Carbamoyl-phosphate synthase (glutamine-hydrolysing)

 

Carbamoyl phosphate synthetase (CPS) catalyses the formation of carbamoyl phosphate, an intermediate in the biosynthesis of pyrimidine nucleotides and arginine, from glutamine, bicarbonate, and two molecules of MgATP. The enzyme from Escherichia coli has three separate active sites, which are connected by a molecular tunnel that is almost 100 Angstroms in length and is a heterodimeric protein that is composed of two subunits of molecular weight ~40 and ~118 kD.

In the proposed reaction mechanism, ATP phosphorylates bicarbonate to form carboxy phosphate, and glutamine is hydrolysed to glutamate and ammonia. The ammonia then reacts with the carboxy phosphate intermediate to form carbamate. In the final step, a second molecule of ATP phosphorylates carbamate to produce the ultimate product, carbamoyl phosphate. Overall, three unstable intermediates (ammonia, carboxy phosphate, and carbamate) and carbamoyl phosphate are formed in a series of four separate reactions.

The hydrolysis of glutamine occurs within the small subunit via a thioester intermediate. Meanwhile, the carboxyphosphate is produced from the phosphorylation of hydrogencarbonate by ATP at a site contained within the N-terminal half of the large subunit. This phosphorylation reaction is thought to trigger the reaction cascade. The ammonia migrates through the interior of the protein, where it reacts with carboxyphosphate to produce the carbamate intermediate. The carbamate intermediate is then transported through the interior of the protein to a second site within the C-terminal half of the large subunit, where it is phosphorylated by another ATP to yield the final product, carbamoyl phosphate.

 

Reference Protein and Structure

Sequences
P00968 UniProt (6.3.5.5)
P0A6F1 UniProt (6.3.5.5) IPR006275, IPR006274 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1bxr - STRUCTURE OF CARBAMOYL PHOSPHATE SYNTHETASE COMPLEXED WITH THE ATP ANALOG AMPPNP (2.1 Å) PDBe PDBsum 1bxr
Catalytic CATH Domains
3.40.50.880 CATHdb 3.30.470.20 CATHdb (see all for 1bxr)
Cofactors
Magnesium(2+) (4)
Click To Show Structure

Enzyme Reaction (EC:6.3.5.5)

hydrogencarbonate
CHEBI:17544ChEBI
+
L-glutamine zwitterion
CHEBI:58359ChEBI
+
water
CHEBI:15377ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
hydron
CHEBI:15378ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
L-glutamate(1-)
CHEBI:29985ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
carbamoyl phosphate(2-)
CHEBI:58228ChEBI
Alternative enzyme names: Carbamoyl-phosphate synthetase (glutamine-hydrolyzing), Carbamoylphosphate synthetase II, Carbamyl phosphate synthetase (glutamine), Glutamine-dependent carbamyl phosphate synthetase, GD-CPSase, Glutamine-dependent carbamoyl-phosphate synthase, Carbamoyl phosphate synthetase, CPS, Carbon-dioxide:L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), CarA (gene name), CarB (gene name), CAD (gene name), Hydrogen-carbonate:L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating),

Enzyme Mechanism

Introduction

Carbamoyl phosphate synthetase (CPS) is a multi-chain, multi-domain protein that catalyses three distinct reactions in three active sites and contains one intermolecular tunnel, and one intramolecular tunnel to transport the reactive intermediates between the various active sites.

The synthetase domain within the N-terminal half of the large subunit is responsible for the phosphorylation of carboxy phosphate and the subsequent nucleophilic attack by ammonia during the formation of carbamate. The phosphorylation of bicarbonate serves as the initial trigger for the rest of the reaction cascade.

The hydrolysis of glutamine (to form the ammonia intermediate) occurs within the small subunit and it has demonstrated that the amide bond is cleaved via the formation of a covalent thioester intermediate with an active site cysteine residue. This ammonia is then transferred to another active site via an intermolecular tunnel.

Finally, the ATP bound at the synthetase domain within the C-terminal half of the large subunit is responsible for the phosphorylation of carbamate and the final formation of carbamoyl phosphate. Finally, kinetic investigations have shown that the ligands bound to the allosteric domain (ornithine and UMP) alter the catalytic properties of CPS primarily by modulating the affinity of the ATP that phosphorylates carbamate.

Although the specific conformational changes that might accompany this process are not fully elucidated, it is assumed that the synchronization mechanisms are allosterically driven.

CPS can catalyze not only its full-forward reaction (reference reaction) but also any of various partial reactions, depending upon the availability of substrates. For example, in the absence of nucleotides, glutamine hydrolysis (reaction 2) occurs within the amidotransferase domain of the small subunit. In the absence of an ammonia source, bicarbonate-dependent ATP hydrolysis (reaction 3) can be monitored independently within the carboxy phosphate domain of the large subunit. Finally, when supplied with the products ADP and carbamoyl phosphate, a reversal of the last step in the mechanism occurs as an ATP synthesis reaction.

Catalytic Residues Roles

UniProt PDB* (1bxr)
His243 His243C Activates the ammonia molecule. Probably also acts as a general acid/base. proton shuttle (general acid/base)
His353 His353D Acts as a general acid/base in the glutaminase reaction. proton shuttle (general acid/base)
Lys202, Glu355 Lys202C, Glu355D Forms part of the triad Lys-Glu-His that is responsible for ensuing the correct orientation and protonation state of the general acid/base histidine (His353). electrostatic stabiliser
Cys269, His353, Lys202, Glu355 Cys269D, His353D, Lys202C, Glu355D Forms the active site of the glutaminase reaction. covalent catalysis, proton shuttle (general acid/base)
Glu215, Glu761 Glu215C, Glu761C Helps position the ATP in the correct orientation. steric role
Glu299, Gln285, Gln829, Asn843 Glu299C, Gln285C, Gln829C, Asn843C Forms part of the metal binding sites. metal ligand
Glu841, Gln829, Glu761, Asn843, Arg715, Gly721 (main-N), Gly722 (main-N), Arg848 Glu841C, Gln829C, Glu761C, Asn843C, Arg715C, Gly721C (main-N), Gly722C (main-N), Arg848C Forms the active site of the final half-reaction. metal ligand
Asn301, Glu299, Gln285, Arg129, Arg169, Glu215, Arg303 Asn301C, Glu299C, Gln285C, Arg129C, Arg169C, Glu215C, Arg303C Forms part of the initial phosphorylation active site. metal ligand
Asn283, Arg129, Arg169, Arg715, Gly721 (main-N), Gly722 (main-N), Arg848, Arg303 Asn283C, Arg129C, Arg169C, Arg715C, Gly721C (main-N), Gly722C (main-N), Arg848C, Arg303C Helps to stabilise the negatively charged intermediates and transition states. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Hart EJ et al. (2008), Protein Sci, 17, 1120-1128. Mutation analysis of carbamoyl phosphate synthetase: Does the structurally conserved glutamine amidotransferase triad act as a functional dyad? DOI:10.1110/ps.073428008. PMID:18458150.
  2. Holden HM et al. (1999), Cell Mol Life Sci, 56, 507-522. Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product. DOI:10.1007/s000180050448.
  3. Lund L et al. (2010), J Am Chem Soc, 132, 3870-3878. Carbamate Transport in Carbamoyl Phosphate Synthetase: A Theoretical and Experimental Investigation. DOI:10.1021/ja910441v. PMID:20187643.
  4. Fan Y et al. (2009), J Am Chem Soc, 131, 10211-10219. A Combined Theoretical and Experimental Study of the Ammonia Tunnel in Carbamoyl Phosphate Synthetase. DOI:10.1021/ja902557r. PMID:19569682.
  5. Johnson JL et al. (2007), Biochemistry, 46, 387-397. Resolving the Fluorescence Response ofEscherichia coliCarbamoyl Phosphate Synthetase:  Mapping Intra- and Intersubunit Conformational Changes†. DOI:10.1021/bi061642n. PMID:17209549.
  6. Kothe M et al. (2005), Protein Sci, 14, 37-44. Direct demonstration of carbamoyl phosphate formation on the C-terminal domain of carbamoyl phosphate synthetase. DOI:10.1110/ps.041041305. PMID:15576558.
  7. Kim J et al. (2002), Biochemistry, 41, 12575-12581. Structural Defects within the Carbamate Tunnel of Carbamoyl Phosphate Synthetase†. DOI:10.1021/bi020421o.
  8. Kim J et al. (2001), Biochemistry, 40, 11030-11036. Allosteric Control of the Oligomerization of Carbamoyl Phosphate Synthetase fromEscherichia coli†. DOI:10.1021/bi011121u.
  9. Miles BW et al. (2000), Biochemistry, 39, 5051-5056. Synchronization of the Three Reaction Centers within Carbamoyl Phosphate Synthetase†. DOI:10.1021/bi992772h.
  10. Thoden JB et al. (1999), Acta Crystallogr D Biol Crystallogr, 55, 8-24. The structure of carbamoyl phosphate synthetase determined to 2.1 Å resolution. DOI:10.1107/s0907444998006234. PMID:10089390.
  11. Thoden JB et al. (1999), Biochemistry, 38, 2347-2357. Carbamoyl Phosphate Synthetase:  Closure of the B-Domain as a Result of Nucleotide Binding†,‡. DOI:10.1021/bi982517h. PMID:10029528.
  12. Thoden JB et al. (1999), Biochemistry, 38, 16158-16166. The Small Subunit of Carbamoyl Phosphate Synthetase:  Snapshots along the Reaction Pathway†. DOI:10.1021/bi991741j.
  13. Thoden JB et al. (1999), J Biol Chem, 274, 22502-22507. The Binding of Inosine Monophosphate to Escherichia coli Carbamoyl Phosphate Synthetase. DOI:10.1074/jbc.274.32.22502.
  14. Hewagama A et al. (1999), J Biol Chem, 274, 28240-28245. Functional Linkage between the Glutaminase and Synthetase Domains of Carbamoyl-phosphate Synthetase: ROLE OF SERINE 44 IN CARBAMOYL-PHOSPHATE SYNTHETASE-ASPARTATE CARBAMOYLTRANSFERASE-DIHYDROOROTASE (CAD). DOI:10.1074/jbc.274.40.28240.
  15. Mullins LS et al. (1999), J Am Chem Soc, 121, 3803-3804. Channeling of Ammonia through the Intermolecular Tunnel Contained within Carbamoyl Phosphate Synthetase. DOI:10.1021/ja990063l.
  16. Huang X et al. (1999), Biochemistry, 38, 15909-15914. Deconstruction of the Catalytic Array within the Amidotransferase Subunit of Carbamoyl Phosphate Synthetase†. DOI:10.1021/bi991805q.
  17. Raushel FM et al. (1999), Biochemistry, 38, 7891-7899. The Amidotransferase Family of Enzymes:  Molecular Machines for the Production and Delivery of Ammonia†. DOI:10.1021/bi990871p. PMID:10387030.
  18. Raushel FM et al. (1998), Curr Opin Chem Biol, 2, 624-632. Carbamoyl phosphate synthetase: a crooked path from substrates to products. DOI:10.1016/s1367-5931(98)80094-x.
  19. Thoden JB et al. (1998), Biochemistry, 37, 8825-8831. Carbamoyl Phosphate Synthetase:  Caught in the Act of Glutamine Hydrolysis†,‡. DOI:10.1021/bi9807761. PMID:9636022.
  20. Stapleton MA et al. (1996), Biochemistry, 35, 14352-14361. Role of Conserved Residues within the Carboxy Phosphate Domain of Carbamoyl Phosphate Synthetase†. DOI:10.1021/bi961183y. PMID:8916922.

Step 1.

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Catalytic Residues Roles

Residue Roles
Cys269D covalent catalysis
His353D proton shuttle (general acid/base)
Glu355D steric role
Lys202C electrostatic stabiliser
Gln285C metal ligand
Glu299C metal ligand
Asn301C metal ligand
Arg129C electrostatic stabiliser
Glu215C steric role
Arg169C electrostatic stabiliser
Arg303C electrostatic stabiliser
Asn283C electrostatic stabiliser
His243C proton shuttle (general acid/base)
Cys269D proton shuttle (general acid/base)
Gly721C (main-N) electrostatic stabiliser
Glu761C steric role
Gln829C metal ligand
Arg715C electrostatic stabiliser
Gly722C (main-N) electrostatic stabiliser
Asn843C metal ligand
Arg848C electrostatic stabiliser
Glu841C metal ligand

Chemical Components

Step 1.

Contributors

Nozomi Nagano, Gemma L. Holliday