Fatty acid amide hydrolase

 

Mammalian fatty acid amide hydrolase (FAAH) degrades fatty acid primary amides and ethanolamides such as anandamide and oleamide. Anandamide binds and activates cannabinoid and vanilloid receptors exerting an analgesic and cannabinoid effects while oleamide has been found to be sleep inducing. Rat FAAH is an integral membrane protein. The wild type protein hydrolyses esters and amides at the same rate but the Lys142Ala mutant hydrolyses esters more rapidly than amides.

 

Reference Protein and Structure

Sequence
P97612 UniProt (3.1.1.-, 3.5.1.99) IPR030560 (Sequence Homologues) (PDB Homologues)
Biological species
Rattus norvegicus (Norway rat) Uniprot
PDB
1mt5 - CRYSTAL STRUCTURE OF FATTY ACID AMIDE HYDROLASE (2.8 Å) PDBe PDBsum 1mt5
Catalytic CATH Domains
3.90.1300.10 CATHdb (see all for 1mt5)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.99)

water
CHEBI:15377ChEBI
+
anandamide
CHEBI:2700ChEBI
→
arachidonate
CHEBI:32395ChEBI
+
ethanolaminium(1+)
CHEBI:57603ChEBI
Alternative enzyme names: FAAH, Oleamide hydrolase, Anandamide amidohydrolase,

Enzyme Mechanism

Introduction

FAAH has a catalytic triad consisting of Ser217, Ser241 and Lys142. Initially Lys142 deprotonates Ser217. Ser241 acts as a nucleophile, attacking the carbonyl carbon of the substrate. The tetrahedral intermediate is formed in a concerted process with the deprotonation of Ser241 by the alkoxide of Ser217. The negative charge on the tetrahedral intermediate is stabilised by strong hydrogen bonds with the backbone NH groups of Ile238, Gly239, Gly240 and Ser241 which form an oxyanion hole. The acylation of the enzyme is the rate limiting step. Deacylation occurs with a water molecule from the solvent acting as the nucleophile. There are four water molecules near the active site. Water-bridging proton transfer is thought to be necessary for the hydrolysis of the acyl intermediate.

Catalytic Residues Roles

UniProt PDB* (1mt5)
Ile238 (main-N), Gly239 (main-N), Gly240 (main-N) Ile238(202)A (main-N), Gly239(203)A (main-N), Gly240(204)A (main-N) Forms part of the oxyanion hole. electrostatic stabiliser
Lys142 Lys142(106)A Lys142 initially acts as a base, deprotonating Ser217 and later as an a acid, protonating Ser217. proton acceptor, proton donor
Ser217 Ser217(181)A Ser217 is deprotonated by Lys142 and then acts as a general base for the deprotonation of Ser241 which is the nucleophile. Then, it acts a general acid by protonating the leaving group of the substrate. Finally, the alkoxide is restored to an OH group by obtaining a proton from Lys142. proton relay, proton acceptor, proton donor
Ser218 Ser218(182)A The exact function of Ser218 is uncertain but it may be hydrogen bonding to Ser217 and stabilising the negative charge on it.
S218A mutants have a decreased kcat with no significant change in Km.		 electrostatic stabiliser
Ser241 Ser241(205)A Ser241 is the nucleophile that attacks the carbonyl carbon of the substrate. The backbone NH group of Ser241 contributes to the oxyanion hole. nucleofuge, nucleophile, 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

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

References

  1. Tubert-Brohman I et al. (2006), J Am Chem Soc, 128, 16904-16913. Elucidation of Hydrolysis Mechanisms for Fatty Acid Amide Hydrolase and Its Lys142Ala Variant via QM/MM Simulations. DOI:10.1021/ja065863s. PMID:17177441.
  2. Chudyk EI et al. (2013), J Phys Chem B, 117, 6656-6666. Nonempirical Energetic Analysis of Reactivity and Covalent Inhibition of Fatty Acid Amide Hydrolase. DOI:https://doi.org/10.1021/jp401834v.
  3. Capoferri L et al. (2011), J Mol Model, 17, 2375-2383. Application of a SCC-DFTB QM/MM approach to the investigation of the catalytic mechanism of fatty acid amide hydrolase. DOI:10.1007/s00894-011-0981-z. PMID:21365225.
  4. Lodola A et al. (2005), Chem Commun (Camb), 4399-. QM/MM modelling of oleamide hydrolysis in fatty acid amide hydrolase (FAAH) reveals a new mechanism of nucleophile activation. DOI:10.1039/b503887a. PMID:16136230.
  5. McKinney MK et al. (2003), J Biol Chem, 278, 37393-37399. Evidence for Distinct Roles in Catalysis for Residues of the Serine-Serine-Lysine Catalytic Triad of Fatty Acid Amide Hydrolase. DOI:10.1074/jbc.m303922200. PMID:12734197.
  6. Patricelli MP et al. (1999), Biochemistry, 38, 9804-9812. Chemical and Mutagenic Investigations of Fatty Acid Amide Hydrolase:  Evidence for a Family of Serine Hydrolases with Distinct Catalytic Properties†. DOI:10.1021/bi990637z. PMID:10433686.

Step 1. Ser217 is deprotonated by Lys142

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Catalytic Residues Roles

Residue Roles
Ser218(182)A electrostatic stabiliser
Ile238(202)A (main-N) electrostatic stabiliser
Gly240(204)A (main-N) electrostatic stabiliser
Gly239(203)A (main-N) electrostatic stabiliser
Lys142(106)A proton acceptor
Ser217(181)A proton donor

Chemical Components

proton transfer

Step 2. The tetrahedral intermediate formed by nucleophilic attack by Ser241 concerted with proton transfer from Ser241 to Ser217.

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Catalytic Residues Roles

Residue Roles
Ser218(182)A electrostatic stabiliser
Ile238(202)A (main-N) electrostatic stabiliser
Gly239(203)A (main-N) electrostatic stabiliser
Gly240(204)A (main-N) electrostatic stabiliser
Ser217(181)A proton acceptor
Ser241(205)A proton donor, nucleophile

Chemical Components

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

Step 3. The nitrogen of the amide bond accepts a proton from Ser217 which initiates an elimination from the oxyanion resulting in the cleavage of the amide bond.

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Catalytic Residues Roles

Residue Roles
Ser218(182)A electrostatic stabiliser
Ile238(202)A (main-N) electrostatic stabiliser
Gly239(203)A (main-N) electrostatic stabiliser
Gly240(204)A (main-N) electrostatic stabiliser
Ser217(181)A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, heterolysis, intermediate collapse, overall product formed

Step 4. Ser217 abstracts a proton from a water which activates it to nucleophilically attack the carbon of the carbonyl group.

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Catalytic Residues Roles

Residue Roles
Ser218(182)A electrostatic stabiliser
Ile238(202)A (main-N) electrostatic stabiliser
Gly239(203)A (main-N) electrostatic stabiliser
Gly240(204)A (main-N) electrostatic stabiliser
Ser217(181)A proton acceptor

Chemical Components

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

Step 5. The oxyanion initiates an elimination which results in the cleavage of the acyl-enzyme intermediate releasing Ser241 which is then protonated by Ser217. Ser 217 then is protonated by Lys142 which returns the active site to its native state.

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Catalytic Residues Roles

Residue Roles
Ser218(182)A electrostatic stabiliser
Ile238(202)A (main-N) electrostatic stabiliser
Gly239(203)A (main-N) electrostatic stabiliser
Gly240(204)A (main-N) electrostatic stabiliser
Ser241(205)A proton acceptor
Ser217(181)A proton acceptor, proton donor
Lys142(106)A proton donor
Ser217(181)A proton relay
Ser241(205)A nucleofuge

Chemical Components

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

Step 1. Ser217 is deprotonated by Lys142

Step 2. The tetrahedral intermediate formed by nucleophilic attack by Ser241 concerted with proton transfer from Ser241 to Ser217.

Step 3. The nitrogen of the amide bond accepts a proton from Ser217 which initiates an elimination from the oxyanion resulting in the cleavage of the amide bond.

Step 4. Ser217 abstracts a proton from a water which activates it to nucleophilically attack the carbon of the carbonyl group.

Step 5. The oxyanion initiates an elimination which results in the cleavage of the acyl-enzyme intermediate releasing Ser241 which is then protonated by Ser217. Ser 217 then is protonated by Lys142 which returns the active site to its native state.

Products.

Contributors

Gemma L. Holliday, Charity Hornby, Trung Nguyen, Marko Babić