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Unmasking the High Affinity of Escherichia coli Glycogen Synthase Towards its Polyglucan Substrate
Author(s) -
Iglesias Alberto Alvaro,
Aleanzi Mabel,
Diez Matias Damian Asencion,
Machtey Matias,
Yep Alejandra,
Ballicora Miguel
Publication year - 2017
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.31.1_supplement.764.18
Subject(s) - glycogen branching enzyme , glycogen , escherichia coli , enzyme , glycogen synthase , chemistry , biochemistry , glycogen debranching enzyme , substrate (aquarium) , atp synthase , stereochemistry , biology , ecology , gene
In bacteria, glycogen synthesis occurs by a 3‐step pathway where the glucosyl donor ADP‐glucose synthesized by ADP‐glucose pyrophosphorylase (EC 2.7.7.27) is used to elongate an α‐1,4‐glucan chain by glycogen‐synthase (EC 2.4.1.21; GlgSase), following by the action of polyglucan branching enzyme (EC 2.4.1.18). The pathway was extensively studied in Escherichia coli where the three enzymes were structurally and functionally characterized. The E. coli GlgSase is an enzyme with a relatively high specific activity (~500 U/mg) but an apparent low affinity towards glycogen, as judged by K m values in the order of 0.65 mg/ml. Interestingly, site‐directed mutants of the E. coli GlgSase with highly reduced V max decreased the K m . To have a K m equal to K s requires the assumption that the enzyme is under rapid equilibrium, where k cat is negligible compared to the dissociation of substrate k off . This scenario sustains the hypothesis that a real (high) affinity towards glycogen by E. coli GlgSase could be masked in the kinetic measurement of K m by a relatively high k cat . It is important to note that the accurate determination of the actual affinity for glycogen is relevant to understand the enzyme action mechanism. For that reason, we conducted saturation curves of glycogen at different temperatures in order to analyze changes in K m under conditions where k cat is significantly reduced. We found that a decrease in one order of magnitude in the V max correlated with a ~200‐fold reduction in K m . This behavior was also observed for GlgSase mutants (H161A and K305A) and when the wild type enzyme used alternative (to ADP‐Glc) substrates. In addition, the high affinity of the enzyme for glycogen was confirmed by measurements of binding in the absence of catalysis, where K s was determined to be in the order of μg/ml. This suggests that the kinetics of this enzyme could be better described by steady state models rather than rapid equilibrium. Support or Funding Information Supported by: ANPCyT [PICT 2014 3362 and PICT 2015 0634 to MDAD, PICT 2014 3256 and PICT 2015 1767 to AAI], NSF MCB‐1616851 to MAB