Carbamoyl phosphate synthetase: Closure of the B-domain as a result of nucleotide binding

Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsi ble for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia col i, CPS is an alpha,beta-heterodimeric protein with its three active sites s eparated by nearly 100 Angstrom. In addition, there are separate binding si tes for the allosteric regulators, ornithine, and UMP. Given the sizable di stances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecu lar communication. Here we describe the structure of CPS from E. coli where , indeed, such a domain movement has occurred in response to nucleotide bin ding. Specifically, the protein was crystallized in the presence of a nonhy drolyzable analogue, AMPPNP, and its structure determined to 2.1 Angstrom r esolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the ac tive-site pocket such that some atoms move by more than 7 Angstrom relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide gro ups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the n ucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reve rse turn, and this type of secondary structural motif is also observed in D -alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometri es of the two active sites contained within the large subunit of CPS are de scribed.