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Tracing an Allosteric Pathway in Escherichia coli ADP‐glucose Pyrophosphorylase
Author(s) -
Hill Benjamin,
Wong Jennifer,
Huerta Fidel,
Manley Tara,
May Brian,
Ballicora Miguel
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.891.10
Subject(s) - allosteric regulation , allosteric enzyme , biochemistry , chemistry , alanine scanning , enzyme , mutant , alanine , mutagenesis , enzyme activator , stereochemistry , biology , amino acid , gene
The synthesis of bacterial glycogen and plant starch is allosterically regulated by the production of ADP‐glucose. The enzyme ADP‐glucose Pyrophosphorylase catalyzes this reaction. We performed a 18 ns molecular dynamics simulation on a model of the Escherichia coli enzyme and analyzed the results using correlated movement analysis and a dynamical network pathway analysis. A loop spanning Pro103‐Arg115 was noted for having strongly correlated movements with parts of the enzyme involved with both substrate binding and allosterism. Dynamical network pathway analysis revealed that the dominant pathway of communication between the activator binding residue (Lys39) and bound ATP mainly involved residues of the Pro103‐Arg115 loop. This and other molecular dynamics data strongly suggested that this loop has an important role in the allosteric response of this enzyme. This hypothesis was biochemically tested by performing alanine scanning mutagenesis. This produced mutants P103A to R115A which were kinetically tested. Several of the mutants had near wild type activity in the absence of allosteric effectors, but they failed to respond to activator FBP. We found that the absence of side chains of Gln106, Arg107, Trp113, Tyr114, and Arg115 disrupted the allosteric signal. In addition, P103A was partially pre‐activated, further highlighting the importance of this region. This loop is a distinct insertional element that is present only in allosterically regulated sugar nucleotide pyrophosphorylases. Such an element may have evolved to form a key part of the triggering mechanism that linked proto allosteric and catalytic sites.