It represents the first committed step in pyrimidine and argininebiosynthesis in prokaryotes and eukaryotes, and in the urea cycle in most terrestrial vertebrates.[2] Most prokaryotes carry one form of CPSase that participates in both arginine and pyrimidine biosynthesis, however certain bacteria can have separate forms.
There are three different forms that serve very different functions:
Carbamoyl phosphate synthase has three main steps in its mechanism and is, in essence, irreversible.[4]
Bicarbonate ion is phosphorylated with ATP to create carboxylphosphate.
The carboxylphosphate then reacts with ammonia to form carbamic acid, releasing inorganic phosphate.
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.[5]
Structure[]
Carbamoyl phosphatesynthase (CPSase) is a heterodimericenzyme 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 synthetasedomains).[2][3][6] CPSase has three active sites, one in the small subunit and two in the large subunit. The small subunit contains the glutaminebinding 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 domaincatalyses the phosphorylation of biocarbonate, while the C-terminal domain catalyses the phosphorylation of the carbamateintermediate.[7] The carboxy phosphate domain found duplicated in the large subunit of CPSase is also present as a single copy in the biotin-dependent enzymesacetyl-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.[8] 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.[9] 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.
References[]
^Simmer JP, Kelly RE, Rinker AG, Scully JL, Evans DR (June 1990). "Mammalian carbamyl phosphate synthetase (CPS). DNA sequence and evolution of the CPS domain of the Syrian hamster multifunctional protein CAD". The Journal of Biological Chemistry. 265 (18): 10395–402. PMID1972379.
^ abHolden HM, Thoden JB, Raushel FM (October 1999). "Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product". Cellular and Molecular Life Sciences. 56 (5–6): 507–22. doi:10.1007/s000180050448. PMID11212301. S2CID23446378.
^ abSaha N, Datta S, Kharbuli ZY, Biswas K, Bhattacharjee A (July 2007). "Air-breathing catfish, Clarias batrachus upregulates glutamine synthetase and carbamyl phosphate synthetase III during exposure to high external ammonia". Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology. 147 (3): 520–30. doi:10.1016/j.cbpb.2007.03.007. PMID17451989.
^Biochemistry, 3rd edition, J.M. Berg, J.L. Tymoczko, L. Stryer
^Raushel FM, Thoden JB, Holden HM (June 1999). "The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia". Biochemistry. 38 (25): 7891–9. doi:10.1021/bi990871p. PMID10387030.
^Stapleton MA, Javid-Majd F, Harmon MF, Hanks BA, Grahmann JL, Mullins LS, Raushel FM (November 1996). "Role of conserved residues within the carboxy phosphate domain of carbamoyl phosphate synthetase". Biochemistry. 35 (45): 14352–61. doi:10.1021/bi961183y. PMID8916922.
^Thoden JB, Raushel FM, Benning MM, Rayment I, Holden HM (January 1999). "The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution". Acta Crystallographica. Section D, Biological Crystallography. 55 (Pt 1): 8–24. doi:10.1107/S0907444998006234. PMID10089390.
^Kim J, Howell S, Huang X, Raushel FM (October 2002). "Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase". Biochemistry. 41 (42): 12575–81. doi:10.1021/bi020421o. PMID12379099.