Equilibrium binding studies of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus

A tryptophan-shifted mutant of phosphofructokinase (PFK) from Bacillus stearothermophilus has been constructed. This mutant, which is functionally similar to wild-type, provides the opportunity to examine the allosteric properties of PFK under equilibrium conditions. The unique fluorescence properties of the tryptophan-shifted mutant enzyme, W179F/F230W, have been utilized to deduce the thermodynamics of ligand binding and the allosteric perturbations in the absence of catalytic turnover. Specifically, phospho(enol)pyruvate (PEP) and MgADP binding to the mutant PFK can be directly observed using tryptophan fluorescence, and dissociation constants for these ligands have been measured to be equal to 2.71 +/- 0.04 and 90.4 +/- 3.5 microM, respectively. In addition, the homotropic couplings for the allosteric ligands have been assessed for the first time. PEP binds cooperatively with a Hill number of 2.9 +/- 0.3, while MgADP binding is not cooperative. The equilibrium couplings between these ligands and the substrate fructose 6-phosphate (Fru-6-P) have also been determined and follow the same trends with temperature observed under steady-state kinetic assay conditions using wild-type PFK, indicating that the presence of bound MgATP has little influence on the allosteric interactions. Like wild-type PFK, the coupling free energies for the mutant result from largely compensating enthalpy and entropy components at 25 degrees C. Furthermore, the sign of each coupling free energy, which signifies the nature of the allosteric effect, is opposite that of the enthalpy contribution and is therefore due to the larger absolute value of the associated entropy change. This characteristic stands in direct contrast to the thermodynamic basis of the allosteric response in the homologous PFK from E. coli in which the sign of the coupling free energy is established by the sign of the coupling enthalpy.     

Kinetic linked-function analysis of the multiligand interactions on Mg(2+)-activated yeast pyruvate kinase.

The multiligand interactions governing the allosteric response of Mg(2+)-activated yeast pyruvate kinase (YPK) during steady-state turnover were quantitated by kinetic linked-function analysis. The substrate, PEP, the enzyme-bound divalent metal, Mg(2+), and the allosteric effector, FBP, positively influence each other's interaction with the enzyme in the presence of saturating concentrations of the second substrate, MgADP. The presence of Mg(2+) enhances the interaction of PEP and of FBP with YPK by -2.0 and -1.0 kcal/mol, respectively. The simultaneous interaction of PEP, Mg(2+), and FBP with YPK is favored by -4.1 kcal/mol over the sum of their independent binding free energies. The coupling free energies measured for Mg(2+)-activated YPK are weaker than the corresponding coupling free energies measured for Mn(2+)-activated YPK [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803], but are consistent with results of thermodynamic measurements with the Mg(2+)-YPK complex [Bollenbach, T. J., and Nowak, T. (2001) Biochemistry 36, 13088-13096]. A comparison of ligand binding data measured by kinetic and thermodynamic linked-function analyses reveals that the MgADP complex modulates both the binding of the other three ligands and the two- and three-ligand coupling interactions between the other three ligands. Enzyme-bound Mg(2+) does not influence the homotropic cooperativity in PEP binding to YPK. It is the MgADP complex that induces homotropic cooperativity in PEP binding. It is the enzyme-bound Mn(2+) that induces homotropic binding of PEP with Mn(2+)-activated YPK. These results lend support to the hypothesis that divalent metals modulate the interactions of ligands on YPK and that divalent metals play a role in regulation of the glycolytic pathway.     

Kinetic and structural characterization of phosphofructokinase from Lactobacillus bulgaricus

Phosphofructokinase from Lactobacillus delbrueckii subspecies bulgaricus (LbPFK) has been reported to be a nonallosteric analogue of phosphofructokinase from Escherichia coli at pH 8.2 [Le Bras et al. (1991) Eur. J. Biochem. 198, 683-687]. A reexamination of the kinetics of this enzyme shows LbPFK to have limited binding affinity toward the allosteric ligands, MgADP and PEP, with dissociation constants of approximately 20 mM for both. Their allosteric effects are observed only at high concentrations of these ligands, with both exhibiting inhibitory effects on substrate binding. No pH dependence was observed for the binding and the influence of MgADP and PEP on the enzyme. To attempt to explain these results, the crystal structure of LbPFK was solved using molecular replacement to 1.86 A resolution. A comparative study of the LbPFK structure with that of phosphofructokinases from E. coli (EcPFK) and Bacillus stearothermophilus (BsPFK) reveals a structure with conserved fold and substrate binding site. The effector binding site, however, shows many differences that could explain the observed decreases in binding affinity for MgADP and PEP in LbPFK as compared to the other two enzymes.     

Identification of substrate contact residues important for the allosteric regulation of phosphofructokinase from Eschericia coli

The side chains of Escherichia coli phosphofructokinase (EcPFK) that interact with bound substrate, fructose 6-phosphate (Fru-6-P), are examined for their potential roles in allosteric regulation. Mutations that severely decrease Fru-6-P affinity and/or k(cat)/K(m) were created at each contact residue, with the exception of the catalytic base, D127. Even though Fru-6-P affinity was greatly decreased for R162E, M169A, E222A/H223A, and R243E, the mutated proteins retained the ability to be activated by MgADP and inhibited by phosphoenolpyruvate (PEP). R252E did not show an allosteric response to either MgADP or PEP. The H249E mutation retained MgADP activation but did not respond to PEP. R72E, T125A, and R171E maintained allosteric inhibition by PEP. Both R72E and T125A displayed a MgADP-dependent decrease in k(cat) but no MgADP-dependent K-type effects. R171E maintained MgADP-dependent K-type activation but also displayed a MgADP-dependent decrease in k(cat). Localization of mutations that alter MgADP activation near the transferred phosphate group indicates the importance of the 1-methoxy region of Fru-6-P in allosteric regulation by MgADP. A region near the 6'-phosphate may be similarly important for PEP inhibition. R252 is uniquely positioned between the 1'- and 6'-phosphates of bound Fru-1,6-BP, and the mutation at this position may alter both allosterically responsive regions. The differential functions of specific regions in the Fru-6-P contact residues support different mechanisms for allosteric activation and inhibition. In addition, the lack of correlation between mutations that decrease Fru-6-P affinity and those that abolish allosteric communications supports the independence of affinity and allosteric coupling.     

Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus

A strategy for isolating each of the four potentially unique heterotropic pairwise allosteric interactions that exist in the homotetramer phosphofructokinase from Bacillus stearothermophilus is described. The strategy involves the construction of hybrid tetramers containing one wild-type subunit and three mutant subunits that have been modified to block binding of both the substrate, fructose 6-phosphate (Fru-6-P), and the allosteric inhibitor, phospho(enol)pyruvate (PEP). Each type of binding site occurs at a subunit interface, and mutations on either side of the interface have been identified that will greatly diminish binding at the respective site. Consequently, four different types of mutant subunits have been created, each containing a different active site and allosteric site modification. The corresponding 1:3 hybrids isolate a different pair of unmodified substrate and allosteric sites with a unique structural disposition located 22, 30, 32, and 45 A apart, respectively. The allosteric inhibition exhibited by the unmodified sites in each of these four hybrids has been quantitatively evaluated in terms of a coupling free energy. Each of the coupling free energies is unique in magnitude, and their relative magnitudes vary with pH. Importantly, the sum of these coupling free energies at each pH is equal to the total heterotropic coupling free energy associated with the tetrameric enzyme. The latter quantity was assessed from the overall inhibition of a control hybrid that removed the homotropic interactions in PEP binding. The results do not agree with either the concerted or sequential models that are often invoked to explain allosteric behavior in oligomeric enzymes.     

Isolation of a single activating allosteric interaction in phosphofructokinase from Escherichia coli

Escherichia coli phosphofructokinase 1 (EcPFK) is a homotetramer with four active and four allosteric sites. Understanding of the structural basis of allosteric activation of EcPFK by MgADP is complicated by the multiplicity of binding sites. To isolate a single heterotropic allosteric interaction, hybrid tetramers were formed between wild-type and mutant EcPFK subunits in which the binding sites of the mutant subunits have decreased affinity for their respective ligands. The 1:3 (wild-type:mutant) hybrid that contained only one native active site and one native allosteric site was isolated. The affinity for the substrate fructose-6-phosphate (Fru-6-P) of a single wild-type active site is greatly decreased over that displayed by the wild-type tetramer due to the lack of homotropic activation. The free energy of activation by MgADP for this heterotropic interaction is -0.58 kcal/mol at 8.5 degrees C. This compares to -2.87 kcal/mol for a hybrid with no homotropic coupling but all four unique heterotropic interactions. Therefore, the isolated interaction contributes 20% of the total heterotropic coupling. By comparison, wild-type EcPFK exhibits a coupling free energy between Fru-6-P and MgADP of -1.56 kcal/mol under these conditions, indicating that the effects of MgADP are diminished by a homotropic activation equal to -1.3 kcal/mol. These data are not consistent with a concerted allosteric mechanism.     

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37 °C. As a function of increasing pH, hL-PYK’s affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and alanine slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to alanine is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for alanine inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response.     

Disentangling the web of allosteric communication in a homotetramer: heterotropic activation in phosphofructokinase from Escherichia coli

Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites and four allosteric sites. Understanding the allosteric activation of EcPFK by MgADP has been complicated by the complex web of possible interactions, including active site homotropic interactions, allosteric site homotropic interactions, and heterotropic interactions between active and allosteric sites. The current work has simplified this web of possible interactions to a series of single heterotropic interactions by forming and isolating hybrid tetramers. Each of the four unique heterotropic interactions have independently been isolated and compared to a control that has all four of the unique heterotropic interactions. If the interactions are labeled with the distances between interacting ligands, the 45-A interaction contributes 20% +/- 1%, the 33-A interaction contributes 34% +/- 1%, the 30-A interaction contributes 21% +/- 1%, and the 23-A interaction contributes 25% +/- 1% with respect to the total free energy of MgADP/fructose 6-phosphate (Fru-6-P) activation in the control. The free energies of the isolated interactions sum to 100% +/- 2% of the total. Therefore, the four unique interactions are all contributors to activation, are nonequivalent, and are additive.     

Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.     

Steady-state fluorescence of Escherichia coli phosphofructokinase reveals a regulatory role for ATP.

We have investigated the effects of ligands and effectors on the intrinsic fluorescence of Escherichia coli phosphofructokinase (PFK). We have found that the substrate fructose 6-phosphate (Fru6P) or the allosteric activator ADP can quench the fluorescence up to 35%. The response is hyperbolic with Ks[Fru6P] of 20 microM and Ks[ADP] of 13 microM. The allosteric inhibitor phosphoenolpyruvate (PEP) converts the hyperbolic response with respect to Fru6P to a sigmoidal response. AMP-PNP, a nonhydrolyzable analogue of ATP, also inhibits the Fru6P fluorescence response. PFK mutant KA213, which is insensitive to effectors, has a decreased fluorescence response with respect to ADP, and PEP does not convert the Fru6P response to sigmoidicity. However, its fluorescence response with respect to Fru6P is decreased by ATP or AMP-PNP. Taken together, these results suggest that, in the absence of effectors or ligands, E. coli PFK exists in a state with high affinity for Fru6P ("R" state). This state can be altered to a low affinity ("T" state) by PEP binding to the allosteric site or by ATP binding to the enzyme.     

Persistent binding of MgADP to the E187A mutant of Escherichia coli phosphofructokinase in the absence of allosteric effects

MgADP binding to the allosteric site enhances the affinity of Escherichia coli phosphofructokinase (PFK) for fructose 6-phosphate (Fru-6-P). X-ray crystallographic data indicate that MgADP interacts with the conserved glutamate at position 187 within the allosteric site through an octahedrally coordinated Mg(2+) ion [Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994]. Lau and Fersht reported that substituting an alanine for this glutamate within the allosteric site of PFK (i.e., mutant E187A) causes MgADP to lose its allosteric effect upon Fru-6-P binding [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. However, these authors later reported that MgADP inhibits Fru-6-P binding in the E187A mutant. The inhibition presumably occurs by preferential binding to the inactive (T) state complex of the Monod-Wyman-Changeux two-state model [Lau, F. T.-K., and Fersht, A. R. (1989) Biochemistry 28, 6841-6847]. The present study provides an alternative explanation of the role of MgADP in the E187A mutant. Using enzyme kinetics, steady-state fluorescence emission, and anisotropy, we performed a systematic linkage analysis of the three-ligand interaction between MgADP, Fru-6-P, and MgATP. We found that MgADP at low concentrations did not enhance or inhibit substrate binding. Anisotropy shows that MgADP binding at the allosteric site occurred even when MgADP produced no allosteric effect. However, as in the wild-type enzyme, the binding of MgADP to the active site in the mutant competitively inhibited MgATP binding and noncompetitively inhibited Fru-6-P binding. These results clarified the mechanism of a three-ligand interaction and offered a nontraditional perspective on allosteric mechanism.     

Linked-function origins of cooperativity in a symmetrical dimer

The thermodynamic origins of substrate binding cooperativity in a dimeric enzyme that can bind one substrate (A) and one allosteric ligand (X) to each of two identical subunits are discussed. It is assumed that maximal activity is not subject to allosteric modification and that the substrates and allosteric ligands achieve binding equilibrium in the steady state. Each uniquely ligated form is assumed to be capable of exhibiting unique binding properties, and only the principles of thermodynamic linkage are used to constrain the system further. The explicit relationship between the Hill coefficient, the concentration of X, and the magnitudes of the relevant coupling free energies and dissociation constants is derived. In the absence of X only the homotropic coupling between substrate sites contributes to a nonhyperbolic substrate saturation profile. An allosteric ligand, X, can alter the cooperativity in two distinct ways, one mechanism being manifested when X is saturating and the only only when X is present at saturating concentrations. By evaluating the concentration of substrate required to produce half-maximal velocity as a function of [X], as well as the Hill coefficients when X is absent and fully saturating, the dissociation and coupling constants most important for understanding the mechanisms of allosteric action in an enzyme of this type can be determined.     

Kinetic mechanism of Ascaris suum phosphofructokinase desensitized to allosteric modulation by diethylpyrocarbonate modification

The kinetic mechanism of phosphofructokinase has been determined at pH 8 for native enzyme and pH 6.8 for an enzyme desensitized to allosteric modulation by diethylpyrocarbonate modification. In both cases, the mechanism is predominantly steady state ordered with MgATP binding first in the direction of fructose 6-phosphate (F6P) phosphorylation and rapid equilibrium random in the direction of MgADP phosphorylation. This is a unique kinetic mechanism for a phosphofructokinase. Product inhibition by MgADP is competitive versus MgATP and noncompetitive versus F6P while fructose 1,6-bisphosphate (FBP) is competitive versus fructose 6-phosphate and uncompetitive versus MgATP. The uncompetitive pattern obtained versus F6P is indicative of a dead-end E.MgATP.FBP complex. Fructose 6-phosphate is noncompetitive versus either FBP or MgADP. Dead-end inhibition by arabinose 5-phosphate or 2,5-anhydro-D-mannitol 6-phosphate is uncompetitive versus MgATP corroborating the ordered addition of MgATP prior to F6P. In the direction of MgADP phosphorylation, inhibition by anhydromannitol 1,6-bisphosphate is noncompetitive versus MgADP, while Mg-adenosine 5'(beta, gamma-methylene)triphosphate is noncompetitive versus FBP. Anhydromannitol 6-phosphate is a slow substrate, while anhydroglucitol 6-phosphate is not. This suggests that the enzyme exhibits beta-anomeric specificity.     

Quantifying the allosteric properties of Escherichia coli carbamyl phosphate synthetase: determination of thermodynamic linked-function parameters in an ordered kinetic mechanism.

The effects of the allosteric ligands UMP, IMP, and ornithine on the partial reactions catalyzed by Escherichia coli carbamyl phosphate synthetase have been examined. Both of these reactions, a HCO3(-)-dependent ATP synthesis reaction and a carbamyl phosphate-dependent ATP synthesis reaction, follow bimolecular ordered sequential kinetic mechanisms. In the ATPase reaction, MgATP binds before HCO3- as established previously for the overall reaction catalyzed by carbamyl phosphate synthetase [Raushel, F. M., Anderson, P. M., & Villafranca, J. J. (1978) Biochemistry 17, 5587-5591]. The initial velocity kinetics for the ATP synthesis reaction indicate that MgADP binds before carbamyl phosphate in an equilibrium ordered mechanism except in the presence of ornithine. Determination of true thermodynamic linked-function parameters describing the impact of allosteric ligands on the binding interactions of the first substrate to bind in an ordered mechanism requires experiments to be performed in which both substrates are varied even if only one is apparently affected by the allosteric ligands. In so doing, we have found that IMP has little effect on the overall reaction of either of these two partial reactions. UMP and ornithine, which have a pronounced effect on the apparent Km for MgATP in the overall reaction, both substantially change the thermodynamic dissociation constant for MgADP from the binary E-MgADP complex, Kia, in the ATP synthesis reaction, with UMP increasing Kia 15-fold and ornithine decreasing Kia by 18-fold. By contrast, only UMP substantially affects the Kia for MgATP in the ATPase reaction, increasing it by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of an individual allosteric interaction in tetrameric phosphofructokinase from Bacillus stearothermophilus

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a model allosteric enzyme system in which the interactions between substrates and allosteric effectors have been extensively studied. However, the oligomeric nature of BsPFK has made it difficult to determine the molecular basis of the allosteric regulation because of the multitude of different types of heterotropic and homotropic interactions that are possible between the four active sites and four allosteric sites in the native tetramer. In an attempt to alleviate the complexity of the system and thereby allow the quantitation of a single interaction between one active site and one allosteric site, site-directed mutagenesis has been coupled with a hybrid-forming scheme to create and isolate a tetramer of BsPFK in which only a single active site and a single allosteric site are capable of binding their respective ligands with high (i.e., near wild type) affinity. Characterization of this single allosteric interaction indicates that the free energy involved in the inhibition by the allosteric effector phosphoenolpyruvate (PEP) is 1.48 +/- 0.15 kcal/mol compared to the 3.58 +/- 0.02 kcal/mol measured for the enzyme.     

Interactive binding to the two principal ligand binding sites of human serum albumin: effect of the neutral-to-base transition.

The relationship between the two principal ligand binding sites, sites I and II, on human serum albumin (HSA) was quantitatively and qualitatively examined by equilibrium dialysis and fluorescence spectroscopy. Among the three subsite markers to site I, only the binding of dansyl-L-asparagine (DNSA), which is a subsite Ib marker (K. Yamasaki et al., Biochim. Biophys. Acta 1295 (1996) 147), was inhibited by the simultaneous binding of a site II ligand, such as ibuprofen and diazepam. This indicates that, in contrast to subsite Ib, subsites Ia and Ic do not strongly interact with site II. The thermodynamic characteristics for the coupling reaction between DNSA and ibuprofen and between DNSA and diazepam, which gave positive coupling free energies and negative values for both coupling enthalpy and entropy, indicated that the reaction process was entropically driven. Increase of pH from 6.5 to 8.2 caused an increase in coupling constant and entropy for the mutual antagonism between DNSA and the site II ligands on binding to HSA. The site II ligand-induced red-shift of lambda(max) and solvent accessibility of DNSA in subsite Ib were decreased when the albumin molecule was isomerized from the neutral (N) to the base (B) conformation in the physiological pH region. Based on these findings, we conclude that a 'competitive' like strong allosteric regulation exists for the binding of these two ligands to the N conformer, whereas for the B conformer this interaction can be classified as nearly 'independent'. Since the distance between Trp-214, which resides within the site I subdomain, and Tyr-411, which is involved in site II, is increased by 6 A during the N-B transition (N.G. Hagag et al., Fed. Proc. 41 (1982) 1189), we propose a mechanism for the pH-dependent antagonistic binding between subsite Ib and site II, which involves the transmission of ligand-induced allosteric effects from one site to another site, modified by changes in the spatial relationship of sites I and II caused by the N-B transition.     

Binding of carbon dioxide to phosphoenolpyruvate carboxykinase deduced from carbon kinetic isotope effects.

Phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] from Chloris gayana Kunth has been purified by a combination of ammonium sulfate fractionation, ion exchange, gel filtration, and affinity chromatography on agarose-hexane-ATP. In the direction of OAA formation, the specific activity of the enzyme was 33 mumol/(min.mg of protein). The carbon isotope effect on carboxylation was measured by successive analysis of remaining CO2 over the course of the reaction. At 22 mM PEP and 1.3 mM MgADP, pH 7.5, the isotope effect is 1.024 +/- 0.001. When the concentration of PEP was reduced to 1 mM, the isotope effect rose to 1.034 +/- 0.004; when the concentration of MgADP was reduced to 60 microM, the value rose to 1.040 +/- 0.006. The variation of the carbon isotope effect on carboxylation with both substrate concentrations indicates that the enzyme operates by a random kinetic mechanism. This in turn requires that the enzyme have a binding site for substrate CO2; this is one of the first enzymes for which such a site has been demonstrated.     

ATP-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme

The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritimawas purified 730-fold to homogeneity. The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits. Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 degrees C. Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant ( S(0.5)) of 0.7 mM and a Hill coefficient of 2.2. The apparent K(m) for ATP was 0.2 mM and the apparent V(max) value was about 360 U/mg. The enzyme also catalyzed in vitro the reverse reaction with an apparent K(m) for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent V(max) of about 13 U/mg. Divalent cations were required for maximal activity; Mg(2+), which was most effective, could partially be replaced by Mn(2+) and Fe(2+). Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP. The enzyme had a temperature optimum of 93 degrees C and showed a significant thermostability up to 100 degrees C. Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli. The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T. maritima. The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family. In accordance with its allosteric properties, ATP-PFK of T. maritima contained the conserved allosteric effector-binding sites for ADP and PEP.     

Plant cytosolic pyruvate kinase: a kinetic study.

The kinetic properties of cytosolic pyruvate kinase (PKc) from germinating castor oil seeds (COS) have been investigated. From experiments in which the free Mg2+ concentration was varied at constant levels of either the complexed or free forms of the substrates it was determined that the true substrates are the free forms of both phosphoenolpyruvate (PEP) and ADP. This conclusion is corroborated by the quenching of intrinsic PKC tryptophan fluorescence by free PEP and ADP. Mg2+ is bound as the free bivalent cation but is likely released as MgATP. The fluorescence data, substrate interaction kinetics, and pattern of inhibition by products and substrate analogues (adenosine 5'-O-(2-thiodiphosphate) for ADP and phenyl phosphate for PEP) are compatible with a sequential, compulsory-ordered, Tri-Bi type kinetic reaction mechanism. PEP is the leading substrate, and pyruvate the last product to abandon the enzyme. The dissociation constant and limiting Km for free PEP (8.2 to 22 and 38 microM, respectively) and the limiting Km for free ADP (2.9 microM) are considerably lower than those reported for the non-plant enzyme. The results indicate that COS PKc exists naturally in an activated state, similar to the fructose 1,6-bisphosphate-activated yeast enzyme. This deduction is consistent with a previous study (F.E. Podestá and W.C. Plaxton (1991) Biochem. J. 279, 495-501) that failed to identify any allosteric activators for the COS PKc, but which proposed a regulatory mechanism based upon ATP levels and pH-dependent alterations in the enzyme's response to various metabolite inhibitors. As plant phosphofructokinases display potent inhibition by PEP, the overall rate of glycolytic flux from hexose 6-phosphate to pyruvate in the plant cytosol will ultimately depend upon variations in PEP levels brought about by the regulation of PKc.