title: Conserved residues of the Pro103-Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 W Sheridan Road, Chicago, Illinois.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Hill
authors.forename: Jennifer
authors.initials: J
authors.lastname: Wong
authors.forename: Brian M
authors.initials: BM
authors.lastname: May
authors.forename: Fidel B
authors.initials: FB
authors.lastname: Huerta
authors.forename: Tara E
authors.initials: TE
authors.lastname: Manley
authors.forename: Peter R F
authors.initials: PR
authors.lastname: Sullivan
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Olsen
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Ballicora
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics*; D000595:Amino Acid Sequence / Q000235:genetics; D001120:Arginine / Q000737:chemistry; D017124:Conserved Sequence / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D013213:Starch / Q000378:metabolism
chemical_list: D013213:Starch; D006003:Glycogen; D001120:Arginine; D011392:Proline; D051599:Glucose-1-Phosphate Adenylyltransferase
keywords: activation signal propagation; alanine scanning mutagenesis; correlated movement analysis; glycogen/starch metabolism; molecular dynamics; network pathway analysis