Carbamate kinase

 

Carbamoyl phosphate (CP) can be synthesised from mixtures of ATP, bicarbonate and ammonia by two types of enzymes: carbamoyl phosphate synthases (CPS) and carbamate kinases (CK). CPS uses a three step mechanism and consumes two molecule of ATP per molecule of CP synthesised; this reaction is essentially irreversible and is the first committed step in the biosynthesis of pyrimidines, arginine and urea. By contrast, carbamate kinases (CK) make CP reversibly in a one-step reaction using one molecule of ATP per CP molecule synthesised. The actual substrate for these enzymes is carbamate which is in equilibrium with bicarbonate and ammonia. The in vivo role of CK from many organisms in which it has been studied (such as Enterococcus faecalis) seems to be to generate ATP from ADP using carbamoyl phosphate derived from the catabolism of arginine. However in some species, such as the hypothermophilic archaea Pyrococcus furiosus and Pyrococcus abyssi the enzyme appears to have an anabolic role. The enzyme from these species has in fact been called a "carbamate kinase-like carbamoyl phosphate synthetase", although studies have shown that is structurally and enzymologically a carbamate kinase.

 

Reference Protein and Structure

Sequence
P0A2X8 UniProt (2.7.2.2) IPR003964 (Sequence Homologues) (PDB Homologues)
Biological species
Enterococcus faecium (Bacteria) Uniprot
PDB
1b7b - Carbamate kinase from Enterococcus faecalis (2.8 Å) PDBe PDBsum 1b7b
Catalytic CATH Domains
3.40.1160.10 CATHdb (see all for 1b7b)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.7.2.2)

carbamoyl phosphate(2-)
CHEBI:58228ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
→
ATP(4-)
CHEBI:30616ChEBI
+
carbamate
CHEBI:13941ChEBI
Alternative enzyme names: CKase, Carbamoyl phosphokinase, Carbamyl phosphokinase,

Enzyme Mechanism

Introduction

Phosphoryl transfer from ATP to carbamate or from CP to ADP is proposed to occur via an in-line displacement reaction. The drawn out mechanism shows the formation of carbamate and ATP but the reverse reaction has the carbonyl on carbamate attacking the electrophilic phosphorous on gamma-phosphate of ATP, with the same catalytic residues and magnesium ion stabilising the transition state. More work is needed to further define the exact roles of the catalytic residues in order to be fully confident in the catalytic mechanism.

Catalytic Residues Roles

UniProt PDB* (1b7b)
Asn12, Gly11 (main-N) Asn12A, Gly11A (main-N) Proposed to neutralise negative charge on the phosphate on the carbamate during the reaction. electrostatic stabiliser, polar interaction
Lys271 Lys271A Proposed to interact with the terminal phosphate of ADP and neutralise the developing negative charge on this group during the reaction. electrostatic stabiliser, polar interaction
Lys128 Lys128A In the direction of CP breakdown, is proposed to interact with the bridging oxygen atom of CP and neutralise the developing negative charge on this oxygen atom as the product carbamate is generated. electrostatic stabiliser, polar interaction
Lys209 Lys209A Proposed to stabilise negative charge in the transition state. electrostatic stabiliser, polar interaction
*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

bimolecular nucleophilic substitution, overall product formed, overall reactant used

References

  1. Ramón-Maiques S et al. (2010), J Mol Biol, 397, 1261-1275. Substrate binding and catalysis in carbamate kinase ascertained by crystallographic and site-directed mutagenesis studies: movements and significance of a unique globular subdomain of this key enzyme for fermentative ATP production in bacteria. DOI:10.1016/j.jmb.2010.02.038. PMID:20188742.
  2. Lim K et al. (2013), PLoS One, 8, e64004-. Crystal structures of carbamate kinase from Giardia lamblia bound with citric acid and AMP-PNP. DOI:10.1371/journal.pone.0064004. PMID:23700444.
  3. Ramón-Maiques S et al. (2002), Biochemistry, 41, 3916-3924. Molecular Physiology of Phosphoryl Group Transfer from Carbamoyl Phosphate by a Hyperthermophilic Enzyme at Low Temperature†. DOI:10.1021/bi011637d. PMID:11900534.
  4. Ramón-Maiques S et al. (2000), J Mol Biol, 299, 463-476. The 1.5 Å resolution crystal structure of the carbamate kinase-like carbamoyl phosphate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus, bound to ADP, confirms that this thermostable enzyme is a carbamate kinase, and provides insight into substrate binding and stability in carbamate kinases. DOI:10.1006/jmbi.2000.3779. PMID:10860751.
  5. Uriarte M et al. (1999), J Biol Chem, 274, 16295-16303. The Carbamoyl-phosphate Synthetase of Pyrococcus furiosus Is Enzymologically and Structurally a Carbamate Kinase. DOI:10.1074/jbc.274.23.16295. PMID:10347186.

Step 1. The non-bridging oxygen on the ADP molecule beta-phosphate nucleophilically attacks the phosphorus on carbamoyl. The transition state observed in this nucleophilic substitution is stabilised by the surrounding residues Lys271, Lys209, Lys128, Gly11 and Asn12 and also Mg2+.

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Catalytic Residues Roles

Residue Roles
Lys209A electrostatic stabiliser
Lys271A electrostatic stabiliser
Lys128A electrostatic stabiliser
Asn12A electrostatic stabiliser
Gly11A (main-N) electrostatic stabiliser
Gly11A (main-N) polar interaction
Asn12A polar interaction
Lys128A polar interaction
Lys209A polar interaction
Lys271A polar interaction

Chemical Components

ingold: bimolecular nucleophilic substitution, overall product formed, overall reactant used
Download: Image, Marvin File

Step 1. The non-bridging oxygen on the ADP molecule beta-phosphate nucleophilically attacks the phosphorus on carbamoyl. The transition state observed in this nucleophilic substitution is stabilised by the surrounding residues Lys271, Lys209, Lys128, Gly11 and Asn12 and also Mg2+.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Morwenna Hall