Diisopropyl-fluorophosphatase

 

Diisopropylfluorophosphatase is a calcium metalloenzyme that catalyses the hydrolysis of diisopropyl fluorophosphate and related acetylcholinesterase inhibitors. The potential for this enzyme to detoxify nerve agents used in chemical warfare such as DFP, sarin and tabun. Only this enzyme and one other are expressed in sufficiently high yields.

 

Reference Protein and Structure

Sequence
Q7SIG4 UniProt (3.1.8.2) IPR013658 (Sequence Homologues) (PDB Homologues)
Biological species
Loligo vulgaris (Common European squid) Uniprot
PDB
1e1a - Crystal structure of DFPase from Loligo vulgaris (1.8 Å) PDBe PDBsum 1e1a
Catalytic CATH Domains
2.120.10.30 CATHdb (see all for 1e1a)
Cofactors
Calcium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.1.8.2)

diisopropyl fluorophosphate
CHEBI:17941ChEBI
+
water
CHEBI:15377ChEBI
→
diisopropyl phosphate(1-)
CHEBI:57896ChEBI
+
fluoride
CHEBI:17051ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: DFPase, OPA anhydrase, Dialkylfluorophosphatase, Diisopropyl phosphorofluoridate hydrolase, Diisopropylfluorophosphonate dehalogenase, Diisopropylphosphofluoridase, Isopropylphosphorofluoridase, Organophosphate acid anhydrase, Organophosphorus acid anhydrolase, Somanase, Tabunase, OPAA,

Enzyme Mechanism

Introduction

This mechanism involves the direct nucleophilic attack of Asp229 to the phosphate. The covalent intermediate formed is then hydrolyzed by a water molecule which is activated by Glu21.

Catalytic Residues Roles

UniProt PDB* (1e1a)
Asp229 Asp229A Directly attacks the phosphate group forming an intermediate. covalently attached, nucleophile, metal ligand, electrofuge, electrophile
Glu21 Glu21A Activates a water molecule which hydrolyzes the intermediate. increase nucleophilicity, metal ligand, proton acceptor
Asn120, Asn175 Asn120A, Asn175A Metal binding residues metal ligand
*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

overall reactant used, overall product formed, intermediate formation, bimolecular nucleophilic substitution, intermediate terminated, proton transfer

References

  1. Blum MM et al. (2006), J Am Chem Soc, 128, 12750-12757. Binding of a designed substrate analogue to diisopropyl fluorophosphatase: implications for the phosphotriesterase mechanism. DOI:10.1021/ja061887n. PMID:17002369.
  2. Purg M et al. (2017), J Am Chem Soc, 139, 17533-17546. Similar Active Sites and Mechanisms Do Not Lead to Cross-Promiscuity in Organophosphate Hydrolysis: Implications for Biotherapeutic Engineering. DOI:10.1021/jacs.7b09384. PMID:29113434.

Step 1. Asp229 performs a nucleophilic attack on the phosphate causing the F-P bond to be cleaved and an intermediate to be formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu21A metal ligand
Asn120A metal ligand
Asn175A metal ligand
Asp229A metal ligand
Asp229A covalently attached
Asp229A nucleophile

Chemical Components

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

Step 2. Glu21 activates a water molecule for nucleophilic attack on Asp229. This causes the bond between Asp229 and the phosphate to be broken.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu21A metal ligand
Asn120A metal ligand
Asn175A metal ligand
Asp229A metal ligand
Glu21A increase nucleophilicity
Glu21A proton acceptor
Asp229A electrophile, electrofuge

Chemical Components

overall product formed, intermediate terminated, proton transfer, ingold: bimolecular nucleophilic substitution
Download: Image, Marvin File

Step 1. Asp229 performs a nucleophilic attack on the phosphate causing the F-P bond to be cleaved and an intermediate to be formed.

Step 2. Glu21 activates a water molecule for nucleophilic attack on Asp229. This causes the bond between Asp229 and the phosphate to be broken.

Products.

Introduction

More recent proposals suggest the catalytic base involved in activating the nucleophilic water is Asp229 rather than the histidine for the one step mechanism. The activated water water then causes a single step substitution reaction similarly to the first proposal.

Catalytic Residues Roles

UniProt PDB* (1e1a)
Asp229 Asp229A Activates water for nucleophilic attack. increase nucleophilicity, metal ligand, proton acceptor
Glu21, Asn120, Asn175 Glu21A, Asn120A, Asn175A Metal binding residues metal ligand
*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 substitution, overall product formed, overall reactant used

References

  1. Blum MM et al. (2006), J Am Chem Soc, 128, 12750-12757. Binding of a designed substrate analogue to diisopropyl fluorophosphatase: implications for the phosphotriesterase mechanism. DOI:10.1021/ja061887n. PMID:17002369.
  2. Purg M et al. (2017), J Am Chem Soc, 139, 17533-17546. Similar Active Sites and Mechanisms Do Not Lead to Cross-Promiscuity in Organophosphate Hydrolysis: Implications for Biotherapeutic Engineering. DOI:10.1021/jacs.7b09384. PMID:29113434.

Step 1. Asp229 activates a water molecule for nucleophilic attack on the phosphate causing the F-P bond to be cleaved in a substitution reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu21A metal ligand
Asn120A metal ligand
Asn175A metal ligand
Asp229A metal ligand
Asp229A increase nucleophilicity
Asp229A proton acceptor

Chemical Components

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

Step 1. Asp229 activates a water molecule for nucleophilic attack on the phosphate causing the F-P bond to be cleaved in a substitution reaction.

Products.

Introduction

The mechanism of diisopropylfluorophosphatase relies on an extensive hydrogen-bonding network of electrostatic interactions within the active site tunnel. The substrate binds the calcium ion which increases the partial positive charge on the phosphorous atom to facilitate nucleophilic attack by the hydrolytic water molecule. The water is itself activated by hydrophobic interactions, particularly from His 287 which can also activate the water molecule for nucleophilic attack by proton abstraction acting as a general base catalyst. His 287 is itself activated by Glu 37. Cleavage of the bond follows via a trigonal bipyramidal transition state, stabilised by calcium and the hydrophobic nature of the pocket and the hydrolysed substrate detaches with loss of the fluoride ion.

Catalytic Residues Roles

UniProt PDB* (1e1a)
His287 His287A Activates water for nucleophilic attack by acting as a general base catalyst. increase nucleophilicity, proton acceptor
Glu37 Glu37A Activates His 287. increase basicity, 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, overall product formed, overall reactant used, bimolecular nucleophilic substitution

References

  1. Hartleib J et al. (2001), Biochim Biophys Acta, 1546, 312-324. Insights into the reaction mechanism of the diisopropyl fluorophosphatase from Loligo vulgaris by means of kinetic studies, chemical modification and site-directed mutagenesis. DOI:10.1016/s0167-4838(01)00153-4. PMID:11295437.
  2. Katsemi V et al. (2005), Biochemistry, 44, 9022-9033. Mutational and Structural Studies of the Diisopropylfluorophosphatase fromLoligo vulgarisShed New Light on the Catalytic Mechanism of the Enzyme†. DOI:10.1021/bi0500675. PMID:15966726.
  3. Koepke J et al. (2003), Acta Crystallogr D Biol Crystallogr, 59, 1744-1754. Statistical analysis of crystallographic data obtained from squid ganglion DFPase at 0.85 Å resolution. DOI:10.1107/s0907444903016135. PMID:14501113.
  4. Scharff EI et al. (2001), Structure, 9, 493-502. Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris. DOI:10.1016/s0969-2126(01)00610-4. PMID:11435114.
  5. Hartleib J et al. (2001), Biochem J, 353, 579-589. Role of calcium ions in the structure and function of thedi-isopropylfluorophosphatase from Loligo vulgaris. DOI:10.1042/bj3530579. PMID:11171055.

Step 1. His287 activates a water molecule for nucleophilic attack on the phosphate causing the F-P bond to be cleaved in a substitution reaction. His287 is itself activated by Glu37.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu21A metal ligand
Asn120A metal ligand
Asn175A metal ligand
Asp229A metal ligand
Glu37A electrostatic stabiliser
Glu37A increase basicity
His287A increase nucleophilicity, proton acceptor

Chemical Components

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

Step 1. His287 activates a water molecule for nucleophilic attack on the phosphate causing the F-P bond to be cleaved in a substitution reaction. His287 is itself activated by Glu37.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, James Willey