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Direct demonstration of carbamoyl phosphate formation on the C-terminal domain of carbamoyl phosphate synthetase

¹ Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA
² Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA

(RECEIVED August 6, 2004; FINAL REVISION September 6, 2004; ACCEPTED September 6, 2004)

Carbamoyl phosphate synthetase synchronizes the utilization of two ATP molecules at duplicated ATP-grasp folds to catalyze carbamoyl phosphate formation. To define the dedicated functional role played by each of the two ATP sites, we have carried out pulse/labeling studies using the synthetases from Aquifex aeolicus and Methanococcus jannaschii, hyperthermophilic organisms that encode the two ATP-grasp folds on separate subunits. These studies allowed us to differentially label each active site with [-³²P]ATP and determine the fate of the labeled -phosphate in the synthetase reaction. Our results provide the first direct demonstration that enzyme-catalyzed transfer of phosphate from ATP to carbamate occurs on the more C-terminal of the two ATP-grasp folds. These findings rule out one mechanism proposed for carbamoyl phosphate synthetase, where one ATP acts as a molecular switch, and provide additional support for a sequential reaction mechanism where the -phosphate groups of both ATP molecules are transferred to reactants. CP synthesis by subunit C in our single turnover pulse/chase assays did not require subunit N, but subunit N was required for detectable CP synthesis in the traditional continuous assay. These findings suggest that cross-talk between domain N and C is required for product release from subunit C.

Keywords: carbamoyl phosphate synthetase; ATP; ATP-grasp; arginine; glutamine; pyrimidine

Abbreviations: aCPS, Aquifex aeolicus carbamoyl phosphate synthetase • cm, carbamate • CP, carbamoyl phosphate • CPS, carbamoyl phosphate synthetase • CPSs, plural form for carbamoyl phosphate synthetase • eCPS, Escherichia coli carbamoyl phosphate synthetase • mCPS, Methanococcus jannaschii carbamoyl phosphate synthetase

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041041305.

Reprint requests to: Susan Powers-Lee, Department of Biology, Northeastern University, Boston, MA 02115, USA; e-mail: spl{at}neu.edu; fax: (617) 373-3724.

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