The Effects of Removing the GAT Domain from E. coli GMP Synthetase
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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 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.
KeywordsGMP Synthetase glutamine amidotransferase ATP-pyrophosphatase Escherichia coli; molecular cloning kinetics polymerase chain reaction
high performance liquid chromatography
polyacrylamide gel electrophoresis
polymerase chain reaction
Phosphate buffered saline
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Portions of this work were supported by a Research Corporation Cottrell College Science Award (Grant # CC5937), a Franklin Research Grant from the American Philosophical Society (2004) and a Council on Undergraduate Summer Undergraduate Research Fellowship (Jordan Newell – 2004).
Portions of this work were also supported by institutional grants to Lebanon Valley College from the Whitaker Foundation (Summer 2002) and Merck-AAAS (Summers 2003–2005).
A SpectraMax® Plus384 Microplate Spectrophotometer used in this work was obtained with funds from a Camille and Henry Dreyfus Scholar/Fellow Program Supplemental Award to Walter A. Patton, PhD.
The authors wish to thank Dr. Owen Moe (Lebanon Valley College) for many useful discussions throughout the course of this work.
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