Investigating the Electrostatic Role of a Critical Arginine for the Catalysis of E. Coli ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the regulatory enzyme of the pathway for starch synthesis in plants and glycogen in mammals and enteric bacteria. It exists as a 200 kDa homotetramer (α4) in enteric bacteria, and as a heterotetramer (α2β2) in plants. In both in vivo and in vitro the substrates (Glucose 1-Phosphate; Glc-1P and Adenosine 5\u27-Triphosphate; ATP) are converted into a glucose donor ADP-Glucose and a pyrophosphate (PPi) via the ADP-Glc PPase enzyme. It has been noted that some residues are conserved in homotetrameric bacterial ADP-Glc PPases, but are not in some plant forms. One of them is Arginine-32 (R32) in the Escherichia coli ADP-Glc PPase. To explore the overall role of this residue and evaluate the structural and electrostatic importance of the Arginine\u27s guanidinium group, we replaced it with Lysine (K, -amino group), Alanine (A, - methyl group), Cysteine (C, -sulfide group), Glutamic (E, - carboxylate group), Glutamine (Q, -amido group) and Leucine (L, -hydrophobic side chain) via site directed mutagenesis. We over-expressed the enzymes, purified them to homogeneity, and measured their kinetic properties. The Specific Activity (U/mg) for the mutants were as follows: WT (90.56), R32A (1.65), R32C (0.57), R32E (0.04), R32K (5.81), R32L (0.65) and R32Q (1.37). Currently, the properties of the R32H (Histidine, -imidizoleum ring) mutant are being investigated. Our results clearly indicate that this guanidinium group of the Arginine-32 residue is critical for catalysis. Modeling of the E. coli enzyme suggests that the two (2) nitrogen atoms of the guanidinium group may interact with the β and γ phosphates of the ATP, helping in the positioning of the substrates, via electrostatic interactions, and making the PPi product a more stable leaving group

Unraveling the Activation Mechanism of the Potato Tuber ADP-glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase regulates the synthesis of glycogen in bacteria and of starch in plants. The enzyme from plants is mainly activated by 3-phosphoglycerate and is a heterotetramer comprising two small and two large subunits. Here, we found that two highly conserved residues are critical for triggering the activation of the potato tuber ADP-glucose pyrophosphorylase, as shown by site-directed mutagenesis. Mutations in the small subunit, which bears the catalytic function in this potato tuber form, had a more dramatic effect on disrupting the allosteric activation than those introduced in the large subunit, which is mainly modulatory. Our results strongly agree with a model where the modified residues are located in loops responsible for triggering the allosteric activation signal for this enzyme, and the sensitivity to this activation correlates with the dynamics of these loops. In addition, previous biochemical data indicates that the triggering mechanism is widespread in the enzyme family, even though the activator and the quaternary structure are not conserved.Fil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Kuhn, Misty L.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Falaschetti, Christine A.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Solamen, Ligin. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Olsen, Kenneth W.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Ballicora, Miguel A.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina

Allosteric Control of Substrate Specificity of the Escherichia coli ADP-Glucose Pyrophosphorylase

The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. However, there is growing evidence that many enzymes can catalyze alternative reactions. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. ADP-glucose pyrophosphorylase from Escherichia coli is an example of an enzyme that needs to select the correct substrate from a broad spectrum of alternatives. This selection will guide the flow of carbohydrate metabolism toward the synthesis of reserve polysaccharides. Here, we show that the allosteric activator fructose-1,6-bisphosphate plays a role in such selection by increasing the catalytic efficiency of the enzyme toward the use of ATP rather than other nucleotides. In the presence of fructose-1,6-bisphosphate, the kcat/S0.5 for ATP was near ~600-fold higher that other nucleotides, whereas in the absence of activator was only ~3-fold higher. We propose that the allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates.Fil: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. University of Chicago; Estados UnidosFil: Solamen, Ligin. University of Chicago; Estados UnidosFil: Hill, Benjamin L.. University of Chicago; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Olsen, Kenneth W.. University of Chicago; Estados UnidosFil: Ballicora, Miguel A.. University of Chicago; Estados Unido

A numerical framework for interstitial fluid pressure imaging in poroelastic MRE

A numerical framework for interstitial fluid pressure imaging (IFPI) in biphasic materials is investigated based on three-dimensional nonlinear finite element poroelastic inversion. The objective is to reconstruct the time-harmonic pore-pressure field from tissue excitation in addition to the elastic parameters commonly associated with magnetic resonance elastography (MRE). The unknown pressure boundary conditions (PBCs) are estimated using the available full-volume displacement data from MRE. A subzone-based nonlinear inversion (NLI) technique is then used to update mechanical and hydrodynamical properties, given the appropriate subzone PBCs, by solving a pressure forward problem (PFP). The algorithm was evaluated on a single-inclusion phantom in which the elastic property and hydraulic conductivity images were recovered. Pressure field and material property estimates had spatial distributions reflecting their true counterparts in the phantom geometry with RMS errors around 20% for cases with 5% noise, but degraded significantly in both spatial distribution and property values for noise levels > 10%. When both shear moduli and hydraulic conductivity were estimated along with the pressure field, property value error rates were as high as 58%, 85% and 32% for the three quantities, respectively, and their spatial distributions were more distorted. Opportunities for improving the algorithm are discussed

Changes in the molecular dynamics simulations due to mutations in the subunits.

<p>The structures are colored according to the difference RMSF values. Colors used are purple, more than 3 standard deviations negative; dark blue, 2 to 3 standard deviations negative; light blue, 1 to 2 standard deviations negative; yellow, +/− one standard deviation; pink, 1 to 2 standard deviations positive; orange, 2 to 3 standard deviations positive; and red, more than 3 standard deviations positive. A. <i>Stu</i>S_Q75A. B. <i>Stu</i>L_Q86A. C. <i>Stu</i>S_W116A. D. <i>Stu</i>L_W128A. The position of the residue mutated to Ala is shown in red, while the non-mutated position is shown in green. The values of the standard deviations were <i>Stu</i>S_Q75A, 0.38; <i>Stu</i>L_Q86A, 0.40; <i>Stu</i>S_W116A, 0.40; and <i>Stu</i>L_W128A, 0.43. Because of the different flexibilities, the Trp-containing loop that was mutated is in different colors. It is shown in red in panel D (arrow).</p

Saturation curves of 3-PGA for the wild type potato tuber ADP-Glc PPase (▪) and mutants <i>Stu</i>S_Q75A/<i>Stu</i>L (•), <i>Stu</i>S/<i>Stu</i>L_Q86A (○), <i>Stu</i>S_W116A/<i>Stu</i>L (▴), <i>Stu</i>S/<i>Stu</i>L_W128A (▵), and <i>Stu</i>S_W116A/<i>Stu</i>L_W128A (▾).

<p>Reactions were performed using <i>Assay B</i>, as stated under “Materials and Methods”.</p

Identification of amino acids important for 3-PGA activation.

<p><b>A.</b> Sequence alignment of ADP-Glc PPases from <i>Escherichia coli</i> (<i>Eco</i>), <i>Anabaena</i> PCC 7120 (<i>Ana</i>), and potato tuber (<i>Stu</i>S: small subunit, <i>Stu</i>L: large subunit). Mutants of the <i>E. coli</i> enzyme that were insensible to Fru-1,6-bisP activation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066824#pone.0066824-Figueroa1" target="_blank">[19]</a> are pointed with arrows. Residues that might be involved in 3-PGA activation of ADP-Glc PPases from <i>Anabaena</i> and potato tuber are highlighted in yellow (Gln) and blue (Trp). <b>B.</b> Insight into the three-dimensional structure of the N-terminal domain (residues 20 to 144) from <i>Stu</i>S. Residues Q75 and W116 are shown as “sticks” and colored by atom type. ATP is shown in red as van der Waals radii. Loops are colored in grey, α-helices in pink, and β-helices in blue.</p

Kinetic parameters obtained for Pi with the potato tuber ADP-Glc PPase and its mutants.

<p>Reactions were performed using <i>Assay B</i> in absence or presence of 5 mM 3-PGA, as stated under “Materials and Methods”.</p