title: Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes.
abstract: Glutamine synthetase brings nitrogen into metabolism by condensing ammonia and glutamate, with the aid of ATP, to yield glutamine, ADP, and inorganic phosphate. Here we present five crystal structures of GS complexed with each of two substrates, Glu and AMPPNP (an ATP analog), with a transition-state analogue, L-methionine-S-sulfoximine, and with each of two products, Gln and ADP. GS of the present study is from Salmonella typhimurium, has Mn2+ bound, and is fully unadenylylated. Protein-metal-substrate interactions and small but significant conformational changes induced by substrate binding are defined by Fourier maps. On the basis of these maps, we propose a tentative structure-based enzymatic mechanism of glutamine synthesis with these steps: (1) ATP binds first at the top of the funnel-shaped active site cavity, adjacent to the n2 Mn2+; Arg 359 moves toward the Glu binding site. (2) Glu binds adjacent to the n1 Mn2+ at the bottom of the active site near a flexible loop (residues 324-328). As proposed earlier by Meister and others, Glu attacks the gamma-phosphorus atom of ATP to produce gamma-glutamyl phosphate and ADP. (3) The presence of ADP (but not ATP) moves Arg 339 toward the Pi site, perhaps stabilizing the gamma-glutamyl phosphate, and moves Asp 50' of the adjacent subunit toward a putative ammonium ion site, enhancing binding of this third substrate. Deprotonation of the ammonium ion, perhaps by Asp 50', permits the resulting active species, ammonia, to attack the gamma-glutamyl phosphate, forming a tetrahedral intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: UCLA-DOE Lab of Structural Biology and Molecular Medicine 90024-1570.
authors.forename: S H
authors.initials: SH
authors.lastname: Liaw
authors.forename: D
authors.initials: D
authors.lastname: Eisenberg
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000378:metabolism*; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism; D008717:Methionine Sulfoximine / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*
chemical_list: D005971:Glutamates; D000644:Quaternary Ammonium Compounds; D005973:Glutamine; D008717:Methionine Sulfoximine; D000266:Adenylyl Imidodiphosphate; D018698:Glutamic Acid; D005974:Glutamate-Ammonia Ligase