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The Effects of Removing the GAT Domain from E. coli GMP Synthetase
Author(s) -
Lam Gary,
Patton Walter
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a278
Subject(s) - glutamine amidotransferase , chemistry , domain (mathematical analysis) , biochemistry , protein domain , escherichia coli , glutamine , hamp domain , amino acid , gene , mathematical analysis , mathematics
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.