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Jorge Babul - One of the best experts on this subject based on the ideXlab platform.
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studying the phosphoryl transfer mechanism of the e coli Phosphofructokinase 2 from x ray structure to quantum mechanics molecular mechanics simulations
Chemical Science, 2019Co-Authors: Juliana A Murillolopez, Jorge Babul, Richard Charles Garratt, Kirill Zinovjev, Humberto Dmuniz Pereira, Andres Caniuguir, Rodrigo Recabarren, Jans Alzatemorales, Julio Caballero, Inaki TunonAbstract:Phosphofructokinases (Pfks) catalyze the ATP-dependent phosphorylation of fructose-6-phosphate (F6P) and they are regulated in a wide variety of organisms. Although numerous aspects of the kinetics and regulation have been characterized for Pfks, the knowledge about the mechanism of the phosphoryl transfer reaction and the transition state lags behind. In this work, we describe the X-ray crystal structure of the homodimeric Pfk-2 from E. coli, which contains products in one site and reactants in the other, as well as an additional ATP molecule in the inhibitory allosteric site adjacent to the reactants. This complex was previously predicted when studying the kinetic mechanism of ATP inhibition. After removing the allosteric ATP, molecular dynamic (MD) simulations revealed conformational changes related to domain packing, as well as stable interactions of Lys27 and Asp256 with donor (ATP) and acceptor (fructose-6-) groups, and of Asp166 with Mg2+. The phosphoryl transfer reaction mechanism catalyzed by Pfk-2 was investigated through Quantum Mechanics/Molecular Mechanics (QM/MM) simulations using a combination of the string method and a path-collective variable for the exploration of its free energy surface. The calculated activation free energies showed that a dissociative mechanism, occurring with a metaphosphate intermediate formation followed by a proton transfer to Asp256, is more favorable than an associative one. The structural analysis reveals the role of Asp256 acting as a catalytic base and Lys27 stabilizing the transition state of the dissociative mechanism.
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Studying the phosphoryl transfer mechanism of the E. coli Phosphofructokinase-2: from X-ray structure to quantum mechanics/molecular mechanics simulations
Chemical science, 2019Co-Authors: Juliana A. Murillo-lópez, Jorge Babul, Richard Charles Garratt, Kirill Zinovjev, Humberto Dmuniz Pereira, Andres Caniuguir, Rodrigo Recabarren, Julio Caballero, Jans Alzate-morales, Inaki TunonAbstract:Phosphofructokinases (Pfks) catalyze the ATP-dependent phosphorylation of fructose-6-phosphate (F6P) and they are regulated in a wide variety of organisms. Although numerous aspects of the kinetics and regulation have been characterized for Pfks, the knowledge about the mechanism of the phosphoryl transfer reaction and the transition state lags behind. In this work, we describe the X-ray crystal structure of the homodimeric Pfk-2 from E. coli, which contains products in one site and reactants in the other, as well as an additional ATP molecule in the inhibitory allosteric site adjacent to the reactants. This complex was previously predicted when studying the kinetic mechanism of ATP inhibition. After removing the allosteric ATP, molecular dynamic (MD) simulations revealed conformational changes related to domain packing, as well as stable interactions of Lys27 and Asp256 with donor (ATP) and acceptor (fructose-6-) groups, and of Asp166 with Mg2+. The phosphoryl transfer reaction mechanism catalyzed by Pfk-2 was investigated through Quantum Mechanics/Molecular Mechanics (QM/MM) simulations using a combination of the string method and a path-collective variable for the exploration of its free energy surface. The calculated activation free energies showed that a dissociative mechanism, occurring with a metaphosphate intermediate formation followed by a proton transfer to Asp256, is more favorable than an associative one. The structural analysis reveals the role of Asp256 acting as a catalytic base and Lys27 stabilizing the transition state of the dissociative mechanism.
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Regulatory network of the allosteric ATP inhibition of E. coli Phosphofructokinase-2 studied by hybrid dimers.
Biochimie, 2016Co-Authors: Pablo Villalobos, Mauricio Baez, Francisco Soto, Jorge BabulAbstract: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.
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Regulatory network of the allosteric ATP inhibition of E. coli Phosphofructokinase-2 studied by hybrid dimers
Biochimie, 2016Co-Authors: Pablo Villalobos, Mauricio Baez, Francisco Soto, Jorge BabulAbstract: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.
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the folding unit of Phosphofructokinase 2 as defined by the biophysical properties of a monomeric mutant
Biophysical Journal, 2015Co-Authors: Cesar A Ramirezsarmiento, Jorge Babul, Mauricio Baez, Elizabeth A Komives, Ricardo A Zamora, Deepa Balasubramaniam, Victoria GuixeAbstract: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 β-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 β-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 β-meander module is not affected by monomerizing mutations.
Victoria Guixe - One of the best experts on this subject based on the ideXlab platform.
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the folding unit of Phosphofructokinase 2 as defined by the biophysical properties of a monomeric mutant
Biophysical Journal, 2015Co-Authors: Cesar A Ramirezsarmiento, Jorge Babul, Mauricio Baez, Elizabeth A Komives, Ricardo A Zamora, Deepa Balasubramaniam, Victoria GuixeAbstract: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 β-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 β-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 β-meander module is not affected by monomerizing mutations.
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A Ribokinase Family Conserved Monovalent Cation Binding Site Enhances the MgATP-induced Inhibition in E. coli Phosphofructokinase-2
Biophysical journal, 2013Co-Authors: Mauricio Baez, Victoria Guixe, Ricardo Cabrera, Richard Charles Garratt, Andres Caniuguir, Humberto M. Pereira, Alejandro Blanco, Pablo Villalobos, César A. Ramírez-sarmiento, Jorge BabulAbstract:The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na+. Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K+ or Cs+. Kinetic characterization of the enzyme shows that K+ and Na+ alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K+ (but not Na+) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K+ (but not Na+) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs+ or K+. These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.
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observation of solvent penetration during cold denaturation of e coli Phosphofructokinase 2
Biophysical Journal, 2013Co-Authors: Cesar A Ramirezsarmiento, Jorge Babul, Mauricio Baez, Christian A M Wilson, Elizabeth A Komives, Victoria GuixeAbstract: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.
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Expanded monomeric intermediate upon cold and heat unfolding of Phosphofructokinase-2 from Escherichia coli.
Biophysical journal, 2012Co-Authors: Mauricio Baez, Victoria Guixe, Christian A M Wilson, César A. Ramírez-sarmiento, Jorge BabulAbstract: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 (N2↔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 N2↔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.
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Divalent metal cation requirements of Phosphofructokinase-2 from E. coli. Evidence for a high affinity binding site for Mn2+.
Archives of biochemistry and biophysics, 2010Co-Authors: Jaime Andrés Rivas-pardo, Jorge Babul, Andres Caniuguir, Christian A M Wilson, Victoria GuixeAbstract:Abstract 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.
Mauricio Baez - One of the best experts on this subject based on the ideXlab platform.
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Regulatory network of the allosteric ATP inhibition of E. coli Phosphofructokinase-2 studied by hybrid dimers.
Biochimie, 2016Co-Authors: Pablo Villalobos, Mauricio Baez, Francisco Soto, Jorge BabulAbstract: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.
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Regulatory network of the allosteric ATP inhibition of E. coli Phosphofructokinase-2 studied by hybrid dimers
Biochimie, 2016Co-Authors: Pablo Villalobos, Mauricio Baez, Francisco Soto, Jorge BabulAbstract: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.
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the folding unit of Phosphofructokinase 2 as defined by the biophysical properties of a monomeric mutant
Biophysical Journal, 2015Co-Authors: Cesar A Ramirezsarmiento, Jorge Babul, Mauricio Baez, Elizabeth A Komives, Ricardo A Zamora, Deepa Balasubramaniam, Victoria GuixeAbstract: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 β-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 β-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 β-meander module is not affected by monomerizing mutations.
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A Ribokinase Family Conserved Monovalent Cation Binding Site Enhances the MgATP-induced Inhibition in E. coli Phosphofructokinase-2
Biophysical journal, 2013Co-Authors: Mauricio Baez, Victoria Guixe, Ricardo Cabrera, Richard Charles Garratt, Andres Caniuguir, Humberto M. Pereira, Alejandro Blanco, Pablo Villalobos, César A. Ramírez-sarmiento, Jorge BabulAbstract:The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na+. Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K+ or Cs+. Kinetic characterization of the enzyme shows that K+ and Na+ alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K+ (but not Na+) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K+ (but not Na+) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs+ or K+. These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.
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observation of solvent penetration during cold denaturation of e coli Phosphofructokinase 2
Biophysical Journal, 2013Co-Authors: Cesar A Ramirezsarmiento, Jorge Babul, Mauricio Baez, Christian A M Wilson, Elizabeth A Komives, Victoria GuixeAbstract: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.
Ricardo Cabrera - One of the best experts on this subject based on the ideXlab platform.
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A Ribokinase Family Conserved Monovalent Cation Binding Site Enhances the MgATP-induced Inhibition in E. coli Phosphofructokinase-2
Biophysical journal, 2013Co-Authors: Mauricio Baez, Victoria Guixe, Ricardo Cabrera, Richard Charles Garratt, Andres Caniuguir, Humberto M. Pereira, Alejandro Blanco, Pablo Villalobos, César A. Ramírez-sarmiento, Jorge BabulAbstract:The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na+. Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K+ or Cs+. Kinetic characterization of the enzyme shows that K+ and Na+ alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K+ (but not Na+) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K+ (but not Na+) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs+ or K+. These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.
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the crystal complex of Phosphofructokinase 2 of escherichia coli with fructose 6 phosphate kinetic and structural analysis of the allosteric atp inhibition
Journal of Biological Chemistry, 2011Co-Authors: Ricardo Cabrera, Richard Charles Garratt, Humberto Dmuniz Pereira, Andres Caniuguir, Mauricio Baez, Jorge BabulAbstract:Abstract 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 A. 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 K0.5 for fructose-6-P and a decrease in the apparent kcat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (nH 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.
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Ribokinase family evolution and the role of conserved residues at the active site of the PfkB subfamily representative, Pfk-2 from Escherichia coli
Archives of biochemistry and biophysics, 2010Co-Authors: Ricardo Cabrera, Jorge Babul, Victoria GuixeAbstract: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.
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crystallographic structure of Phosphofructokinase 2 from escherichia coli in complex with two atp molecules implications for substrate inhibition
Journal of Molecular Biology, 2008Co-Authors: Ricardo Cabrera, Victoria Guixe, Andre Luis Berteli Ambrosio, Richard Charles Garratt, Jorge BabulAbstract: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 ATPdependent 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 nonWatson-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.
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Unfolding pathway of the dimeric and tetrameric forms of Phosphofructokinase-2 from Escherichia coli.
Biochemistry, 2007Co-Authors: Mauricio Baez, Ricardo Cabrera, Victoria Guixe, Jorge BabulAbstract: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.
Andres Caniuguir - One of the best experts on this subject based on the ideXlab platform.
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studying the phosphoryl transfer mechanism of the e coli Phosphofructokinase 2 from x ray structure to quantum mechanics molecular mechanics simulations
Chemical Science, 2019Co-Authors: Juliana A Murillolopez, Jorge Babul, Richard Charles Garratt, Kirill Zinovjev, Humberto Dmuniz Pereira, Andres Caniuguir, Rodrigo Recabarren, Jans Alzatemorales, Julio Caballero, Inaki TunonAbstract:Phosphofructokinases (Pfks) catalyze the ATP-dependent phosphorylation of fructose-6-phosphate (F6P) and they are regulated in a wide variety of organisms. Although numerous aspects of the kinetics and regulation have been characterized for Pfks, the knowledge about the mechanism of the phosphoryl transfer reaction and the transition state lags behind. In this work, we describe the X-ray crystal structure of the homodimeric Pfk-2 from E. coli, which contains products in one site and reactants in the other, as well as an additional ATP molecule in the inhibitory allosteric site adjacent to the reactants. This complex was previously predicted when studying the kinetic mechanism of ATP inhibition. After removing the allosteric ATP, molecular dynamic (MD) simulations revealed conformational changes related to domain packing, as well as stable interactions of Lys27 and Asp256 with donor (ATP) and acceptor (fructose-6-) groups, and of Asp166 with Mg2+. The phosphoryl transfer reaction mechanism catalyzed by Pfk-2 was investigated through Quantum Mechanics/Molecular Mechanics (QM/MM) simulations using a combination of the string method and a path-collective variable for the exploration of its free energy surface. The calculated activation free energies showed that a dissociative mechanism, occurring with a metaphosphate intermediate formation followed by a proton transfer to Asp256, is more favorable than an associative one. The structural analysis reveals the role of Asp256 acting as a catalytic base and Lys27 stabilizing the transition state of the dissociative mechanism.
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Studying the phosphoryl transfer mechanism of the E. coli Phosphofructokinase-2: from X-ray structure to quantum mechanics/molecular mechanics simulations
Chemical science, 2019Co-Authors: Juliana A. Murillo-lópez, Jorge Babul, Richard Charles Garratt, Kirill Zinovjev, Humberto Dmuniz Pereira, Andres Caniuguir, Rodrigo Recabarren, Julio Caballero, Jans Alzate-morales, Inaki TunonAbstract:Phosphofructokinases (Pfks) catalyze the ATP-dependent phosphorylation of fructose-6-phosphate (F6P) and they are regulated in a wide variety of organisms. Although numerous aspects of the kinetics and regulation have been characterized for Pfks, the knowledge about the mechanism of the phosphoryl transfer reaction and the transition state lags behind. In this work, we describe the X-ray crystal structure of the homodimeric Pfk-2 from E. coli, which contains products in one site and reactants in the other, as well as an additional ATP molecule in the inhibitory allosteric site adjacent to the reactants. This complex was previously predicted when studying the kinetic mechanism of ATP inhibition. After removing the allosteric ATP, molecular dynamic (MD) simulations revealed conformational changes related to domain packing, as well as stable interactions of Lys27 and Asp256 with donor (ATP) and acceptor (fructose-6-) groups, and of Asp166 with Mg2+. The phosphoryl transfer reaction mechanism catalyzed by Pfk-2 was investigated through Quantum Mechanics/Molecular Mechanics (QM/MM) simulations using a combination of the string method and a path-collective variable for the exploration of its free energy surface. The calculated activation free energies showed that a dissociative mechanism, occurring with a metaphosphate intermediate formation followed by a proton transfer to Asp256, is more favorable than an associative one. The structural analysis reveals the role of Asp256 acting as a catalytic base and Lys27 stabilizing the transition state of the dissociative mechanism.
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A Ribokinase Family Conserved Monovalent Cation Binding Site Enhances the MgATP-induced Inhibition in E. coli Phosphofructokinase-2
Biophysical journal, 2013Co-Authors: Mauricio Baez, Victoria Guixe, Ricardo Cabrera, Richard Charles Garratt, Andres Caniuguir, Humberto M. Pereira, Alejandro Blanco, Pablo Villalobos, César A. Ramírez-sarmiento, Jorge BabulAbstract:The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na+. Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K+ or Cs+. Kinetic characterization of the enzyme shows that K+ and Na+ alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K+ (but not Na+) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K+ (but not Na+) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs+ or K+. These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.
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the crystal complex of Phosphofructokinase 2 of escherichia coli with fructose 6 phosphate kinetic and structural analysis of the allosteric atp inhibition
Journal of Biological Chemistry, 2011Co-Authors: Ricardo Cabrera, Richard Charles Garratt, Humberto Dmuniz Pereira, Andres Caniuguir, Mauricio Baez, Jorge BabulAbstract:Abstract 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 A. 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 K0.5 for fructose-6-P and a decrease in the apparent kcat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (nH 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.
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Divalent metal cation requirements of Phosphofructokinase-2 from E. coli. Evidence for a high affinity binding site for Mn2+.
Archives of biochemistry and biophysics, 2010Co-Authors: Jaime Andrés Rivas-pardo, Jorge Babul, Andres Caniuguir, Christian A M Wilson, Victoria GuixeAbstract:Abstract 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.