Asparaginase

 

Asparaginase (EC 3.5.1.1) and glutaminaase (EC 3.5.1.38) remove amino groups from asparagine and glutamine respectively leaving aspartate and glutamate. This entry covers both enzymes, though is derived from asparaginase. Enzymes from both EC families have some activity in the other. Two forms exist in E.coli, type I is cytoplasmic, type II is periplasmic. The type II form has a higher affinity for the substrate and its production is induced under anaerobic conditions when amino acids are the primary carbon source. Type II L-asparaginase is the most studied, because it is an important anti-cancer drug due to its ability to lower the levels of asparagine available to cancerous cells lacking in asparaginase synthase.

 

Reference Protein and Structure

Sequence
P00805 UniProt (3.5.1.1) IPR004550 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
3eca - CRYSTAL STRUCTURE OF ESCHERICHIA COLI L-ASPARAGINASE, AN ENZYME USED IN CANCER THERAPY (2.4 Å) PDBe PDBsum 3eca
Catalytic CATH Domains
3.40.50.40 CATHdb 3.40.50.1170 CATHdb (see all for 3eca)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.1)

water
CHEBI:15377ChEBI
+
L-asparagine zwitterion
CHEBI:58048ChEBI
→
L-aspartate(1-)
CHEBI:29991ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: Alpha-asparaginase, L-asparaginase, Asparaginase II, Colaspase, Crasnitin, Elspar, Leunase,

Enzyme Mechanism

Introduction

In the double displacement (ping-pong) mechanism proposal, the reaction proceeds via a nucleophilic attack of Thr12 on the CG/CD of asparagine/glutamine forming a covalent intermediate. After the intermediate is formed, the amine group is eliminated, followed by a second nucleophilic attack on the substrate, this time by a water molecule, which ultimately leads to the collapse of the covalent intermediate and regeneration of the active site. The existence of a crystallographic structure (PDB ID:6v26) of an acyl-enzyme intermediate (post ammonia elimination) is a strong piece of experimental evidence in favour of this mechanism. Although computational studies did not support a previous version of this mechanism, it has been suggested that two water molecules present in the active site but not taken into account in the computational studies might account for additional stabilisation.

Catalytic Residues Roles

UniProt PDB* (3eca)
Thr34 Thr12A Acts as a catalytic nucleophile. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor
Thr111 (main-N) Thr89A (main-N) The main chain amide forms part of the oxyanion hole along with the active site water molecule. electrostatic stabiliser
Tyr47 Tyr25A Activates Thr12 via proton abstraction. proton relay, increase nucleophilicity, proton acceptor, proton donor
Thr111, Asp112, Lys184 Thr89A, Asp90A, Lys162A Acts as the general acid/base. proton relay, proton acceptor, proton donor
*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, proton transfer, unimolecular elimination by the conjugate base

References

  1. Lubkowski J et al. (2020), Biochemistry, 59, 1927-1945. Mechanism of Catalysis by l-Asparaginase. DOI:10.1021/acs.biochem.0c00116. PMID:32364696.
  2. Borek D et al. (2014), FEBS J, 281, 4097-4111. Crystal structure of active site mutant of antileukemicl-asparaginase reveals conserved zinc-binding site. DOI:10.1111/febs.12906. PMID:25040257.
  3. Gesto DS et al. (2013), J Am Chem Soc, 135, 7146-7158. Unraveling the Enigmatic Mechanism ofl-Asparaginase II with QM/QM Calculations. DOI:10.1021/ja310165u. PMID:23544711.
  4. Aung HP et al. (2000), Biochim Biophys Acta, 1481, 349-359. Dynamics of a mobile loop at the active site of Escherichia coli asparaginase. DOI:10.1016/s0167-4838(00)00179-5. PMID:11018727.
  5. Lubkowski J et al. (1996), Eur J Biochem, 241, 201-207. Crystal Structure and Amino Acid Sequence of Wolinella Succinogenesl-Asparaginase. DOI:10.1111/j.1432-1033.1996.0201t.x. PMID:8898907.
  6. Palm GJ et al. (1996), FEBS Lett, 390, 211-216. A covalently bound catalytic intermediate inEscherichia coliasparaginase : Crystal structure of a Thr-89-Val mutant. DOI:10.1016/0014-5793(96)00660-6. PMID:8706862.
  7. Swain AL et al. (1993), Proc Natl Acad Sci U S A, 90, 1474-1478. Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. DOI:10.1073/pnas.90.4.1474. PMID:8434007.
  8. Harms E et al. (1991), FEBS Lett, 285, 55-58. A catalytic role for threonine-12 of E. coli asparaginase II as established by site-directed mutagenesis. PMID:1906013.

Step 1. Thr12 is activated by Tyr25 and Asp90 and performs a nucleophilic attack on the substrate, forming the covalent intermediate.

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Catalytic Residues Roles

Residue Roles
Asp90A proton acceptor
Tyr25A proton acceptor, proton donor, proton relay
Thr12A nucleophile, proton donor
Asp90A increase nucleophilicity
Tyr25A increase nucleophilicity
Thr89A (main-N) electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 2. Ammonia is released while accepting a proton from Thr89 which in turn deprotonates Lys162.

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Catalytic Residues Roles

Residue Roles
Lys162A proton acceptor
Thr12A covalently attached
Thr89A proton acceptor, proton donor, proton relay
Thr89A (main-N) electrostatic stabiliser

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base

Step 3. A water molecule performs a nucleophilic attack on the acyl-enzyme intermediate, forming again a tetrahedral intermediate. A proton from the nucleophilic water molecule is transferred to Thr89 which in turn looses a proton to Lys162.

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Catalytic Residues Roles

Residue Roles
Lys162A proton acceptor
Thr89A proton acceptor, proton donor, proton relay
Thr89A (main-N) electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 4. The covalent intermediate collapses, as Thr12 is protonated by Tyr25, which in turn is re-protonated by Asp90 through a bridging water. The final product is formed.

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Catalytic Residues Roles

Residue Roles
Thr12A nucleofuge, proton acceptor
Tyr25A proton acceptor, proton donor, proton relay
Asp90A proton donor
Thr89A (main-N) electrostatic stabiliser

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base
Download: Image, Marvin File

Step 1. Thr12 is activated by Tyr25 and Asp90 and performs a nucleophilic attack on the substrate, forming the covalent intermediate.

Step 2. Ammonia is released while accepting a proton from Thr89 which in turn deprotonates Lys162.

Step 3. A water molecule performs a nucleophilic attack on the acyl-enzyme intermediate, forming again a tetrahedral intermediate. A proton from the nucleophilic water molecule is transferred to Thr89 which in turn looses a proton to Lys162.

Step 4. The covalent intermediate collapses, as Thr12 is protonated by Tyr25, which in turn is re-protonated by Asp90 through a bridging water. The final product is formed.

Products.

Introduction

In this direct displacement mechanism proposal, the Thr89-Lys162-Asp90 triad activates a water molecule to perform the nucleophilic attack in the first step of reaction, while the second triad, Thr12-Tyr25-Glu283, stabilizes the tetrahedral intermediate formed in the first step. The Thr89-Lys162-Asp90 triad is situated in a rigid part of the structure and mutational and crystallographic studies indicate that all three residues, which are absolutely conserved, are involved in the catalytic reaction.

Catalytic Residues Roles

UniProt PDB* (3eca)
Thr34, Glu305, Tyr47 Thr12A, Glu283C, Tyr25A Forms the Thr-Tyr-Glu triad that is responsible for stabilising the intermediates of the reaction. electrostatic stabiliser
Thr111, Asp112 Thr89A, Asp90A Activates Lys162 for the initial proton abstraction from the water molecule. proton acceptor, proton donor, proton relay, electrostatic stabiliser, increase basicity
Asp112, Lys184 Asp90A, Lys162A Forms the Thr-Lys-Asp triad that activates the Thr as a general acid/base. increase basicity, electrostatic stabiliser, increase acidity
Lys184 Lys162A Acts as a general acid/base. proton acceptor, proton donor
*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, proton transfer, unimolecular elimination by the conjugate base, native state of enzyme regenerated

References

  1. Gesto DS et al. (2013), J Am Chem Soc, 135, 7146-7158. Unraveling the Enigmatic Mechanism ofl-Asparaginase II with QM/QM Calculations. DOI:10.1021/ja310165u. PMID:23544711.
  2. Schalk AM et al. (2016), J Biol Chem, 291, 5088-5100. Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases. DOI:10.1074/jbc.m115.699884. PMID:26733195.
  3. Borek D et al. (2014), FEBS J, 281, 4097-4111. Crystal structure of active site mutant of antileukemicl-asparaginase reveals conserved zinc-binding site. DOI:10.1111/febs.12906. PMID:25040257.

Step 1. Lys162, activated by Asp90 and Thr89, abstracts a proton from water, which initiates a nucleophilic attack on the carbonyl carbon, resulting in a negatively charged intermediate, stabilises by Thr12.

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Catalytic Residues Roles

Residue Roles
Glu283C electrostatic stabiliser
Tyr25A electrostatic stabiliser
Thr12A electrostatic stabiliser
Asp90A electrostatic stabiliser, increase basicity
Thr89A increase basicity, electrostatic stabiliser
Lys162A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 2. The amine group of the intermediate abstracts a proton from Thr89, which in turn abstracts a proton from Lys162.

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Catalytic Residues Roles

Residue Roles
Asp90A increase acidity
Thr12A electrostatic stabiliser
Glu283C electrostatic stabiliser
Tyr25A electrostatic stabiliser
Thr89A proton relay
Thr89A proton donor, proton acceptor
Lys162A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer

Step 3. The tetrahedral intermediate collapses, eliminating ammonia.

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Catalytic Residues Roles

Residue Roles
Thr12A electrostatic stabiliser
Tyr25A electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Lys162, activated by Asp90 and Thr89, abstracts a proton from water, which initiates a nucleophilic attack on the carbonyl carbon, resulting in a negatively charged intermediate, stabilises by Thr12.

Step 2. The amine group of the intermediate abstracts a proton from Thr89, which in turn abstracts a proton from Lys162.

Step 3. The tetrahedral intermediate collapses, eliminating ammonia.

Products.

Contributors

Gemma L. Holliday, Craig Porter, Yordanos Abeje