Aspartate-ammonia ligase

 

L-asparagine synthetase in Escherichia coli converts L-aspartate and ammonia to L-asparagine, with the simultaneous hydrolysis of ATP to AMP and diphosphate. The reaction involves the formation of an aminoacyl-adenylate intermediate.

 

Reference Protein and Structure

Sequence
P00963 UniProt (6.3.1.1) IPR004618 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
12as - ASPARAGINE SYNTHETASE MUTANT C51A, C315A COMPLEXED WITH L-ASPARAGINE AND AMP (2.2 Å) PDBe PDBsum 12as
Catalytic CATH Domains
3.30.930.10 CATHdb (see all for 12as)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.3.1.1)

ammonia
CHEBI:16134ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
L-aspartate(1-)
CHEBI:29991ChEBI
→
hydron
CHEBI:15378ChEBI
+
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
L-asparagine zwitterion
CHEBI:58048ChEBI
+
diphosphate(4-)
CHEBI:18361ChEBI
Alternative enzyme names: L-asparagine synthetase, Asparagine synthetase,

Enzyme Mechanism

Introduction

The carboxylate group of the L-aspartate side chain acts as a nucleophile and attacks the alpha-phosphate of ATP in a substitution reaction that liberates pyrophosphate. Arg100 and Gln116 stabilise the intermediates formed. Asp46 deprotonates the ammonia molecule, which acts as a nucleophile to attack the carbonyl group (attached to the phosphate) of the L-aspartate in an addition reaction. Arg100 and Gln116 stabilise the intermediates formed. The tetrahedral intermediate collapses to reform the carbonyl, cleaving the P-O bond and liberating AMP and the L-asparagine. Asp46 donates its proton back to the free AMP. Arg100 and Gln116 stabilise the intermediates formed.

An Mg(II) ion is also believed to be involved in catalysis, stabilising negative charges along with Arg100 and Gln116 in each transition state or intermediate, but this is not present in the crystal structure.

Catalytic Residues Roles

UniProt PDB* (12as)
Arg100 Arg100A Forms part of oxyanion hole to stabilise the negative charge on substrate oxygen during the transition state. hydrogen bond donor, electrostatic stabiliser
Asp235, Glu248 Asp235A, Glu248A Forms part of the magnesium binding site. metal ligand
Asp46 Asp46A Deprotonates the ammonia molecule to activate it as a nucleophile, and donates the proton back to AMP. Also stabilises the positive charge on the substrate nitrogen during the transition state. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Gln116 Gln116A The NH2 group forms part of oxyanion hole to stabilise the negative charge on oxygen during the transition state. The oxygen of Gln116 also stabilises the positive charge on the substrate nitrogen during the transition state. hydrogen bond donor, electrostatic stabiliser
Ser251, Ser251 (main-C) Ser251A, Ser251A (main-C) Helps position and stabilise the intermediates during the course of the reaction. 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

bimolecular nucleophilic substitution, overall reactant used, overall product formed, dephosphorylation, intermediate formation, proton transfer, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, intermediate collapse, intermediate terminated, native state of enzyme regenerated

References

  1. Koizumi M et al. (1999), J Am Chem Soc, 121, 5799-5800. A Potent Transition-State Analogue Inhibitor ofEscherichia coliAsparagine Synthetase A. DOI:10.1021/ja990851a.
  2. Manhas R et al. (2014), J Biol Chem, 289, 12096-12108. Identification and Functional Characterization of a Novel Bacterial Type Asparagine Synthetase A: A tRNA SYNTHETASE PARALOG FROM LEISHMANIA DONOVANI. DOI:10.1074/jbc.m114.554642. PMID:24610810.
  3. Kolodkin-Gal I et al. (2007), Science, 318, 652-655. A Linear Pentapeptide Is a Quorum-Sensing Factor Required for mazEF-Mediated Cell Death in Escherichia coli. DOI:10.1126/science.1147248. PMID:17962566.
  4. Nakatsu T et al. (1998), Nat Struct Biol, 5, 15-19. Crystal structure of asparagine synthetase reveals a close evolutionary relationship to class II aminoacyl-tRNA synthetase. DOI:10.1038/nsb0198-15. PMID:9437423.
  5. Hinchman SK et al. (1992), J Biol Chem, 267, 144-149. A relationship between asparagine synthetase A and aspartyl tRNA synthetase. PMID:1346128.

Step 1. The carboxylate group of the L-aspartate side chain acts as a nucleophile and attacks the alpha-phosphate of ATP in a substitution reaction that liberates pyrophosphate. Mg(II), Arg100 and Gln116 stabilise the intermediates formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg100A hydrogen bond donor, electrostatic stabiliser
Gln116A hydrogen bond donor, electrostatic stabiliser
Ser251A electrostatic stabiliser
Ser251A (main-C) electrostatic stabiliser
Glu248A metal ligand
Asp235A metal ligand

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, dephosphorylation, intermediate formation

Step 2. Asp46 deprotonates the ammonia molecule, which acts as a nucleophile to attack the carbonyl group (attached to the phosphate) of the L-aspartate in an addition reaction. Mg(II), Arg100 and Gln116 stabilise the intermediates formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp46A hydrogen bond acceptor
Arg100A hydrogen bond donor, electrostatic stabiliser
Gln116A hydrogen bond donor, electrostatic stabiliser
Ser251A electrostatic stabiliser
Ser251A (main-C) electrostatic stabiliser
Glu248A metal ligand
Asp235A metal ligand
Asp46A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation

Step 3. The oxyanion formed re-forms the carbonyl group, cleaving the P-O bond in a conjugate base elimination reaction liberating AMP and the L-asparagine. Asp46 donates its proton back to the liberated AMP. Mg(II), Arg100 and Gln116 stabilise the intermediates formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln116A hydrogen bond donor, electrostatic stabiliser
Asp46A hydrogen bond donor
Arg100A hydrogen bond donor, electrostatic stabiliser
Glu248A metal ligand
Asp235A metal ligand
Ser251A electrostatic stabiliser
Ser251A (main-C) electrostatic stabiliser
Asp46A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, overall product formed, intermediate collapse, intermediate terminated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The carboxylate group of the L-aspartate side chain acts as a nucleophile and attacks the alpha-phosphate of ATP in a substitution reaction that liberates pyrophosphate. Mg(II), Arg100 and Gln116 stabilise the intermediates formed.

Step 2. Asp46 deprotonates the ammonia molecule, which acts as a nucleophile to attack the carbonyl group (attached to the phosphate) of the L-aspartate in an addition reaction. Mg(II), Arg100 and Gln116 stabilise the intermediates formed.

Step 3. The oxyanion formed re-forms the carbonyl group, cleaving the P-O bond in a conjugate base elimination reaction liberating AMP and the L-asparagine. Asp46 donates its proton back to the liberated AMP. Mg(II), Arg100 and Gln116 stabilise the intermediates formed.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Emma LR Compton, Craig Porter