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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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Intrauterine growth restriction (IUGR) associates with fetal and placental vascular dysfunction, and increased cardiovascular risk later on life. We hypothesize that endothelial cells derived from IUGR umbilical veins present significant changes in the proteome which could be involved in the endothelial dysfunction associated to this conditions. To address this the proteome profile of human umbilical endothelial cells (HUVEC) isolated from control and IUGR pregnancies was compared by 2D-Differential In Gel Electrophoresis (DIGE) and further protein identification by MALDI-TOF MS. Using 2D-DIGE 124 spots were identified as differentially expressed between control and IUGR HUVEC, considering a cut-off of 2 fold change, which represented ∼10% of the total spots detected. Further identification by MALDI-TOF MS and in silico clustering of the proteins showed that those differentially expressed proteins between control and IUGR HUVEC were mainly related with cytoskeleton organization, proteasome degradation, oxidative stress response, mRNA processing, chaperones and vascular function. Finally Principal Component analysis of the identified proteins showed that differentially expressed proteins allow distinguishing between control and IUGR HUVEC based on their proteomic profile. This study demonstrates for the first time that IUGR-derived HUVEC maintained in primary culture conditions present an altered proteome profile, which could reflect an abnormal programming of endothelial function in this fetal condition.

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Arginase-2 counteracts endothelial nitric oxide synthase (eNOS) activity in human endothelium, and its expression is negatively controlled by histone deacetylase (HDAC2). Conversely NO inhibits HDAC and previous studies suggest that arginase-2 is up-regulated by NO. We studied whether NO regulates arginase-2 expression in umbilical artery endothelial cells (HUAEC) increasing ARG2 promoter accessibility. HUAEC exposed to NOC-18 (NO donor, 1-100 µM, 0-24 h) showed an increase in arginase-2 but a decrease in eNOS mRNA levels in a time-dependent manner, with a maximal effect at 100 µM (24 h). Conversely NOS inhibition with L-NAME (100 µM) reduced arginase-2 mRNA and protein levels, an effect reverted by co-incubation with NOC-18. Treatment with TSA paralleled the effects of NO on arginase-2 and eNOS at mRNA and protein levels, with maximal effect at 10 µM. Co-incubation of NOC-18 (100 µM) with a sub-maximal concentration of TSA (1 µM) potentiated the increase in arginase-2 mRNA levels, whilst L-NAME prevented TSA-dependent arginase-2 induction. The effects on arginase-2 mRNA were paralleled by changes in chromatin accessibility, as well as increased levels of H3K9 and H4K12 acetylation, at ARG2 proximal (-579 to -367 and -280 to -73 bp from TSS) and core (-121 to +126 bp from TSS) promoter. Finally NO-dependent arginase-2 induction was prevented by pre-incubation for 10 min with the cysteine blocker MMTS (10 mM). These data showed for the first time that NO up-regulates arginase-2 expression in primary cultured human endothelial cells by an epigenetic-mediated mechanism increasing ARG2 promoter accessibility suggesting a negative regulatory loop for eNOS activity.

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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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In some archaea, glucose degradation proceeds through a modified version of the Embden-Meyerhof pathway where glucose and fructose-6-P phosphorylation is carried out by kinases that use ADP as the phosphoryl donor. Unlike their ATP-dependent counterparts these enzymes have been reported as non-regulated. Based on the three dimensional structure determination of several ADP-dependent kinases they can be classified as members of the ribokinase superfamily. In this work, we have studied the role of divalent metal cations on the catalysis and regulation of ADP-dependent glucokinases and phosphofructokinase from hyperthermophilic archaea by means of initial velocity assays as well as molecular dynamics simulations. The results show that a divalent cation is strictly necessary for the activity of these enzymes and they strongly suggest that the true substrate is the metal-nucleotide complex. Also, these enzymes are promiscuous in relation to their metal usage where the only considerations for metal assisted catalysis seem to be related to the ionic radii and coordination geometry of the cations. Molecular dynamics simulations strongly suggest that this metal is bound to the highly conserved NXXE motif, which constitutes one of the signatures of the ribokinase superfamily. Although free ADP cannot act as a phosphoryl donor it still can bind to these enzymes with a reduced affinity, stressing the importance of the metal in the proper binding of the nucleotide at the active site. Also, data show that the binding of a second metal to these enzymes produces a complex with a reduced catalytic constant. On the basis of these findings and considering evolutionary information for the ribokinase superfamily, we propose that the regulatory metal acts by modulating the energy difference between the protein-substrates complex and the reaction transition state, which could constitute a general mechanism for the metal regulation of the enzymes that belong this superfamily.

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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.

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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.

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Some hyperthermophilic archaea use a modified glycolytic pathway that employs an ADP-dependent glucokinase (ADP-GK) and an ADP-dependent phosphofructokinase (ADP-PFK) or, in the case of Methanococcus jannaschii, a bifunctional ADP-dependent glucophosphofructokinase (ADP-GK/PFK). The crystal structures of three ADP-GKs have been determined. However, there is no structural information available for ADP-PFKs or the ADP-GK/PFK. Here, we present the first crystal structure of an ADP-PFK from Pyrococcus horikoshii OT3 (PhPFK) in both apo- and AMP-bound forms determined to 2.0-A and 1.9-A resolution, respectively, along with biochemical characterization of the enzyme. The overall structure of PhPFK maintains a similar large and small alpha/beta domain structure seen in the ADP-GK structures. A large conformational change accompanies binding of phosphoryl donor, acceptor, or both, in all members of the ribokinase superfamily characterized thus far, which is believed to be critical to enzyme function. Surprisingly, no such conformational change was observed in the AMP-bound PhPFK structure compared with the apo structure. Through comprehensive site-directed mutagenesis of the substrate binding pocket we identified residues that were critical for both substrate recognition and the phosphotransfer reaction. The catalytic residues and many of the substrate binding residues are conserved between PhPFK and ADP-GKs; however, four key residues differ in the sugar-binding pocket, which we have shown determine the sugar-binding specificity. Using these results we were able to engineer a mutant PhPFK that mimics the ADP-GK/PFK and is able to phosphorylate both fructose 6-phosphate and glucose.

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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.

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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.

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