The effects of removing the GAT domain from E. coli GMP synthetase.
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.
Abbott
Jessica L
JL
Department of Chemistry, Lebanon Valley College, 101 N. College Ave., Annville, PA 17003-1400, USA.
Newell
Jordan M
JM
Lightcap
Christine M
CM
Olanich
Mary E
ME
Loughlin
Danielle T
DT
Weller
Melanie A
MA
Lam
Gary
G
Pollack
Sidney
S
Patton
Walter A
WA
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
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)