Conserved residues of the Pro103-Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase

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...

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Bibliographic Details
Published in:	Protein science Vol. 24; no. 5; pp. 714 - 728
Main Authors:	Hill, Benjamin L, Wong, Jennifer, May, Brian M, Huerta, Fidel B, Manley, Tara E, Sullivan, Peter R F, Olsen, Kenneth W, Ballicora, Miguel A
Format:	Journal Article
Language:	English
Published:	United States Wiley Subscription Services, Inc 01-05-2015 BlackWell Publishing Ltd
Subjects:	Active sites Adenosine diphosphate Adenosine triphosphate Alanine Allosteric properties Allosteric Regulation - genetics Amino Acid Sequence - genetics Arginine - chemistry ATP Binding Computer applications Conserved Sequence - genetics E coli Escherichia coli Escherichia coli - chemistry Escherichia coli - enzymology Escherichia coli - genetics Fructose Glucose Glucose-1-Phosphate Adenylyltransferase - chemistry Glucose-1-Phosphate Adenylyltransferase - genetics Glucose-1-Phosphate Adenylyltransferase - metabolism Glycogen Glycogen - metabolism Glycogens Kinetics Molecular dynamics Molecular Dynamics Simulation Mutagenesis Mutagenesis, Site-Directed Mutants Mutation Network analysis Nucleotides Proline - chemistry Residues Site-directed mutagenesis Starch - metabolism glycogen/starch metabolism network pathway analysis correlated movement analysis molecular dynamics activation signal propagation alanine scanning mutagenesis
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Grant sponsor: National Science Foundation; Grant number: MCB 1024945 (to M.A.B.); Grant sponsor: National Science Foundation; Grant number: CHE-1004430 (to K.W.O.).
ISSN:	0961-8368 1469-896X
DOI:	10.1002/pro.2644