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Pyruvate Revisited as a Synergistic Secondary Activator of the Escherichia coli ADP‐glucose Pyrophosphorylase
Author(s) -
Ballicora Miguel A.,
Diez Matías Asención,
Iglesias Alberto A.
Publication year - 2013
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.27.1_supplement.1004.8
Subject(s) - allosteric regulation , biochemistry , activator (genetics) , chemistry , effector , enzyme , escherichia coli , fructose , intracellular , receptor , gene
Bacterial glycogen synthesis is allosterically regulated at the level of ADP‐Glc pyrophosphorylase (ADP‐Glc PPase; EC 2.7.7.27) by intracellular metabolites. In Agrobacterium tumefaciens ( Atu ‐) the enzyme is activated by both pyruvate (Pyr) and fructose‐6P (Fru‐6P) while in Escherichia coli ( Eco‐ ) only fructose‐1,6‐bisP (Fru‐1,6‐bisP) was described as the main activator. Previous switching of the N‐ and C‐terminal domains of Eco‐ and Atu‐ enzymes supported the hypothesis of Pyr prevalence, since both constructs were activated by it. Here, we found that Eco‐ ADP‐Glc PPase is activated by Pyr with an A 0.5 of 25 mM increasing affinity for substrates (ATP). The high A 0.5 may seem to suggest that Pyr is a poor effector; however, it has a synergistic role in Fru‐1,6‐bisP activation. Sub‐saturated Fru‐1,6‐bisP concentrations remarkably augmented Pyr affinity (5‐fold at 10 μM Fru‐1,6‐bisP) and vice versa , and 2.5 mM Pyr duplicated the Eco‐ ADP‐Glc PPase affinity for Fru‐1,6‐bisP. In addition, Pyr reversed AMP inhibition with an A 0.5 value of 2.76 mM. Pyr and Fru‐1,6‐bisP were synergistic and did not compete, which indicated that both effectors bind to different sites. Results with Eco ‐ and Atu ‐ enzymes may sustain the idea that two binding sites may be involved in allosteric regulation of other bacterial ADP‐Glc PPases. One of these regulatory sites is related to Pyr, which behaves as a fine modulator governing enzyme activity and/or sensitivity to other allosteric effectors. Supported by grants to AAI from CONICET [PIP 2519], UNL [CAI+D Orientado and Redes], and ANPCyT [PICT’08 1754]; and to MAB from the NSF [MCB‐1024945].