Glutamin-(asparagin-)ase

 

Glutaminase-asparaginase catalyses the hydrolysis of D and L isomers of glutamine and asparagine.

Glutaminase-asparaginase belongs to a family of related amidohydrolases, in a less specific class that catalyses the hydrolysis of glutamine to glutamic acid and asparagine to aspartic acid with similar efficiency. They are mechanistically similar to trypsin.

 

Reference Protein and Structure

Sequence
P10182 UniProt (3.5.1.38) IPR004550 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas sp. ATCC29598 (Bacteria) Uniprot
PDB
1djo - Crystal structure of Pseudomonas 7A Glutaminase-asparaginase with the inhibitor donv covalently bound in the active site (2.0 Å) PDBe PDBsum 1djo
Catalytic CATH Domains
3.40.50.40 CATHdb 3.40.50.1170 CATHdb (see all for 1djo)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.38)

L-asparagine zwitterion
CHEBI:58048ChEBI
+
water
CHEBI:15377ChEBI
→
L-aspartate(1-)
CHEBI:29991ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: L-asparagine/L-glutamine amidohydrolase, L-ASNase/L-GLNase, Glutaminase-asparaginase, AnsB (gene name),

Enzyme Mechanism

Introduction

Tyr 1034 is polarised by Glu 3294 B and facilitates proton abstraction from Thr 1020 and its transfer to the substrate. Nucleophilic attack by Thr 1020 proceeds on the carbonyl carbon and this forms a tetrahedral intermediate, which collapses using Tyr 1034 as a general acid/base catalyst, and activation by Glu 3294 B with release of ammonia as a by-product. Hydrolysis occurs using Thr 1100 to activate water as a nucleophile to attack the carbonyl carbon, which is activated by Tyr 1034. By general acid/base catalysis of Tyr 1034 and activation by Glu 3294 B the tetrahedral intermediate again collapses to break the acyl-enzyme linkage and release the product.

Catalytic Residues Roles

UniProt PDB* (1djo)
Glu294 Glu3294(287)B Activates the substrate and Tyr 1034 proton relay, hydrogen bond acceptor, proton acceptor, proton donor
Thr20 Thr1020(13)A Acts as a nucleophile to attack the substrate and form an acyl-enzyme intermediate. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, nucleofuge, proton donor, proton acceptor
Tyr34 Tyr1034(27)A Acts as a general acid/base catalyst in activating Thr 1020 and the substrate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Thr100 Thr1100(93)A Activates water for nucleophilic attack on the substrate. hydrogen bond acceptor, hydrogen bond donor
Asp101, Lys173 Asp1101(94)A, Lys1173(166)A Part of the charge relay system that activates Thr1100 hydrogen bond acceptor
*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 addition, overall reactant used, enzyme-substrate complex formation, proton relay, intermediate formation, proton transfer, bimolecular elimination, overall product formed, deamination, enzyme-substrate complex cleavage, intermediate collapse, native state of enzyme regenerated, intermediate terminated

References

  1. Sanches M et al. (2007), Curr Chem Biol, 1, 75-86. Structure, Substrate Complexation and Reaction Mechanism of Bacterial Asparaginases. DOI:10.2174/2212796810701010075.
  2. Ortlund E et al. (2000), Biochemistry, 39, 1199-1204. Reactions ofPseudomonas7A Glutaminase-Asparaginase with Diazo Analogues of Glutamine and Asparagine Result in Unexpected Covalent Inhibitions and Suggests an Unusual Catalytic Triad Thr-Tyr-Glu†,‡. DOI:10.1021/bi991797d. PMID:10684596.

Step 1. Glu3294B deprotonates Tyr1034, which in turn deprotonates Thr1020. Thr1020 then initiates a nucleophilic attack upon the amide carbon of L-asparagine in an addition reaction. The oxyanion formed deprotonates Glu3294B.

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Catalytic Residues Roles

Residue Roles
Tyr1034(27)A proton relay, hydrogen bond donor, hydrogen bond acceptor
Lys1173(166)A hydrogen bond donor, hydrogen bond acceptor
Glu3294(287)B hydrogen bond acceptor, proton relay
Asp1101(94)A hydrogen bond acceptor
Thr1100(93)A hydrogen bond donor, hydrogen bond acceptor
Thr1020(13)A hydrogen bond donor
Thr1020(13)A proton donor
Glu3294(287)B proton acceptor
Tyr1034(27)A proton acceptor
Glu3294(287)B proton donor
Thr1020(13)A nucleophile
Tyr1034(27)A proton donor

Chemical Components

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

Step 2. Glu1294B deprotonates the hydroxyl formed from the oxyanion, initiating the elimination of ammonia, which gains a proton from Glu1294B.

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Catalytic Residues Roles

Residue Roles
Tyr1034(27)A hydrogen bond acceptor, hydrogen bond donor
Lys1173(166)A hydrogen bond donor, hydrogen bond acceptor
Glu3294(287)B hydrogen bond acceptor, proton relay
Asp1101(94)A hydrogen bond acceptor
Thr1100(93)A hydrogen bond donor, hydrogen bond acceptor
Thr1020(13)A covalently attached, hydrogen bond acceptor
Glu3294(287)B proton donor, proton acceptor

Chemical Components

proton transfer, ingold: bimolecular elimination, overall product formed, proton relay, deamination, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation

Step 3. The Asp-Lys-Thr triad activates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently attached intermediate. The formed oxyanion deprotonates the water which attacked.

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Catalytic Residues Roles

Residue Roles
Tyr1034(27)A hydrogen bond acceptor, hydrogen bond donor
Lys1173(166)A hydrogen bond donor, hydrogen bond acceptor
Glu3294(287)B hydrogen bond acceptor
Asp1101(94)A hydrogen bond acceptor
Thr1100(93)A hydrogen bond donor, hydrogen bond acceptor
Thr1020(13)A covalently attached, hydrogen bond acceptor

Chemical Components

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

Step 4. Glu1294B deprotonates the hydroxyl formed from the oxyanion, initiating the elimination of Thr1020, which in turn deprotonates Tyr1034, which then deprotonates Glu1294B.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr1034(27)A hydrogen bond acceptor, hydrogen bond donor, proton relay
Lys1173(166)A hydrogen bond donor, hydrogen bond acceptor
Glu3294(287)B hydrogen bond acceptor, proton relay
Asp1101(94)A hydrogen bond acceptor
Thr1100(93)A hydrogen bond donor
Thr1020(13)A hydrogen bond acceptor
Glu3294(287)B proton donor, proton acceptor
Tyr1034(27)A proton acceptor
Thr1020(13)A nucleofuge, proton acceptor
Tyr1034(27)A proton donor

Chemical Components

ingold: bimolecular elimination, proton transfer, proton relay, overall product formed, native state of enzyme regenerated, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated
Download: Image, Marvin File

Step 1. Glu3294B deprotonates Tyr1034, which in turn deprotonates Thr1020. Thr1020 then initiates a nucleophilic attack upon the amide carbon of L-asparagine in an addition reaction. The oxyanion formed deprotonates Glu3294B.

Step 2. Glu1294B deprotonates the hydroxyl formed from the oxyanion, initiating the elimination of ammonia, which gains a proton from Glu1294B.

Step 3. The Asp-Lys-Thr triad activates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently attached intermediate. The formed oxyanion deprotonates the water which attacked.

Step 4. Glu1294B deprotonates the hydroxyl formed from the oxyanion, initiating the elimination of Thr1020, which in turn deprotonates Tyr1034, which then deprotonates Glu1294B.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Fiona J. E. Morgan, Katherine Ferris