The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103-Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103-Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites.
Biophysical and enzymatic analyses show that UDP- and ADP-glucose are more stable than claimed in a controversial study that questions the generally accepted pathway of starch synthesis in plants.
The enzyme ADP-glucose pyrophosphorylase (ADP-Glc PPase) controls the biosynthesis of glycogen in bacteria and starch in plants. It is regulated by various activators in different organisms according to their metabolic characteristics. In Escherichia coli, the major allosteric activator is fructose 1,6-bisphosphate (FBP). Other potent activator analogs include 1,6-hexanediol bisphosphate (HBP) and pyridoxal 5′-phosphate (PLP). Recently, a crystal structure with FBP bound was reported (PDB ID: 5L6S). However, it is possible that the FBP site found is not directly responsible for the activation of the enzyme. We hypothesized FBP activates by binding one of its phosphate groups to another site (“P1”) in which a sulfate molecule was observed. In the E. coli enzyme, Arg40, Arg52, and Arg386 are part of this “P1” pocket and tightly complex this sulfate, which is also present in the crystal structures of ADP-Glc PPases from Agrobacterium tumefaciens and Solanum tuberosum. To test this hypothesis, we modeled alternative binding conformations of FBP, HBP, and PLP into “P1.” In addition, we performed a scanning mutagenesis of Arg residues near potential phosphate binding sites (“P1,” “P2,” “P3”). We found that Arg40 and Arg52 are essential for FBP and PLP binding and activation. In addition, mutation of Arg386 to Ala decreased the apparent affinity for the activators more than 35-fold. We propose that the activator binds at this “P1” pocket, as well as “P2.” Arg40 and Arg52 are highly conserved residues and they may be a common feature to complex the phosphate moiety of different sugar phosphate activators in the ADP-Glc PPase family.
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.
ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step for the synthesis of glycogen in cyanobacteria and starch in green algae and plants. The enzyme from cyanobacteria is homotetrameric (α4), while that from green algae and plants is heterotetrameric (α2β2). These ADP-Glc PPases are allosterically regulated by 3-phosphoglycerate (3PGA, activator) and inorganic orthophosphate (Pi, inhibitor). Previous studies on the cyanobacterial and plant enzymes showed that 3PGA binds to two highly conserved Lys residues located in the C-terminal domain. We observed that both Lys residues are present in the small (α) subunit of the Ostreococcus tauri enzyme; however, one of these Lys residues is replaced by Arg in the large (β) subunit. In this work, we obtained the K443R and R466K mutants of the O. tauri small and large subunits, respectively, and co-expressed them together or with their corresponding wild type counterparts. Our results show that restoring the Lys residue in the large subunit enhanced 3PGA affinity, whereas introduction of an Arg residue in the small subunit reduced 3PGA affinity of the heterotetramers. Inhibition kinetics also showed that heterotetramers containing the K443R small subunit mutant were less sensitive to Pi inhibition, but only minor changes were observed for those containing the R466K large subunit mutant, suggesting a leading role of the small subunit for Pi inhibition of the heterotetramer. We conclude that, during evolution, the ADP-Glc PPase large subunit from green algae and plants acquired mutations in its regulatory site. The rationale for this could have been to accommodate sensitivity to particular metabolic needs of the cell or tissue.
Novel copolymers of trisubstituted ethylene monomers, alkyl ring-substituted 2-phenyl-1,1-dicyanoethylenes, RC 6 H 4 CH=C(CN) 2 (where R is 2-methyl, 3-methyl, 4-methyl, 4-ethyl, 4-i -propyl, 4-butyl, 4-i -butyl, and 4-t-butyl) and 4-fluorostyrene were prepared at equimolar monomer feed composition by solution copolymerization in the presence of a radical initiator (ABCN) at 70 • C. The composition of the copolymers was calculated from nitrogen analysis, and the structures were analyzed by IR, 1 H and 13 C-NMR. The order of relative reactivity (1/r 1 ) for the monomers is 4-ethyl (42.6) > 4-butyl (29.4) > 4-t-butyl (26.7) > 4-i -butyl (1.6) > 4-i -propyl (1.29) > 3-methyl (1.26) > 2-methyl (0.8) > 4-methyl (0.4). High T g of the copolymers, in comparison with that of poly(4-fluorostyrene) indicates a substantial decrease in chain mobility of the copolymer due to the high dipolar character of the trisubstituted ethylene monomer unit. Decomposition of the copolymers in nitrogen occurred in two steps, first in 183-500 • C range with residue (5-30% wt.), which then decomposed in the 500-800 • C range.
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