title: The effects of removing the GAT domain from E. coli GMP synthetase.
abstract: E. coli GMP synthetase (GMPS) catalyzes the conversion of XMP to GMP. Ammonia, generated in the amino-terminal glutamine amidotransferase (GAT) domain, is transferred by an unknown mechanism to the ATP-pyrophosphatase (ATPP) domain, where it attacks a highly reactive adenyl-XMP intermediate, leading to GMP formation. To study the structural requirements for the activity of E. coli GMPS, we used PCR to generate a protein expression construct that contains the ATPP domain as well as the predicted dimerization domain (DD). The ATPP/DD protein is active in solution, utilizing NH (4) (+) as an NH(3) donor. Size-exclusion chromatography demonstrates a dimeric mass for the ATPP/ DD protein, providing the first evidence in solution for the structural organization of the intact GMPS. Kinetic characterization of the ATPP/DD domain protein provides evidence that the presence of the GAT domain can regulate the activity of the ATPP domain.
authors.affiliation: Department of Chemistry, Lebanon Valley College, 101 N. College Ave., Annville, PA 17003-1400, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Abbott
authors.forename: Jordan M
authors.initials: JM
authors.lastname: Newell
authors.forename: Christine M
authors.initials: CM
authors.lastname: Lightcap
authors.forename: Mary E
authors.initials: ME
authors.lastname: Olanich
authors.forename: Danielle T
authors.initials: DT
authors.lastname: Loughlin
authors.forename: Melanie A
authors.initials: MA
authors.lastname: Weller
authors.forename: Gary
authors.initials: G
authors.lastname: Lam
authors.forename: Sidney
authors.initials: S
authors.lastname: Pollack
authors.forename: Walter A
authors.initials: WA
authors.lastname: Patton
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000641:Ammonia / Q000737:chemistry; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012265:Ribonucleotides / Q000737:chemistry; D012996:Solutions; D019820:Xanthine
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012265:Ribonucleotides; D012996:Solutions; D019820:Xanthine; C011141:xanthosine monophosphate; D000641:Ammonia; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing)