carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase has three main steps in its mechanism and is, in essence, irreversible.Biochemistry, 3rd edition, J.M. Berg, J.L. Tymoczko, L. Stryer

1. Bicarbonate ion is phosphorylated with ATP to create {{chem name|carboxylphosphate}}.
2. The {{chem name|carboxylphosphate}} then reacts with ammonia to form carbamic acid, releasing inorganic phosphate.
3. A second molecule of ATP then phosphorylates carbamic acid, creating carbamoyl phosphate.

The activity of the enzyme is known to be inhibited by both Tris and HEPES buffers.{{cite journal | vauthors = Lund P, Wiggins D | title = Inhibition of carbamoyl-phosphate synthase (ammonia) by Tris and Hepes. Effect on Ka for N-acetylglutamate | journal = The Biochemical Journal | volume = 243 | issue = 1 | pages = 273–6 | date = April 1987 | pmid = 3606575 | pmc = 1147843 | doi = 10.1042/bj2430273 }}

Carbamoyl phosphate synthase (CPSase) is a heterodimeric enzyme composed of a small and a large subunit (with the exception of CPSase III, which is composed of a single polypeptide that may have arisen from gene fusion of the glutaminase and synthetase domains).{{cite journal | vauthors = Raushel FM, Thoden JB, Holden HM | title = The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia | journal = Biochemistry | volume = 38 | issue = 25 | pages = 7891–9 | date = June 1999 | pmid = 10387030 | doi = 10.1021/bi990871p }} 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, which is in turn used by the large chain to synthesize carbamoyl phosphate. The small subunit has a 3-layer beta/beta/alpha structure, and is thought to be mobile in most proteins that carry it. The C-terminal domain of the small subunit of CPSase has glutamine amidotransferase activity. 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 {{chem name|biocarbonate}}, while the C-terminal domain catalyses the phosphorylation of the carbamate intermediate.{{cite journal | vauthors = Stapleton MA, Javid-Majd F, Harmon MF, Hanks BA, Grahmann JL, Mullins LS, Raushel FM | title = Role of conserved residues within the carboxy phosphate domain of carbamoyl phosphate synthetase | journal = Biochemistry | volume = 35 | issue = 45 | pages = 14352–61 | date = November 1996 | pmid = 8916922 | doi = 10.1021/bi961183y }} 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 (ACC), propionyl-CoA carboxylase (PCCase), pyruvate carboxylase (PC) and urea carboxylase.

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.{{cite journal | vauthors = Thoden JB, Raushel FM, Benning MM, Rayment I, Holden HM | title = The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution | journal = Acta Crystallographica. Section D, Biological Crystallography | volume = 55 | issue = Pt 1 | pages = 8–24 | date = January 1999 | pmid = 10089390 | doi = 10.1107/S0907444998006234 }} 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.{{cite journal | vauthors = Kim J, Howell S, Huang X, Raushel FM | title = Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase | journal = Biochemistry | volume = 41 | issue = 42 | pages = 12575–81 | date = October 2002 | pmid = 12379099 | doi = 10.1021/bi020421o }} 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.

{{reflist}}

• [https://www.ncbi.nlm.nih.gov/books/NBK1217/ GeneReviews/NCBI/NIH/UW entry on Urea Cycle Disorders Overview]

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{{Ligases CO CS and CN}}

{{Enzymes}}

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Category:Urea cycle

Category:Protein domains

Category:EC 6.3.4