Direct demonstration of carbamoyl phosphate formation on the C-terminal domain of carbamoyl phosphate synthetase.
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.
Kothe
Michael
M
Department of Biology, Northeastern University, Boston, MA 02115, USA.
Purcarea
Cristina
C
Guy
Hedeel I
HI
Evans
David R
DR
Powers-Lee
Susan G
SG
D000255:Adenosine Triphosphate / Q000737:chemistry; D001419:Bacteria / Q000201:enzymology; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000302:isolation & purification; D002221:Carbamyl Phosphate / Q000138:chemical synthesis*; D002384:Catalysis; D004789:Enzyme Activation; D017017:Methanococcus / Q000201:enzymology; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D002221:Carbamyl Phosphate; D000255:Adenosine Triphosphate; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)