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Direct demonstration of carbamoyl phosphate formation on the C‐terminal domain of carbamoyl phosphate synthetase
Author(s) -
Kothe Michael,
Purcarea Cristina,
Guy Hedeel I.,
Evans David R.,
PowersLee Susan G.
Publication year - 2005
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.041041305
Subject(s) - carbamoyl phosphate synthetase , aquifex aeolicus , protein subunit , carbamyl phosphate , methanococcus , atp synthase , phosphate , reaction intermediate , biochemistry , enzyme , atp hydrolysis , stereochemistry , adenosine triphosphate , biology , biophysics , chemistry , catalysis , escherichia coli , gene , atpase
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 [γ‐ 32 P]ATP and determine the fate of the labeled γ‐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 γ‐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.