Structure of Carbamoyl Phosphate Synthetase: A Journey of 96 Å from Substrate to Product †, ‡

ABSTRACT: Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. As isolated from Escherichia coli, the enzyme has a total molecular weight of ∼160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal structure of this enzyme determined to 2.8 Å resolution in the presence of ADP, Mn 2+ , phosphate, and ornithine. The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH 2 -and COOH-terminal domains. Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components. Each component contains four distinct domains. Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase. A linear distance of ∼80 Å separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit. The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein. Carbamoyl phosphate synthetase (CPS) catalyzes one of the most remarkable reactions ever described in biological chemistry. This enzyme assembles carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP coupled with the production of glutamate, phosphate, and two molecules of MgADP. On the basis of isotopic labeling studies and the discovery of three additional partial reactions, Anderson and Meister (1) postulated that carbamoyl phosphate is constructed within the active site(s) of this enzyme by a series of four separate chemical reactions as illustrated below: There are thus a minimum of three discrete, highly reactive, and unstable intermediates in this reaction scheme: carboxyphosphate, carbamate, and ammonia. The existence of these intermediates and the order of events have been supported by steady state and rapid mixing kinetic analyses The synthesis of carbamoyl phosphate is critical for the initiation of two separate biosynthetic pathways. In one, carbamoyl phosphate is coupled to aspartate in a reaction catalyzed by aspartate transcarbamoylase. The carbon and nitrogen nuclei of carbamoyl phosphate are ultimately incorporated in the aromatic moieties of pyrimidine nucleotides such as UMP. In the second pathway, carbamoyl phosphate is condensed with ornithine at the start of the urea cycle and is utilized for the detoxification of ammonia and the biosynthesis of arginine. As would be anticipated, CPS therefore displays a complex pattern of metabolic regulation. The precise set of allosteric ligands depends on the particular biological and physiological niche of the cell where CPS resides. With the CPS from Escherichia coli, where there is a single enzyme for both pathways, the protein is allosterically activated by ornithine and inhibited by UMP