Influence of phosphate on the allosteric behavior of yeast phosphofructokinase.

Initial rate data obtained with purified yeast phosphofructokinase (PFK) show an ATP dependent kinetic cooperativity with respect to fructose-6-phosphate. In the presence of 25 mM phosphate, the cooperativity index (Hill number) is related to the half saturation concentration of fructose-6-phosphate as predicted by the concerted allosteric model in the case of a “K-system”. In the absence of phosphate, however, the kinetic behavior of yeast PFK is more complex and the cooperativity index is invariant with respect to the half saturation concentration of fructose-6-phosphate which is increased by ATP. In both cases, 5′AMP behaves as a strong activator of the enzyme. These kinetic data suggest that the two distinct functions of ATP as phosphate donnor and as allosteric inhibitor, respectively, are supported by different binding sites. These regulatory properties of yeast PFK are discussed in relation to glycolytic oscillations.     

An equilibrium binding study of the interaction of fructose 6-phosphate and fructose 1,6-bisphosphate with rabbit muscle phosphofructokinase.

Equilibrium binding studies of the interaction of rabbit muscle phosphofructokinase with fructose 6-phosphate and fructose 1,6-bisphosphate have been carried out at 5 degrees in the presence of 1-10 mM potassium phosphate (pH 7.0 and 8.0), 5 mM citrate (pH 7.0), or 0.22 mm adenylyl imidodiphosphate (pH 7.0 and 8.0). The binding isotherms for both fructose 6-phosphate and fructose 1,6-bisphosphate exhibit negative cooperativity at pH 7.0 and 8.0 in the presence of 1-10 mM potassium phosphate at protein concentrations where the enzyme exists as a mixture of dimers and tetramers (pH 7.0) or as tetramers (pH 8.0) and at pH 7.0 in the presence of 5 mM citrate where the enzyme exists primarily as dimers. The enzyme binds 1 mol of either fructose phosphate/mol of enzyme monomer (molecular weight 80,000). When enzyme aggregation states smaller than the tetramer are present, the saturation of the enzyme with either ligand is paralleled by polymerization of the enzyme to tetramer, by an increase in enzymatic activity and by a quenching of the protein fluorescence. At protein concentrations where aggregates higher than the tetramer predominate, the fructose 1,6-bisphosphate binding isotherms are hyperbolic. These results can be quantitatively analyzed in terms of a model in which the dimer is associated with extreme negative cooperativity in binding the ligands, the tetramer is associated with less negative cooperativity, and aggregates larger than the tetramer are associated with little or no cooperativity in the binding process. Phosphate is a competitive inhibitor of the fructose phosphate sites at both pH 7.0 and 8.0, while citrate inhibits binding in a complex, noncompetitive manner. In the presence of the ATP analog adenylyl imidodiphosphate, the enzyme-fructose 6-phosphate binding isotherm is sigmoidal at pH 7.0, but hyperbolic at pH 8.0. The characteristic sigmoidal initial velocity-fructose 6-phosphate isotherms for phosphofructokinase at pH 7.0, therefore, are due to an heterotropic interaction between ATP and fructose 6-phosphate binding sites which alters the homotropic interactions between fructose 6-phosphate binding sites. Thus the homotropic interactions between fructose 6-phosphate binding sites can give rise to positive, negative, or no cooperativity depending upon the pH, the aggregation state of the protein, and the metabolic effectors present. The available data suggest the regulation of phosphofructokinase involves a complex interplay between protein polymerization and homotropic and heterotropic interactions between ligand binding sites.     

A conformational transition involved in antagonistic substrate binding to the allosteric phosphofructokinase from Escherichia coli.

The binding of fructose 6-phosphate, ATP or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate), ADP, and phosphoenolpyruvate to Escherichia coli phosphofructokinase has been studied by changes in the protein fluorescence and/or equilibrium dialysis. The results lead to the following conclusions: (1) tetrameric phosphofructokinase can bind four ATP but only two fructose-6-phosphate, and this binding occurs without cooperativity; (2) only two conformational states, T and R, with respectively a high and a low fluorescence, seem accessible to phosphofructokinase, which exists as a mixture of one-third R and two-third T states in the absence of ligand; (3) the substrate fructose 6-phosphate and the allosteric activator ADP bind preferentially to the low-fluorescence R state, while the other substrate, ATP [or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate)], and the allosteric inhibitor phosphoenolpyruvate bind to the high-fluorescence T state; (4) the binding of a given ligand is cooperative, with a Hill coefficient of 2, only when this binding is accompanied by a complete shift from one state to the other; for instance, the binding of the ATP analogue adenylyl 5'-(beta,gamma-methylenediphosphonate) to the T state is cooperative only in the presence of fructose 6-phosphate which favors the R state. This behavior is qualitatively consistent with a concerted transition, but quite different from that described earlier for phosphofructokinase from steady-state activity measurements (Blangy et al., 1968). This discrepancy suggests that the allosteric properties of phosphofructokinase are due in part to ligand binding and in part to the kinetics of the enzymatic reaction.     

Interaction of calmodulin with muscle phosphofructokinase. Interplay with metabolic effectors of the enzyme under physiological conditions

The hysteretic calmodulin-induced inactivation of muscle phosphofructokinase and the calmodulin-mediated reactivation are essentially dependent on environmental conditions. The interplay of calmodulin during these reactions and at allosteric conditions with Mg . ATP, fructose 6-phosphate, adenosine 5'-[beta, gamma-imido]triphosphate and with the allosteric effectors AMP, ADP, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate was studied by two techniques. (a) A two-step technique with a preincubation of enzyme, calmodulin and effectors in close to physiological concentrations before dilution into an optimal activity assay. It reveals aggregation and slowly reversible conformation changes. (b) A direct assay of dilute enzyme at allosteric conditions. Dominating in the interplay of calmodulin with metabolic effectors is the competitive-like action of calmodulin on Mg . ATP binding to the regulatory sites of the enzyme. At high enzyme concentrations in the absence of hexose phosphates, i.e. at noncatalytic conditions calmodulin counteracts the stabilization of the highly active tetrameric form caused by Mg . ATP. In the allosteric assay it counteracts the ATP-induced allosteric inhibition. In both cases calmodulin acts synergistic with AMP and ADP. To a minor degree calmodulin also counteracts the stabilization of the tetrameric form caused by fructose 6-phosphate and hexose bisphosphate, now however antagonistically to AMP and ADP. By the demonstrated interactions the enzyme can be slowly and hysteretically shifted between an active tetrameric and an inactive dimeric state under control metabolic conditions and of Ca2+ and calmodulin. Resting conditions will inactivate and high contractile activity reactivate available enzyme.     

Effects of AMP and Cibacron blue F3G-A on the fructose 6-phosphate binding of yeast phosphofructokinase.

The positive effector 5′-AMP of yeast phosphofructokinase does not influence the binding of fructose 6-phosphate to the enzyme. Cibacron blue F3G-A considered an ATP analogue decreases the affinity of the enzyme to fructose 6-phosphate without exerting an effect on the cooperativity of fructose 6-phosphate binding. The peculiarities of the interactions of AMP and Cibacron blue with fructose 6-phosphate binding demonstrate compatibility of the allosteric kinetics with the binding behavior of the enzyme.     

pH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli.

The pH dependence of the activity of the allosteric phosphofructokinase from Escherichia coli has been studied in the pH range from 6 to 9, in the absence or presence of allosteric effectors. The sigmoidal cooperative saturation of phosphofructokinase by fructose 6-phosphate has been analyzed according to the Hill equation, and the following results have been obtained: (i) the apparent affinity for Fru-6P, as measured by the half-saturating concentration, [Fru-6P]0.5, does not change with pH; (ii) the cooperativity, as measured empirically by the Hill coefficient, nH, increases markedly with pH and reaches a value of 5.5-6 at pH 9; (iii) the catalytic rate constant, kcat, is controlled by the ionization of a critical group which has a pK of 7 in the absence of effector and must be deprotonated for phosphofructokinase to be active. The observation that pH affects both the cooperativity and the maximum velocity suggests that the catalytic efficiency of a given active site could be modified by the binding of fructose 6-phosphate to other remote sites. Finding values of the cooperativity coefficient larger than the number of substrate binding sites indicates that slow conformational changes may occur in phosphofructokinase. The cooperative saturation of phosphofructokinase by fructose 6-phosphate appears more complex than described by the classical concerted model at steady state and could involve two slowly interconverting states which differ in both their turnover rate constants and their affinities for fructose 6-phosphate. The presence of GDP shifts the pK of the critical group which controls kcat from 7 to 6.6.(ABSTRACT TRUNCATED AT 250 WORDS)     

A proteolyzed derivative of Escherichia coli phosphofructokinase is no longer sensitive to allosteric effectors and still shows cooperativity in substrate binding

Limited proteolysis of Escherichia coli phosphofructokinase by subtilisin yields a homogeneous derivative. This proteolyzed protein is composed of four polypeptide chains, with a molecular weight of 32 000 as compared to 37 000 for the original enzyme. Removal on each chain of about 5 kdaltons maintains the enzymatic activity and does not change the apparent affinity for the substrates ATP and fructose 6-phosphate. Limited proteolysis, however, affects the cooperativity of fructose 6-phosphate binding: the Hill coefficient is reduced from almost 4 in the native enzyme to only 2 in its proteolyzed derivative. Also, the proteolyzed protein is no longer sensitive to allosteric effectors, activator, or inhibitor. These changes in regulatory properties upon proteolysis are apparently due to the destruction of the effector binding site. The allosteric effector GDP protects phospho-fructokinase against proteolysis and irreversible thermal inactivation; GDP is, however, inefficient in protecting the proteolyzed protein against thermal denaturation. These results suggest that phosphofructokinase may function as a dimer of dimers, in which homotropic and heterotropic allosteric effects are not mediated by the same sets of quaternary interactions.     

Heart phosphofructokinase: allosteric kinetics with fructose 6-sulfate.

The allosteric regulation of heart phosphofructokinase was studied at pH 6.9 with an alternative substrate, fructose 6-sulfate. The alternative substrate allowed kinetic studies to be carried out at high enzyme concentrations (0.1 mg/ml) where the effect of allosteric ligands on enzyme physical structure has been studied. A Km for ATP binding (8-10 muM) in the presence of saturating AMP concentrations was found which agreed well with the value obtained at pH 8.2, ATP inhibitory effects closely followed saturation of its substrate site. Hill plots for ATP inhibition gave an interaction coefficient of 3.5 indicating cooperatively between at least four enzyme subunits. Neither AMP nor fructose 6-sulfate affected the cooperativity between the ATP inhibitory sites but only increased the inhibitory threshold. As the ATP concentration was increased from suboptimal to inhibitory levels, interaction coefficients for AMP and fructose 6-sulfate changed from 1 to 2. Increasing citrate concentration resulted in an increase in the interaction coefficient for fructose 6-sulfate to a value of 1.9. Citrate inhibition was synergistic with ATP inhibition with an interaction coefficient of 2. The data indicate that allosteric kinetics of the enzyme can be shown at high enzyme concentrations with the alternative substrate. ATP inhibition appears to involve interaction between at least four subunits, while citrate, AMP, and fructose 6-sulfate interact minimally with two subunits.     

Phosphofructokinase from bumblebee flight muscle. Molecular and catalytic properties and role of the enzyme in regulation of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle

Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 25 degrees C or rest at 5 degrees C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 25 and 7.5 degrees C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bisphosphate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.     

Phosphofructokinase. II. Role of ligands in pH-dependent structural changes of the rabbit muscle enzyme.

The effect of ligands, including substrates and allosteric effectors, on the pH-dependent inactivation and reactivation of rabbit muscle phosphofructokinase has been examined in terms of the mechanism proposed previously (Bock, P.E. and Fireden, C. (1976) J. Biol. Chem. 251, 5630-5636). It is concluded thatt many ligands exert their effect by binding preferentially to either protonated or unprotonated forms of the enzyme and thus shifting an apparent pK for the inactivation or reactivation process. ATP and fructose 6-phosphate influence the apparent pK to different extents and in different directions, with ATP binding preferentially to the protonated forms and fructose 6-phosphate to the unprotonated forms. Enzyme inactivated by ATP can be reactivated by the addition of fructose 6-phosphate. The experiments indicate that inactivation and reactivation in the presence of these ligands can occur by kinetically different pathways as has been found for these processes in the absence of ligands. The results are discussed in relation to what might be expected for ligand binding properties of the enzyme as a function of pH, temperature, and enzyme concentration. The effect of ATP and MgATP is complex, perhaps representing more than one site of binding. Citrate appears to bind preferentially to protonated forms of the enzyme while fructose 1,6-bisphosphate and AMP bind preferentially to the unprotonated forms. ADP, K+, and NH4+ appear to have little or no preference in binding to different enzyme forms.     

Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC₅₀) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen Cryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.     

Spectroscopic studies on effector-induced and substrate-induced conformation changes of phosphofructokinase.

The interaction of phosphofructokinase with NH4+, AMP, ATP, citrate, MgATP or fructose 6-phosphate, and in part with their mixtures forming either binary or ternary complexes has been studied by means of ultraviolet difference spectroscopy and circular dichroism spectroscopy in the wavelength range 265-300 nm with the aim of characterizing the conformational corollaries of the ligand effects on phosphofructokinase. The positive as well as the negative effectors change phosphofructokinase conformation in different ways, not easily interpretable in terms of one active and one inactive enzyme conformation. The spectroscopic equivalents of phosphofructokinase conformation changes resulting from catalytic activity are similar to those produced by the reaction products. The ligand concentration-dependent changes of absorption differences in the tryptophyl, tyrosyl and phenylalanyl region parallel each other, i.e. the interactions of the ligands with phosphofructokinase are not confined to specific aromatic side chains, but involve conformation changes of the large domains of the protein. ATP affinity to the enzyme shows temperature-dependent biphasic changes so that ATP binding appears to be either an entropy-driven or enthalpy-driven process. The dissociation constants of the ligands derived from spectroscopic titration of binary complex formation are comparable to those calculated from kinetic experiments. MgATP and fructose 6-phosphate each alone change phosphofructokinase conformation by binary complex formation in keeping with a random order of reaction sequence.     

Solution X-ray scattering studies of the yeast phosphofructokinase allosteric transition. Characterization of an ATP-induced conformation distinct in quaternary structure from the R and T states of the enzyme

The allosteric transition of yeast phosphofructokinase has been studied by solution x-ray scattering. The scattering curves corresponding to the native enzyme (T conformation) were found to be similar to the curves recorded in the presence of saturating concentrations of fructose 6-phosphate (R conformation) or AMP (R or R' conformation). However, the curves obtained in the presence of ATP are clearly different: the radius of gyration increases and the secondary minima and maxima are systematically shifted to lower angles, suggesting a swelling of the enzyme in the presence of ATP. These results give the first direct evidence for the existence of an ATP-induced T' conformation, distinct in quaternary structure from the R and T states of the enzyme oligomer, in agreement with our previous modeling of yeast phosphofructokinase regulation. X-ray scattering data are discussed in relation to the distinct molecular mechanisms of the ATP and fructose 6-phosphate allosteric effects involving, respectively, sequential and concerted conformational changes of the enzyme oligomer.     

Reaction path of phosphofructo-1-kinase is altered by mutagenesis and alternative substrates

Escherichia coli phosphofructokinase (PFK) has been proposed to have a random, nonrapid equilibrium mechanism that produces nonallosteric ATP inhibition as a result of substrate antagonism. The consequences of such a mechanism have been investigated by employing alternative substrates and mutants of the enzyme that produce a variety of nonallosteric kinetic patterns demonstrating substrate inhibition and sigmoid velocity curves. Mutations of a methionine residue in the sugar phosphate binding site produced apparent cooperativity in the interaction of fructose 6-phosphate. Cooperativity could also be seen with native enzyme using a poorly binding substrate, fructose 1-phosphate. With an alternative nucleotide, 1-carboxymethyl-ATP, coupled with a mutation that introduced a negative charge in the nucleotide binding site, one could observe substrate inhibition by fructose 6-phosphate and apparent cooperativity in the interaction with nucleotide. Furthermore, the use of a phosphoryl donor, gamma-thiol-ATP, which greatly reduced the catalytic rate, apparently facilitated the equilibration of all binding reactions and eliminated ATP inhibition. These unusual kinetic patterns could be interpreted within the random, steady-state model as reflecting changes in the rates of particular binding and catalytic events.     

The kinetics of effector binding to phosphofructokinase. The influence of effectors on the allosteric conformational transition.

1. The extent of the allosteric transition from the R into the T conformation of rabbit skeletal muscle phosphofructokinase induced by Mg2+-1,N6-etheno-ATP was determined by stopped-flow fluorimetry from the amplitude of the slow phase of the Mg2+-1,N6-etheno-ATP fluorescence enhancement [Roberts & Kellet (1979) Biochem. J. 183, 349--360]. 2. The amplitude of the slow phase was decreased by low concentrations of the activators cyclic AMP and fructose 1,6-bisphosphate, but increased in a complex manner by the inhibitor citrate. 3. Mg2+-1,N6-etheno-ATP and Mg2+-ATP are unable to induce the T conformation to a detectable extent in the presence of saturating cyclic AMP, but can do so readily in the presence of saturating fructose 1,6-bisphosphate. 4. The conformational transitions induced in enzyme alone by different ligands were observed by changes in intrinsic protein fluorescence. In general, an R-type conformation has diminished protein fluorescence compared with a T-type conformation. 5. Mg2+-ATP exerts a complex effect on protein fluorescence; both the enhancement at low concentrations and the quenching at high concentrations of Mg2+-ATP result from the binding of Mg2+-ATP to the inhibitory site and the ensuing allosteric transition. Enhancement reflects the extent of the allosteric transition and involves both tyrosine and tryptophan, probably in the region of the active site; quenching reflects occupation of the inhibitory site and involves tyrosine at the inhibitory site. 6. The mechanism of the allosteric transition from the R into the T conformation induced by Mg2+-1,N6-etheno-ATP at low concentrations occurs predominantly by a 'prior-isomerization' pathway; at higher concentrations a limited contribution from a 'substrate-guided' pathway occurs. 7. The allosteric behaviour of phosphofructokinase with respect to Mg2+-ATP and Mg2+-1,N6-ethenol-ATP binding may be accounted for in terms of the simple, concerted model.     

Regulation of adult and fetal myocardial phosphofructokinase. Relief of cooperativity and competition between fructose 2,6-bisphosphate, ATP, and citrate

To clarify the physiological role of fructose 2,6-bisphosphate in the perinatal switching of myocardial fuels from carbohydrate to fatty acids, the kinetic effects of fructose 2,6-bisphosphate on phosphofructokinase purified from fetal and adult rat hearts were compared. For both enzymes at physiological pH and ATP concentrations, 1 microM fructose 2,6-bisphosphate induced a greater than 10-fold reduction in S0.5 for fructose 6-phosphate and it completely eliminated subunit cooperativity. Fructose 2,6-bisphosphate may thereby reduce the influence of changes in fructose 6-phosphate concentration on phosphofructokinase activity. Based on double-reciprocal plots and ATP inhibition studies, adult heart phosphofructokinase activity is more sensitive to physiological changes in ATP and citrate concentrations than to changes in fructose 2,6-bisphosphate concentrations. Fetal heart phosphofructokinase is less sensitive to ATP concentration above 5 mM and equally sensitive to citrate inhibition. The fetal enzyme has up to a 15-fold lower affinity for fructose 2,6-bisphosphate, rendering it more sensitive to changes in fructose 2,6-bisphosphate concentration than adult heart phosphofructokinase. Together, these factors allow greater phosphofructokinase activity in fetal heart while retaining sensitive metabolic control. In both fetal and adult heart, fructose 2,6-bisphosphate is primarily permissive: it abolishes subunit cooperativity and in its presence phosphofructokinase activity is extraordinarily sensitive to both the energy balance of the cell as reflected in ATP concentration and the availability of other fuels as reflected in cytosolic citrate concentration.     

Kinetic model of phosphofructokinase-1 from Escherichia coli

This paper presents a kinetic model of phosphofructokinase-1 from Escherichia coli. A complete catalytic cycle has been reconstructed based on available information on the oligomeric structure of the enzyme and kinetic mechanism of its monomer. Applying the generalization of the Monod-Wyman-Changeux approach proposed by Popova and Sel'kov(35-37) to the reconstructed catalytic cycle rate equation has been derived. Dependence of the reaction rate on pH, magnesium, and effectors has been taken into account. Kinetic parameters have been estimated via fitting the rate equation against experimentally measured dependencies of initial rate on substrates, products, effectors, and pH available from the literature. The model of phosphofructokinase-1 predicts (1) cooperativity of binding both fructose-6-phosphate and ATPMg(2-), (2) significant inhibition of the enzyme resulting from an increase in total concentration of ATP under the condition of fixed concentration of Mg(2+) ions, and (3) dual effect of ADP consisting of allosteric activation and product inhibition of the enzyme. Moreover, the model developed can be used in the kinetic modeling of biochemical pathways containing phosphofructokinase-1.     

Effect of substrates and effectors on the reversible inactivation of pig spleen phosphofructokinase by adenosine triphosphate

1. To investigate the mechanism of the reversible inactivation of pig spleen phosphofructokinase by ATP, the effect of order of addition of reactants (substrates, effectors and enzyme solution) was studied by preincubating the enzyme before assay with various combinations of its substrates and effectors. 2. Preincubation of the enzyme with MgATP or ATP at pH7.0 before addition of fructose 6-phosphate caused a rapid and much greater inhibition of activity than that observed when the reaction (carried out at identical substrate concentrations) was initiated with enzyme. 3. The rapid inhibition caused by preincubation with ATP, together with the sigmoidal response to fructose 6-phosphate and activation by AMP, were all blocked by prior photo-oxidation of the enzyme with Methylene Blue, which selectively destroys the inhibitory binding site for ATP [Ahlfors & Mansour (1969) J. Biol. Chem.244, 1247-1251]. 4. Fructose 6-phosphate, but not Mg(2+), protected phosphofructokinase from inhibition during preincubation with ATP in a manner that was sigmoidally dependent on the fructose 6-phosphate concentration. 5. Mg(2+), by protecting the enzyme from the inhibitory effect of preincubation at low pH (7.0) and by preventing its activation during preincubation with fructose 6-phosphate, demonstrated both a weak activating effect in the absence of the other substrates and a stronger inhibitory effect in the presence of fructose 6-phosphate. 6. Positive effectors (K(+), NH(4) (+), AMP and aspartate) protected the enzyme from inhibition during preincubation with MgATP in proportion to their potency as activators, but citrate potentiated the ATP inhibition. P(i) significantly slowed the inactivation process without itself acting as a positive effector. 7. The non-linear dependence of the initial rate of the unmodified enzyme on protein concentration (associated with increased positive homotropic co-operativity to fructose 6-phosphate) was intensified by preincubation with ATP and abolished by photo-oxidation. 8. The results are interpreted in terms of an association-dissociation model which postulates that protonation, at low pH, of a photo-oxidation-sensitive inhibitory site for ATP allows more rapid dissociation of an active tetramer to an inactive dimeric species.     

Allosteric and non-allosteric phosphofructokinases from Lactobacilli. Purification and properties of phosphofructokinases from L. plantarum and L. acidophilus.

Phosphofructokinase (ATP : D-fructose-6-phosphate 1 phosphotransferase, EC 2.7.1.11) from two different lactobacilli, Lactobacillus plantarum and Lactobacillus acidophilus were isolated and purified. Both enzymes have a molecular weight of 154 000 and consist of four subunits of identical size. Antisera from sheep immunized against the purified phosphofructokinase from L. plantarum showed immunologic cross reaction with the enzyme from L. acidophilus. In spite of the close molecular relationship indicated by the immunologic cross reaction, the kinetic behaviour of the two enzymes was strikingly different. Phosphofructokinase from L. plantarum showed pure Michaelis-Menten behaviour. Phosphofructokinase from L. acidophilus, however, showed sigmoidal substrate saturation curves for fructose 6-phosphate in the presence of slightly alkaline pH and high ATP concentrations; it was activated by fructose 1,6-biphosphate and inhibited by ADP. The results indicate that even enzymes which are structurally very similar may differ greatly with respect to their kinetic and regulatory properties and suggest that allosteric and non-allosteric phosphofructokinases have the same origin in evolution.