Asparagine synthase (glutamine-hydrolysing)

 

Asparagine synthetase B catalyses the ATP dependent formation of asparagine from aspartate using either glutamine or ammonia as a nitrogen source. The enzyme is a homodimer with two active sites [PMID:10587437] in each subunit, one responsible for glutamine hydrolysis and one for asparagine synthesis, separated by a hydrophobic intramolecular tunnel around 20A long [PMID:12706338]. Sequencing analysis has shown the synthetase to belong to the larger glutamine transferase family.

 

Reference Protein and Structure

Sequence
P22106 UniProt (6.3.5.4) IPR006426 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1ct9 - CRYSTAL STRUCTURE OF ASPARAGINE SYNTHETASE B FROM ESCHERICHIA COLI (2.0 Å) PDBe PDBsum 1ct9
Catalytic CATH Domains
3.60.20.10 CATHdb 3.40.50.620 CATHdb (see all for 1ct9)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.3.5.4)

water
CHEBI:15377ChEBI
+
L-glutamine zwitterion
CHEBI:58359ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
L-aspartate(1-)
CHEBI:29991ChEBI
→
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
hydron
CHEBI:15378ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
+
L-glutamate(1-)
CHEBI:29985ChEBI
+
L-asparagine zwitterion
CHEBI:58048ChEBI
Alternative enzyme names: Asparagine synthetase (glutamine-hydrolyzing), Glutamine-dependent asparagine synthetase, Asparagine synthetase B, AS, AS-B,

Enzyme Mechanism

Introduction

The enzyme catalyses three distinct chemical reactions: glutamine hydrolysis to yield ammonia (which is then channelled to the second active site) takes place in the N-terminal domain. The C-terminal active site mediates both the synthesis of a beta-aspartyl-AMP intermediate and its subsequent reaction with ammonia. However, the exact order of the partial reactions is still somewhat unclear [PMID:9748330, PMID:12706338], here we show the hydrolysis of the glutamate occurring first.

Glutamine hydrolysis occurs at the N terminal. A nucleophilic Cys residue attacks the glutamine substrate, displacing ammonia, forming a substrate-enzyme intermediate which is then hydrolysed. The ammonia diffuses though the interdomain tunnel from the site of production to the site of utilisation: the synthetase component in the C terminal.

A two site ping pong mechanism for the reaction between the aspartic acid, ATP and ammonia has been implicated with several residues thought to be responsible for the binding the substrates through hydrogen bonding interactions.

Kinetic analysis has shown the rate of catalysis at each site to be independent of one another, and so the stoichiometry between the sites must be maintained by the catalytic efficiency of the two sites.

Catalytic Residues Roles

UniProt PDB* (1ct9)
Cys2 (N-term) Ala1A (N-term) Acts as a general acid/base to activate the cysteine nucleophile. proton acceptor, proton donor
Leu51 (main-C) Leu50A (main-C) Helps stabilise the reactive intermediates formed. hydrogen bond acceptor, electrostatic stabiliser
Thr322, Arg325 Thr321A, Arg324A Bind and stabilise the phosphate groups of the ATP and reactive intermediates formed. hydrogen bond donor, electrostatic stabiliser
Cys2 Ala1A Acts as a catalytic nucleophile in the glutaminase domain reaction. covalently attached, hydrogen bond acceptor, nucleofuge, nucleophile, proton acceptor, proton donor
Gly76 (main-N), Asn75 Gly75A (main-N), Asn74A Forms the oxyanion hole that stabilises the reactive intermediates and transition states formed. hydrogen bond donor, 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

proton transfer, bimolecular nucleophilic addition, proton relay, enzyme-substrate complex formation, overall reactant used, intermediate formation, unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, deamination, intermediate collapse, overall product formed, native state of enzyme regenerated

References

  1. Tesson AR et al. (2003), Arch Biochem Biophys, 413, 23-31. Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli. DOI:10.1016/s0003-9861(03)00118-8. PMID:12706338.
  2. Fresquet V et al. (2004), Bioorg Chem, 32, 63-75. Kinetic mechanism of asparagine synthetase from Vibrio cholerae. DOI:10.1016/j.bioorg.2003.10.002. PMID:14990305.
  3. Larsen TM et al. (2000), Biochemistry, 39, 7330-. Three-dimensional structure of escherichia coli asparagine synthetase B: A short journey from substrate to product. PMID:10852734.
  4. Larsen TM et al. (1999), Biochemistry, 39, 7330-7330. Three-Dimensional Structure ofEscherichia coliAsparagine Synthetase B:  A Short Journey from Substrate to Product. DOI:10.1021/bi005109y. PMID:10587437.
  5. Boehlein SK et al. (1998), Biochemistry, 37, 13230-13238. Kinetic Mechanism ofEscherichiacoliAsparagine Synthetase B†. DOI:10.1021/bi981058h. PMID:9748330.
  6. Boehlein SK et al. (1997), J Biol Chem, 272, 12384-12392. Mutagenesis and Chemical Rescue Indicate Residues Involved in beta -Aspartyl-AMP Formation by Escherichia coli Asparagine Synthetase B. DOI:10.1074/jbc.272.19.12384. PMID:9139684.

Step 1. This reaction takes place in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which deprotonates the thiol group of Cys1, initiating a nucleophilic attack on the amide carbon in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Leu50A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Gly75A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn74A hydrogen bond donor, electrostatic stabiliser
Ala1A (N-term) proton acceptor
Ala1A nucleophile, proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, proton relay, enzyme-substrate complex formation, overall reactant used, intermediate formation

Step 2. This reaction takes place in the glutaminase domain. The oxyanion initiates an elimination that cleaves ammonia from the bound L-glutamine substrate. Ammonia deprotonates water, which deprotonates the N-terminus of Cys1. The ammonia product is then transferred to the second domain of the enzyme through an internal ion channel

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Catalytic Residues Roles

Residue Roles
Ala1A covalently attached
Leu50A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Gly75A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn74A hydrogen bond donor, electrostatic stabiliser
Ala1A (N-term) proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, proton relay, intermediate formation, enzyme-substrate complex cleavage, deamination

Step 3. This reaction occurs in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound intermediate in an addition reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala1A covalently attached, hydrogen bond acceptor
Leu50A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Gly75A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn74A hydrogen bond donor, electrostatic stabiliser
Ala1A (N-term) proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, overall reactant used, intermediate formation

Step 4. This reaction occurs in the glutaminase domain. The oxyanion initiates an elimination that cleaves the C-S bond, the thiolate of Cys1 deprotonates water, which deprotonates the N-terminus of Cys1.

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Catalytic Residues Roles

Residue Roles
Ala1A hydrogen bond acceptor
Leu50A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Gly75A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn74A hydrogen bond donor, electrostatic stabiliser
Ala1A (N-term) proton donor
Ala1A nucleofuge, proton acceptor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, proton relay, intermediate collapse, overall product formed, enzyme-substrate complex cleavage

Step 5. This reaction occurs in the ligase domain of the enzyme. The aspartate substrate initiates a nucleophilic attack on the alpha phosphate of ATP in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Arg324A hydrogen bond donor, electrostatic stabiliser
Thr321A hydrogen bond donor, electrostatic stabiliser

Chemical Components

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

Step 6. This reaction occurs in the ligase domain of the enzyme. The pentavalent intermediate collapses to eliminate the diphosphate product and the beta-Asp-AMP intermediate.

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Catalytic Residues Roles

Residue Roles
Arg324A hydrogen bond donor, electrostatic stabiliser
Thr321A hydrogen bond donor, electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, intermediate formation

Step 7. This reaction occurs in the ligase domain of the enzyme. The ammonia nitrogen initiates a nucleophilic attack on the carbonyl carbon of the beta-Asp-AMP intermediate in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Arg324A hydrogen bond donor, electrostatic stabiliser
Thr321A hydrogen bond donor, electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 8. This reaction occurs in the ligase domain of the enzyme. The oxyanion initiates an elimination that cleaves the C-O bond, the phosphate of AMP deprotonates the ammonium group, releasing the AMP and Asparagine products.

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Catalytic Residues Roles

Residue Roles
Arg324A hydrogen bond donor, electrostatic stabiliser
Thr321A hydrogen bond donor, electrostatic stabiliser

Chemical Components

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

Step 1. This reaction takes place in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which deprotonates the thiol group of Cys1, initiating a nucleophilic attack on the amide carbon in an addition reaction.

Step 2. This reaction takes place in the glutaminase domain. The oxyanion initiates an elimination that cleaves ammonia from the bound L-glutamine substrate. Ammonia deprotonates water, which deprotonates the N-terminus of Cys1. The ammonia product is then transferred to the second domain of the enzyme through an internal ion channel

Step 3. This reaction occurs in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound intermediate in an addition reaction.

Step 4. This reaction occurs in the glutaminase domain. The oxyanion initiates an elimination that cleaves the C-S bond, the thiolate of Cys1 deprotonates water, which deprotonates the N-terminus of Cys1.

Step 5. This reaction occurs in the ligase domain of the enzyme. The aspartate substrate initiates a nucleophilic attack on the alpha phosphate of ATP in an addition reaction.

Step 6. This reaction occurs in the ligase domain of the enzyme. The pentavalent intermediate collapses to eliminate the diphosphate product and the beta-Asp-AMP intermediate.

Step 7. This reaction occurs in the ligase domain of the enzyme. The ammonia nitrogen initiates a nucleophilic attack on the carbonyl carbon of the beta-Asp-AMP intermediate in an addition reaction.

Step 8. This reaction occurs in the ligase domain of the enzyme. The oxyanion initiates an elimination that cleaves the C-O bond, the phosphate of AMP deprotonates the ammonium group, releasing the AMP and Asparagine products.

Products.

Contributors

Gemma L. Holliday, Angela Malumbe, Craig Porter