Elucidation of the reaction mechanisms involved in the catalysis mediated by glutamine synthetase in Escherichia coli

Master Thesis


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University of Cape Town

Structural and molecular dynamics analysis of the glutamine synthetase from E. coli indicates that a possible mechanism by which the adenylylation/deadenylylation of the enzyme affects the enzyme specificity for either MgATP or Mn2ATP and NH4+ or NH3, is by switching between two putative serine protease-like catalytic triads. Site-directed mutagenesis of a number of residues identiï¬ ed as playing a role in these catalytic triads, led to the following observations. Both Ser52 and Ser53 were important for the catalytic activity of the enzyme. It was determined that the Ser52 residue appeared to have adenylylated functionality and the Ser53 residue, deadenylylated functionality. Evaluating these serine mutant enzymes in the presence of the serine protease inhibitors, AEBSF and PMSF, led to the conclusion that Ser52 and Ser53, did, indeed, appear to form part of a catalytic triad, as the activity of the enzymes were inhibited in the presence of the inhibitors. When both serine residues were removed in a single mutant, activity was not signiï¬ cantly inhibited by either inhibitor. His210 and His211 were found to be equally important to the functionality of the enzyme, and the results, in consultation with the enzyme model, led to the conclusion that the His210 residue had deadenylylated activity and the His211 residue had adenylylated activity. All the potential acid residues, when removed, had an effect on activity, but this was to be expected as all had previously been identified in the literature as important in the active site of the glutamine synthetase from E. coli. Again, consultation of the model led to the conclusion that the two acid residues that filled the function of the acid residue in each catalytic triad, were Glu129 for the adenylylated from of the enzyme, and Glu357 for the deadenylylated form of the enzyme. These catalytic triads are believed to be comprised of Ser52', His211 and Glu129 for the adenylylated form of the enzyme, and Ser53', His210 and Glu357 for the deadenylylated form of the enzyme. Possible model mechanisms for the both the adenylylated and deadenylylated forms of the enzyme are proposed.

Includes bibliographical references (leaves 117-125).