The Effects of Removing the GAT Domain from E. coli GMP Synthetase |
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Authors: | Jessica L. Abbott Jordan M. Newell Christine M. Lightcap Mary E. Olanich Danielle T. Loughlin Melanie A. Weller Gary Lam Sidney Pollack Walter A. Patton |
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Affiliation: | (1) Department of Chemistry, Lebanon Valley College, 101 N. College Ave., Annville, PA 17003-1400, USA;(2) Department of Biology, Lebanon Valley College, 101 N. College Ave., Annville, PA 17003-1400, USA |
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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 NH4+ as an NH3 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. |
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Keywords: | GMP Synthetase glutamine amidotransferase ATP-pyrophosphatase Escherichia coli molecular cloning kinetics polymerase chain reaction |
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