The structure of carbamoyl phosphate synthetase determined to 2.1 angstromresolution

Carbamoyl phosphate synthetase catalyzes the formation of carbamoyl phospha te from one molecule of bicarbonate, two molecules of Mg(2+)ATP and one mol ecule of glutamine or ammonia depending upon the particular form of the enz yme under investigation. As isolated from Escherichia coli, the enzyme is a n alpha,beta-heterodimer consisting of a small subunit that hydrolyzes glut amine and a large subunit that catalyzes the two required phosphorylation e vents, Here the three-dimensional structure of carbamoyl phosphate syntheta se from E. coli refined to 2.1 Angstrom resolution with an R factor of 17.9 % is described. The small subunit is distinctly bilobal with a catalytic tr iad (Cys269, His353 and Glu355) situated between the two structural domains . As observed in those enzymes belonging to the alpha/beta-hydrolase family , the active-site nucleophile, Cys269, is perched at the top of a tight tur n. The large subunit consists of four structural units: the carboxyphosphat e synthetic component, the oligomerization domain, the carbamoyl phosphate synthetic component and the allosteric domain. Both the carboxyphosphate an d carbamoyl phosphate synthetic components bind Mn(2+)ADP. In the carboxyph osphate synthetic component, the two observed Mn2+ ions are both octahedral ly coordinated by oxygen-containing ligands and are bridged by the carboxyl ate side chain of Glu299. Glu215 plays a key allosteric role by coordinatin g to the physiologically important potassium ion and hydrogen bonding to th e ribose hydroxyl groups of ADP, In the carbamoyl phosphate synthetic compo nent, the single observed Mn2+ ion is also octahedrally coordinated by oxyg en-containing ligands and Glu761 plays a similar role to that of Glu215, Th e carboxyphosphate and carbamoyl phosphate synthetic components, while topo logically equivalent, are structurally different, as would be expected in l ight of their separate biochemical functions.