title: The central loop of Escherichia coli glutamine synthetase is flexible and functionally passive.
abstract: Bacterial glutamine synthetases (GSs) are dodecameric aggregates comprised of two face-to-face hexameric rings, which form a cylindrical aqueous channel. Available crystal structures indicate that each subunit provides a 'central loop' that protrudes into this channel. Residues on either side of this loop contribute directly to substrate or metal ion cofactor binding. Although it has been suggested that this conspicuous structural feature may be functionally important, a systematic structure-function analysis of this loop has not been done. Here, we examine the behavior of a cysteine mutant, E165C, which yields inter-subunit disulfide bonds connecting the central loops. The inter-subunit disulfide bonds are readily detected by electrospray ionization mass spectrometry. Based on molecular models, the disulfide bonds would form only if the engineered cysteines on adjacent subunits moved approximately 5 A. Surprisingly, inter-subunit disulfide bonds between the central loops caused no detectable changes in the KMs for glutamate or ATP, nor the KD for either ATP or the transition state analog (L)-methionine sulfoximine (MSOX). Furthermore, covalent and quantitative adduction of the E165C mutant with iodo-acetamido-pyrene yielded nearly fully active enzyme bearing fluorescent pyrene excimers. The relative contribution of pyrene monomers to excimers in the steady state fluorescence is temperature dependent, suggesting thermal equilibrium between loop conformational states. However, the monomer-excimer ratio is independent of ligands such as MSOX, glutamate, or Mn2+. These results validate the suspected flexibility of the central loop, but raise significant doubt about its direct functional role in GS catalysis via conformational switching, including the proposed regulation of GS via ADP-ribosylation within this loop.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610, USA.
authors.forename: Josh T
authors.initials: JT
authors.lastname: Pearson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Dabrowski
authors.forename: Irene
authors.initials: I
authors.lastname: Kung
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D002384:Catalysis; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011721:Pyrenes / Q000737:chemistry; D012266:Ribose / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature
chemical_list: D004220:Disulfides; D011721:Pyrenes; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D012266:Ribose; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium