The small subunit of carbamoyl phosphate synthetase: Snapshots along the reaction pathway

Carbamoyl phosphate synthetase (CPS) plays a key role in both arginine and pyrimidine biosynthesis by catalyzing the production of carbamoyl phosphate . The enzyme from Escherichi coli consists of two polypeptide chains referr ed to as the small and large subunits. On the basis of both amino acid sequ ence analyses and X-ray structural studies, it is known that the small subu nit belongs to the Triad or Type I class of amidotransferases, all of which contain a cysteine-histidine (Cys269 and His353) couple required for activ ity. The hydrolysis of glutamine by the small subunit has been proposed to occur via two tetrahedral intermediates and a glutamyl-thioester moiety, He re, we describe the three-dimensional structures of the C269S/glutamine and CPS/glutamate gamma-semialdehyde complexes, which serve as mimics for the Michaelis complex and the tetrahedral intermediates, respectively. In conju nction with the previously solved glutamyl-thioester intermediate complex, the stereochemical course of glutamine hydrolysis in CPS has been outlined. Specifically, attack by the thiolate of Cys269 occurs at the Si face of th e carboxamide group of the glutamine substrate leading to a tetrahedral int ermediate with an S-configuration, Both the backbone amide groups of Gly241 and Leu270, and O-gamma of Ser47 play key roles in stabilizing the develop ing oxyanion. Collapse of the tetrahedral intermediate leads to formation o f the,glutamyl-thioester intermediate, which is subsequently attacked at th e Si face by an activated water molecule positioned near His353. The result s described here serve as a paradigm for other members of the Triad class o f amidotranferases.