RESUMEN

Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.

Asunto(s)

Penaeidae/enzimología , Penaeidae/genética , Fosfofructoquinasa-2/genética , Fosfofructoquinasa-2/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Branquias/metabolismo , Hipoxia/enzimología , Hipoxia/genética , Ácido Láctico/metabolismo , Modelos Moleculares , Fosfofructoquinasa-2/química , Filogenia , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of different forms of the enzyme together with a kinetic enzyme analysis. Here we complement the mechanism by using hybrid oligomers of the homodimeric enzyme to get insights about the allosteric communication pathways between the same sites or different ones located in different subunits. Kinetic analysis of the hybrid enzymes indicate that homotropic interactions between allosteric sites for ATP or between substrate sites for fructose-6-P have a minor effect on the enzymatic inhibition induced by ATP. In fact, the sigmoid response for fructose-6-P observed at elevated ATP concentrations can be eliminated even though the enzymatic inhibition is still operative. Nevertheless, leverage coupling analysis supports heterotropic interactions between the allosteric ATP and fructose-6-P binding occurring between and within each subunit.

Asunto(s)

Adenosina Trifosfato/metabolismo , Proteínas de Escherichia coli/metabolismo , Fructosafosfatos/metabolismo , Fosfofructoquinasa-2/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/farmacología , Regulación Alostérica , Sitio Alostérico , Sitios de Unión/genética , Biocatálisis/efectos de los fármacos , Simulación por Computador , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fructosafosfatos/química , Cinética , Modelos Moleculares , Estructura Molecular , Mutación , Fosfofructoquinasa-2/antagonistas & inhibidores , Fosfofructoquinasa-2/química , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Especificidad por Sustrato

RESUMEN

Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened ß-barrel. Although isolated monomers of E. coli Pfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30 µM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240-309. These residues correspond to most of the ß-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that the ß-meander module is not affected by monomerizing mutations.

Asunto(s)

Proteínas de Escherichia coli/química , Fosfofructoquinasa-2/química , Pliegue de Proteína , Multimerización de Proteína , Secuencia de Aminoácidos , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Mutación , Fosfofructoquinasa-2/genética , Fosfofructoquinasa-2/metabolismo , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo

RESUMEN

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner.

Asunto(s)

Frío , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Fosfofructoquinasa-2/química , Desnaturalización Proteica , Solventes/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Escherichia coli/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fosfofructoquinasa-2/metabolismo , Multimerización de Proteína , Estructura Terciaria de Proteína , Solventes/metabolismo

RESUMEN

Folding studies have been focused mainly on small, single-domain proteins or isolated single domains of larger proteins. However, most of the proteins present in biological systems are composed of multiple domains, and to date, the principles that underlie its folding remain elusive. The unfolding of Pfk-2 induced by GdnHCl has been described by highly cooperative three-state equilibrium (N(2)↔2I↔2U). This is characterized by a strong coupling between the subunits' tertiary structure and the integrity of the dimer interface because "I" represents an unstructured and expanded monomeric intermediate. Here we report that cold and heat unfolding of Pfk-2 resembles the N(2)↔2I step of chemically induced unfolding. Moreover, cold unfolding appears to be as cooperative as that induced chemically and even more so than its heat-unfolding counterpart. Because Pfk-2 is a large homodimer of 66 kDa with a complex topology consisting of well-defined domains, these results are somewhat unexpected considering that cold unfolding has been described as a special kind of perturbation that decouples the cooperative unfolding of several proteins.

Asunto(s)

Frío , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Calor , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/metabolismo , Desplegamiento Proteico , Dicroismo Circular , Estabilidad de Enzimas/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Guanidina/farmacología , Luz , Desnaturalización Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Desplegamiento Proteico/efectos de los fármacos , Dispersión de Radiación

RESUMEN

Phosphofructokinase-2 is a 66 kD homodimer whose subunits are associated by means of a bimolecular domain, the ß-clasp, which is linked to the larger portion of each subunit by a reentrant chain topology. To investigate how this structural organization determines the folding pathway of Pfk-2, unfolding and folding kinetic experiments were performed. The folding pathway shows an unstructured monomeric intermediate and that most part of the dimer structure is reached as a slow concerted folding/association step with a quite folded transition state in terms of solvent exposure. Unfolding kinetics show a transient intermediate, probably a partially unfolded dimer. We propose that these characteristics arise by a mutual constrain between the large domain and the ß-clasp domain imposed by their interrupted chain connectivity.

Asunto(s)

Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Fosfofructoquinasa-2/química , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Dimerización , Modelos Moleculares , Desplegamiento Proteico , Termodinámica

RESUMEN

The reaction catalyzed by E. coli Pfk-2 presents a dual-cation requirement. In addition to that chelated by the nucleotide substrate, an activating cation is required to obtain full activity of the enzyme. Only Mn(2+) and Mg(2+) can fulfill this role binding to the same activating site but the affinity for Mn(2+) is 13-fold higher compared to that of Mg(2+). The role of the E190 residue, present in the highly conserved motif NXXE involved in Mg(2+) binding, is also evaluated in this behavior. The E190Q mutation drastically diminishes the kinetic affinity of this site for both cations. However, binding studies of free Mn(2+) and metal-Mant-ATP complex through EPR and FRET experiments between the ATP analog and Trp88, demonstrated that Mn(2+) as well as the metal-nucleotide complex bind with the same affinity to the wild type and E190Q mutant Pfk-2. These results suggest that this residue exert its role mainly kinetically, probably stabilizing the transition state and that the geometry of metal binding to E190 residue may be crucial to determine the catalytic competence.

Asunto(s)

Escherichia coli/enzimología , Magnesio/metabolismo , Manganeso/metabolismo , Fosfofructoquinasa-2/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Cationes Bivalentes/química , Cationes Bivalentes/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/genética , Cinética , Magnesio/química , Manganeso/química , Mutación , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/genética

RESUMEN

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K(0.5) for fructose-6-P and a decrease in the apparent k(cat) as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n(H) of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.

Asunto(s)

Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Fructosafosfatos/química , Fosfofructoquinasa-2/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Dominio Catalítico , Cristalografía por Rayos X , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Fructosafosfatos/metabolismo , Cinética , Fosfofructoquinasa-1/química , Fosfofructoquinasa-1/metabolismo , Fosfofructoquinasa-2/metabolismo

RESUMEN

Phosphofructokinase-2 (Pfk-2) belongs to the ribokinase family and catalyzes the ATP-dependent phosphorylation of fructose-6-phosphate, showing allosteric inhibition by a second ATP molecule. Several structures have been deposited on the PDB for this family of enzymes. A structure-based multiple sequence alignment of a non-redundant set of these proteins was used to infer phylogenetic relationships between family members with different specificities and to dissect between globally conserved positions and those common to phosphosugar kinases. We propose that phosphosugar kinases appeared early in the evolution of the ribokinase family. Also, we identified two conserved sequence motifs: the TR motif, not described previously, present in phosphosugar kinases but not in other members of the ribokinase family, and the globally conserved GXGD motif. Site-directed mutagenesis of R90 and D256 present in these motifs, indicate that R90 participates in the binding of the phosphorylated substrate and that D256 is involved in the phosphoryl transfer mechanism.

Asunto(s)

Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Escherichia coli/genética , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico/genética , Secuencia Conservada , ADN Bacteriano/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Genes Bacterianos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosfofructoquinasa-2/clasificación , Fosfofructoquinasa-2/metabolismo , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

Escherichia coli phosphofructokinase-2 (Pfk-2) is a homodimer whose subunits consist of a large domain and an additional beta-sheet that provides the interfacial contacts between the subunits, creating a beta-barrel flattened-like structure with the adjacent subunit's beta-sheet. To determine how the structural organization of Pfk-2 determines its stability, the reversible unfolding of the enzyme was characterized under equilibrium conditions by enzymatic activity, circular dichroism, fluorescence and hydrodynamic measurements. Pfk-2 undergoes a cooperative unfolding/dissociation process with the accumulation of an expanded and unstructured monomeric intermediate with a marginal stability and a large solvent accessibility with respect to the native dimer.

Asunto(s)

Escherichia coli/enzimología , Fosfofructoquinasa-2/química , Dicroismo Circular , Dimerización , Estabilidad de Enzimas , Guanidina , Luz , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína , Dispersión de Radiación , Espectrometría de Fluorescencia

RESUMEN

Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the substrate and allosteric ATPs are also participating in the dimer-dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli.

Asunto(s)

Adenosina Trifosfato , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/metabolismo , Estructura Cuaternaria de Proteína , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Proteínas de Escherichia coli/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ligandos , Magnesio/metabolismo , Modelos Moleculares , Fosfofructoquinasa-2/genética , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Especificidad por Sustrato

RESUMEN

Escherichia coli phosphofructokinase-2 (Pfk-2) is an oligomeric enzyme characterized by two kinds of interfaces: a monomer-monomer interface, critical for enzymatic activity, and a dimer-dimer interface formed upon tetramerization due to allosteric binding of MgATP. In this work, Pfk-2 was denatured by guanidine hydrochloride (GdnHCl) and the impact of ligand binding on the unfolding pathway of the dimeric and the tertrameric forms of the enzyme was examined. The unligated dimeric form unfolds and dissociates from 0.15 to 0.8 M GdnHCl without the accumulation of native monomers, as indicated by circular dichroism and size exclusion chromatography measurements. However, a monomeric intermediate with an expanded volume and residual secondary structure accumulates above 0.8 M GdnHCl. The dimeric fructose-6-P-enzyme complex shows a shift in the simultaneous dissociation and unfolding process to elevated GdnHCl concentrations (from 0.8 to 1.4 M) together with the expulsion of the ligand detected by intrinsic fluorescence measurements. The unfolding pathway of the tetrameric MgATP-enzyme complex shows the accumulation of a tetrameric intermediate with altered fluorescence properties at about 0.4 M GdnHCl. Above this concentration a sharp transition from tetramers to monomers, without the accumulation of either compact dimers or monomers, was detected by light scattering measurements. Indeed, the most populated species was a partially unfolded monomer about 0.7 M GdnHCl. On the basis of these results, we suggest that the subunit contacts are critical for the maintenance of the overall structure of Pfk-2 and for the binding of ligands, explaining the reported importance of the dimeric state for enzymatic activity.

Asunto(s)

Proteínas de Escherichia coli/química , Fosfofructoquinasa-2/química , Pliegue de Proteína , Transducción de Señal , Naftalenosulfonatos de Anilina/metabolismo , ATPasa de Ca(2+) y Mg(2+)/química , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Cromatografía en Gel , Dicroismo Circular , Dimerización , Activación Enzimática , Proteínas de Escherichia coli/metabolismo , Fructosafosfatos/química , Fructosafosfatos/metabolismo , Luz , Fosfofructoquinasa-2/metabolismo , Unión Proteica , Desnaturalización Proteica , Dispersión de Radiación , Transducción de Señal/fisiología , Espectrometría de Fluorescencia

RESUMEN

Escherichia coli contains two phosphofructokinases, Pfk-1 and Pfk-2, which belong to unrelated protein families. In addition to catalytic function, the enzymes have converged in showing substrate inhibition by the nucleotide MgATP. However, although both Pfk-1 and Pfk-2 have been extensively characterized biochemically, only the structure of the former has been solved by X-ray diffraction. In order to fully understand how the same function has evolved on different structural folds, Pfk-2 has been crystallized by the hanging-drop vapour-diffusion method using PEG 6000 as precipitant. Single crystals were grown in the presence of MgATP and diffracted to 1.98 A. The crystals belong to the orthorhombic system, space group P222(1), with unit-cell parameters a = 42.8, b = 86.8, c = 171.3 A. The calculated Matthews coefficient of 2.45 A(3) Da(-1) indicates the presence of two monomers in the asymmetric unit, corresponding to a solvent content of 49%. Structure determination is ongoing.

Asunto(s)

Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Familia de Multigenes , Fosfofructoquinasa-2/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Cristalización , Cristalografía por Rayos X/métodos , Escherichia coli/genética , Fosfofructoquinasa-2/genética

RESUMEN

Phosphofructokinase-2 (Pfk-2) from Escherichia coli belongs to the ribokinase family of sugar kinases. One of the signatures observed in amino acid sequences from the ribokinase familiy members is the NXXE motif, which locates at the active site in the ribokinase fold. It has been suggested that the effect of Mg2+ and phosphate ions on enzymatic activity, observed in several adenosine kinases and ribokinases, would be a widespread feature in the ribokinase family, with the conserved amino acid residues in the NXXE motif playing a role in the binding of these ions at the active site [Maj, M. C., et al. (2002) Biochemistry 41, 4059-4069]. In this work we study the effect of Mg2+ and phosphate ions on Pfk-2 activity and the involvement of residue E190 from the NXXE motif in this behavior. The kinetic data are in agreement with the requirement of a Mg2+ ion, besides the one present in the metal-nucleotide complex, for catalysis in the wild-type enzyme. Since the response to free Mg2+ concentration is greatly affected in the E190Q mutant, we conclude that this residue is required for the proper binding of the catalytic Mg2+ ion at the active site. The E190Q mutant presents a 50-fold decrease in the kcat value and a 15-fold increment in the apparent Km for MgATP(2-). Inorganic phosphate, typically considered an activator of adenosine kinases, ribokinases, and phosphofructokinases (nonhomologous to Pfk-2) acted as an inhibitor of wild-type and E190Q mutant Pfk-2. We suggest that phosphate can bind to the allosteric site of Pfk-2, producing an inhibition pattern qualitatively similar to MgATP(2-), which can be reversed to some extent by increasing the concentration of fructose-6-P. Given that the E190Q mutant presents alterations in the inhibition by MgATP(2-) and phosphate, we conclude that the E190 residue has a role not only in catalysis but also in allosteric regulation.

Asunto(s)

Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Magnesio/química , Fosfatos/química , Fosfofructoquinasa-2/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Regulación Alostérica/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Catálisis , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ácido Glutámico/genética , Glutamina/genética , Magnesio/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Fosfatos/metabolismo , Fosfofructoquinasa-2/antagonistas & inhibidores , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética

RESUMEN

In a previous work, chemical modification of Cys-238 of Escherichia coli Pfk-2 raised concerns on the importance of the dimeric state of Pfk-2 for enzyme activity, whereas modification of Cys-295 impaired the enzymatic activity and the MgATP-induced tetramerization of the enzyme. The results presented here demonstrate that the dimeric state of Pfk-2 is critical for the stability and the activity of the enzyme. The replacement of Cys-238 by either Ala or Phe shows no effect on the kinetic parameters, allosteric inhibition, dimer stability and oligomeric structure of Pfk-2. However, the mutation of Cys-295 by either Ala or Phe provokes a decrease in the k(cat) value and an increment in the K(m) values for both substrates. We suggest that the Cys-295 residue participates in intersubunit interactions in the tetramer since the Cys-295-Phe mutant exhibits higher tetramer stability, which in turn results in an increase in the fructose-6-P concentration required for the reversal of the MgATP inhibition relative to the wild type enzyme.

Asunto(s)

Cisteína/metabolismo , Escherichia coli/enzimología , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/metabolismo , Subunidades de Proteína/metabolismo , Regulación Alostérica , Cisteína/genética , Dimerización , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Escherichia coli/genética , Fructosafosfatos/metabolismo , Guanidina/farmacología , Cinética , Mutación/genética , Fosfofructoquinasa-2/antagonistas & inhibidores , Fosfofructoquinasa-2/genética , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Especificidad por Sustrato

RESUMEN

Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with pyrene maleimide (PM) results in a rapid inactivation of the enzyme. The loss of enzyme activity correlates with the incorporation of 2 mol of PM/mol of subunit and the concomitant dissociation of the dimeric enzyme. The two modified residues were identified as Cys-238 and Cys-295. In the presence of the negative allosteric effector, MgATP, Cys-238 was the only modified cysteine residue. Kinetic characterization of the Cys-238-labelled Pfk-2 indicates that the enzyme is fully active, with the kinetic constants ( K(m), kcat) being almost identical to the ones obtained for the native enzyme. The modified enzyme is a monomer in the absence of ligands and, like the native enzyme, behaves as a tetramer in the presence of the nucleotide. However, in the presence of fructose-6-phosphate (fru-6-P) and ATP(-4), the enzyme behaves as a dimer, suggesting that the monomers undergo re-association in the presence of the substrates and that the active species is a dimer. Modification of Pfk-2 with eosin-5-maleimide (EM) results in the labelling of Cys-295. This modified enzyme is inactive and is not able to bind to the allosteric effector, remaining as a dimer in its presence. Nonetheless, Cys-295-labelled Pfk-2 is able to bind to the substrate fru-6-P in an hyperbolic fashion with a K(d) value that is 6-fold higher than the one determined for the native enzyme. These are the first residues to be implicated in the activity and/or structure of the Pfk-2.

Asunto(s)

Cisteína/química , Cisteína/fisiología , Eosina Amarillenta-(YS)/análogos & derivados , Escherichia coli/enzimología , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Dimerización , Eosina Amarillenta-(YS)/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Maleimidas/química , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Solventes/química , Reactivos de Sulfhidrilo/química

RESUMEN

The binding of MgATP and fructose-6-phosphate to phosphofructokinase-2 from Escherichia coli induces conformational changes that result in significant differences in the x-ray-scattering profiles compared with the unligated form of the enzyme. When fructose- 6-phosphate binds to the active site of the enzyme, the pair distribution function exhibits lower values at higher distances, indicating a more compact structure. Upon binding of MgATP to the allosteric site of the enzyme, the intensity at lower angles increases as a consequence of tetramer formation, but differences along higher angles also suggest changes at the tertiary structure level. We have used homology modeling to build the native dimeric form of phosphofructokinase-2 and fitted the experimental scattering curves by using rigid body movements of the domains in the model, similar to those observed in known homologous structures. The best fit with the experimental data of the unbound protein was achieved with open conformations of the domains in the model, whereas domain closure improves the agreement with the scattering of the enzyme-fructose-6-phosphate complex. Using the same approach, we utilized the scattering curve of the phosphofructokinase-2-MgATP complex to model the arrangement and conformation of dimers in the tetramer. We observed that, along with tetramerization, binding of MgATP to the allosteric site induces domain closure. Additionally, we used the scattering data to restore the low resolution structure of phosphofructokinase-2 (free and bound forms) by an ab initio procedure. Based on these findings, a proposal is made to account for the inhibitory effect of MgATP on the enzymatic activity.

Asunto(s)

Escherichia coli/enzimología , Fosfofructoquinasa-2/química , Estructura Cuaternaria de Proteína , Ligandos , Modelos Moleculares , Fosfofructoquinasa-2/aislamiento & purificación , Fosfofructoquinasa-2/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Soluciones , Difracción de Rayos X

RESUMEN

Binding of MgATP to the allosteric site of phosphofructokinase-2 promotes a dimer to tetramer conversion. In the presence of Fru-6-P the enzyme remains as a dimer. Limited proteolysis in the presence of MgATP completely protects the enzyme against inactivation and cleavage, while Fru-6-P provides a partial protection. A 28-kDa proteolytic fragment containing the N-terminus of the protein is inactive, but retains the ability to bind Fru-6-P and the allosteric effector MgATP. The fragment remains as a dimer but does not form a tetramer in the presence of MgATP. The results suggest major conformational changes of the enzyme upon ligand binding that confer a higher degree of compactness to the monomers in the dimer and in the tetramer, demonstrate the presence of the active and allosteric sites in this N-terminus fragment, and stress the importance of the C-terminus region of the protein for catalytic activity and ligand-induced oligomerization.

Asunto(s)

Escherichia coli/enzimología , Fosfofructoquinasa-2/metabolismo , Adenosina Trifosfato/metabolismo , Sitio Alostérico , Sitios de Unión , Cromatografía en Gel , Dimerización , Hidrólisis , Cinética , Ligandos , Fragmentos de Péptidos/metabolismo , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/aislamiento & purificación , Subtilisina/metabolismo