InterPro: IPR001317 Carbamoyl phosphate synthase, GATase domain

Carbamoyl phosphate synthase (CPSase) is a heterodimeric enzyme composed of a small and a large subunit (with the exception of CPSase III, see below). CPSase catalyses the synthesis of carbamoyl phosphate from biocarbonate, ATP and glutamine (EC:6.3.5.5) or ammonia (EC:6.3.4.16), and represents the first committed step in pyrimidine and arginine biosynthesis in prokaryotes and eukaryotes, and in the urea cycle in most terrestrial vertebrates [1, 2]. CPSase has three active sites, one in the small subunit and two in the large subunit. The small subunit contains the glutamine binding site and catalyses the hydrolysis of glutamine to glutamate and ammonia. The large subunit has two homologous carboxy phosphate domains, both of which have ATP-binding sites; however, the N-terminal carboxy phosphate domain catalyses the phosphorylation of biocarbonate, while the C-terminal domain catalyses the phosphorylation of the carbamate intermediate [3]. The carboxy phosphate domain found duplicated in the large subunit of CPSase is also present as a single copy in the biotin-dependent enzymes acetyl-CoA carboxylase (EC:6.4.1.2) (ACC), propionyl-CoA carboxylase (EC:6.4.1.3) (PCCase), pyruvate carboxylase (EC:6.4.1.1) (PC) and urea carboxylase (EC:6.3.4.6).

Most prokaryotes carry one form of CPSase that participates in both arginine and pyrimidine biosynthesis, however certain bacteria can have separate forms. The large subunit in bacterial CPSase has four structural domains: the carboxy phosphate domain 1, the oligomerisation domain, the carbamoyl phosphate domain 2 and the allosteric domain [4]. CPSase heterodimers from Escherichia coli contain two molecular tunnels: an ammonia tunnel and a carbamate tunnel. These inter-domain tunnels connect the three distinct active sites, and function as conduits for the transport of unstable reaction intermediates (ammonia and carbamate) between successive active sites [5]. The catalytic mechanism of CPSase involves the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain.

Eukaryotes have two distinct forms of CPSase: a mitochondrial enzyme (CPSase I) that participates in both arginine biosynthesis and the urea cycle; and a cytosolic enzyme (CPSase II) involved in pyrimidine biosynthesis. CPSase II occurs as part of a multi-enzyme complex along with aspartate transcarbamoylase and dihydroorotase; this complex is referred to as the CAD protein [6]. The hepatic expression of CPSase is transcriptionally regulated by glucocorticoids and/or cAMP [7]. There is a third form of the enzyme, CPSase III, found in fish, which uses glutamine as a nitrogen source instead of ammonia [8]. CPSase III is closely related to CPSase I, and is composed of a single polypeptide that may have arisen from gene fusion of the glutaminase and synthetase domains [9].

This entry represents the domain responsible for GATase (glutamine amidotransferase) activity (EC:6.3.5.2) in CPSases, which catalyses the hydrolysis of glutamine to glutamate and ammonia. This reaction occurs at the active site on the small subunit of CPSases. This function has been detected in some other enzymes, including aminodeoxychorismate synthase (EC:2.6.1.85) and anthranilate synthase component II (EC:4.1.3.27), all of which show sequence similarity in the area thought to contain the GATase activity. The active site contains a conserved Cys residue, which is necessary for catalytic activity, and several conserved residues in the areas surrounding this Cys have also been found to be important.