen activator complexes are compared and a model for the interaction of group A streptococci with the plasminogen system in an infected host is presented.