carbamoyl phosphate synthetase

{{Short description|Class of enzymes}}

{{infobox enzyme

| Name = Carbamoyl phosphate synthetase (ammonia)

| EC_number = 6.3.4.16

| CAS_number = 37318-69-7

| GO_code =

| image =

| width =

| caption =

}}

Image:CarbPhos.png]]

{{Infobox protein family

| Symbol = CPSase_L_D2

| Name = CPSase large subunit ATP-binding domain

| image = PDB 1dv2 EBI.jpg

| width =

| caption = the structure of biotin carboxylase, mutant e288k, complexed with atp

| Pfam = PF02786

| Pfam_clan = CL0179

| InterPro = IPR005479

| SMART =

| PROSITE = PDOC00676

| MEROPS =

| SCOP = 1bnc

| TCDB =

| OPM family =

| OPM protein =

| CAZy =

| CDD =

}}

{{Infobox protein family

| Symbol = CPSase_L_D3

| Name = CPSase large subunit oligomerisation domain

| image = PDB 1bxr EBI.jpg

| width =

| caption = structure of carbamoyl phosphate synthetase complexed with the atp analog amppnp

| Pfam = PF02787

| Pfam_clan =

| InterPro = IPR005480

| SMART =

| PROSITE = PDOC00676

| MEROPS =

| SCOP = 1bnc

| TCDB =

| OPM family =

| OPM protein =

| CAZy =

| CDD =

}}

{{Infobox protein family

| Symbol = CPSase_L_chain

| Name = CPSase large subunit N-terminal domain

| image = PDB 1ulz EBI.jpg

| width =

| caption = crystal structure of the biotin carboxylase subunit of pyruvate carboxylase

| Pfam = PF00289

| Pfam_clan =

| InterPro = IPR005481

| SMART =

| PROSITE = PDOC00676

| MEROPS =

| SCOP = 1bnc

| TCDB =

| OPM family =

| OPM protein =

| CAZy =

| CDD =

}}

{{Infobox protein family

| Symbol = CPSase_sm_chain

| Name = CPSase small subunit N-terminal domain

| image = PDB 1kee EBI.jpg

| width =

| caption = inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin

| Pfam = PF00988

| Pfam_clan =

| InterPro = IPR002474

| SMART =

| PROSITE = PDOC00676

| MEROPS =

| SCOP = 1jdb

| TCDB =

| OPM family =

| OPM protein =

| CAZy =

| CDD =

}}

Carbamoyl phosphate synthetase catalyzes the ATP-dependent synthesis

of carbamoyl phosphate from glutamine ({{EnzExplorer|6.3.5.5}}) or ammonia ({{EnzExplorer|6.3.4.16}}) and bicarbonate.{{cite journal | vauthors = Simmer JP, Kelly RE, Rinker AG, Scully JL, Evans DR | title = Mammalian carbamyl phosphate synthetase (CPS). DNA sequence and evolution of the CPS domain of the Syrian hamster multifunctional protein CAD | journal = The Journal of Biological Chemistry | volume = 265 | issue = 18 | pages = 10395–402 | date = June 1990 | doi = 10.1016/S0021-9258(18)86959-9 | pmid = 1972379 | doi-access = free }} This ATP-grasp enzyme catalyzes the reaction of ATP and bicarbonate to produce carboxy phosphate and ADP. Carboxy phosphate reacts with ammonia to give carbamic acid. In turn, carbamic acid reacts with a second ATP to give carbamoyl phosphate plus ADP.

It represents the first committed step in pyrimidine and arginine biosynthesis in prokaryotes and eukaryotes, and in the urea cycle in most terrestrial vertebrates.{{cite journal | vauthors = Holden HM, Thoden JB, Raushel FM | title = Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product | journal = Cellular and Molecular Life Sciences | volume = 56 | issue = 5–6 | pages = 507–22 | date = October 1999 | pmid = 11212301 | doi = 10.1007/s000180050448 | s2cid = 23446378 | pmc = 11147029 }} 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:

Mechanism

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 }}

Structure

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.

References

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