PMID:15576558
| Citation |
Kothe, M, Purcarea, C, Guy, HI, Evans, DR and Powers-Lee, SG (2005) Direct demonstration of carbamoyl phosphate formation on the C-terminal domain of carbamoyl phosphate synthetase. Protein Sci. 14:37-44 |
|---|---|
| Abstract |
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 [gamma-(32)P]ATP and determine the fate of the labeled gamma-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 gamma-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. |
| Links |
PubMed PMC2253338 Online version:10.1110/ps.041041305 |
| Keywords |
Adenosine Triphosphate/chemistry; Bacteria/enzymology; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/chemistry; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/isolation & purification; Carbamyl Phosphate/chemical synthesis; Catalysis; Enzyme Activation; Methanococcus/enzymology; Protein Folding; Protein Structure, Tertiary |
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