Carboxylesterase

 

Carboxylesterases hydrolyse carboxylic ester bonds with relatively broad substrate specificity and are useful for stereospecific synthesis and hydrolysis of esters.

 

Reference Protein and Structure

Sequence
Q0ZPV7 UniProt (3.1.1.1) IPR029058 (Sequence Homologues) (PDB Homologues)
Biological species
Actinidia eriantha (Kiwi) Uniprot
PDB
2o7r - Plant carboxylesterase AeCXE1 from Actinidia eriantha with acyl adduct (1.4 Å) PDBe PDBsum 2o7r
Catalytic CATH Domains
3.40.50.1820 CATHdb (see all for 2o7r)
Click To Show Structure

Enzyme Reaction (EC:3.1.1.1)

water
CHEBI:15377ChEBI
+
propyl acetate
CHEBI:40116ChEBI
→
hydron
CHEBI:15378ChEBI
+
propan-1-ol
CHEBI:28831ChEBI
+
acetate
CHEBI:30089ChEBI
Alternative enzyme names: Alpha-carboxylesterase, B-esterase, Ali-esterase, Butyrate esterase, Butyryl esterase, Carboxyesterase, Carboxyl ester hydrolase, Carboxylate esterase, Carboxylic acid esterase, Carboxylic esterase, Cocaine esterase, Esterase A, Esterase B, Esterase D, Methylbutyrase, Methylbutyrate esterase, Monobutyrase, Nonspecific carboxylesterase, Procaine esterase, Propionyl esterase, Serine esterase, Triacetin esterase, Vitamin A esterase,

Enzyme Mechanism

Introduction

Carboxylesterases use a Ser-His-Asp catalytic triad. Serine, deprotonated by histidine which is stabilised in its charged form by aspartate, carries out a nucleophilic attack on the carbonyl of the substrate. The resulting tetrahedral transition state is stabilised by an oxyanion hole. When it collapses, histidine protonates the leaving group and the result is a covalent enzyme-substrate intermediate. A water molecule, deprotonated by histidine, attacks the covalent intermediate to give another tetrahedral intermediate, which collapses to give the product and the enzyme in its native state.

Catalytic Residues Roles

UniProt PDB* (2o7r)
Gly92 (main-N), Gly93 (main-N), Ala170 (main-N) Gly92A (main-N), Gly93A (main-N), Ala170A (main-N) Oxyanion hole formed by the backbone amine groups of Gly92, Gly93 and Ala170. The ester substrate is stabilised in the active site by three hydrogen bonds from the NH groups of the oxyanion hole. These bonds align the substrate with Ser169 which is essential for initiating the catalytic process. hydrogen bond donor, electrostatic stabiliser
Ser169, His306, Asp276 Ser169A, His306A, Asp276A Ser169, Asp276 and His306 form a conserved catalytic triad of residues. Ser169 performs a nucleophilic attack on the substrate and forms the acyl-enzyme complex. His306 acts as a general acid/base, facilitating the formation and cleavage of the two tetrahedral intermediates. Asp276 is hydrogen-bonded to His306, Gln282 and Met278. hydrogen bond acceptor, hydrogen bond donor, nucleophile, nucleofuge, covalent catalysis
Gln282, Met278 (main-N) Gln282A, Met278A (main-N) Gln282 and Met278 are hydrogen-bonded to Asp276. hydrogen bond 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, overall reactant used, intermediate formation, enzyme-substrate complex formation, intermediate collapse, unimolecular elimination by the conjugate base, overall product formed, heterolysis, enzyme-substrate complex cleavage, native state of enzyme regenerated

References

  1. Aranda J et al. (2014), Biochemistry, 53, 5820-5829. The catalytic mechanism of carboxylesterases: a computational study. DOI:10.1021/bi500934j. PMID:25101647.
  2. Yan M et al. (2021), Comput Theor Chem, 1199, 113198-. Catalytic hydrolysis mechanism of aminocarboxylester substrate by human carboxylesterase 1: A theoretical study on methylphenidate hydrolysis. DOI:10.1016/j.comptc.2021.113198.
  3. Zhan H et al. (2020), Environ Res, 182, 109138-. New insights into the microbial degradation and catalytic mechanism of synthetic pyrethroids. DOI:10.1016/j.envres.2020.109138. PMID:32069744.
  4. Yan M et al. (2019), Molecules, 24,Catalytic Hydrolysis Mechanism of Cocaine by Human Carboxylesterase 1: An Orthoester Intermediate Slows Down the Reaction. DOI:10.3390/molecules24224057. PMID:31717501.
  5. Kim KK et al. (1997), Structure, 5, 1571-1584. Crystal structure of carboxylesterase from Pseudomonas fluorescens, an α/β hydrolase with broad substrate specificity. DOI:10.1016/s0969-2126(97)00306-7. PMID:9438866.

Step 1. The carbonyl group forms three hydrogen bonds to the backbone nitrogen atoms of Gly92 and Gly93 of the oxyanion hole region and the backbone nitrogen of two of them with the backbone nitrogen atoms of Ala170. His306 facilitates the nucleophilic attack of Ser169 to the substrate. The first tetrahedral intermediate forms.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly92A (main-N) electrostatic stabiliser, hydrogen bond donor
Gly93A (main-N) electrostatic stabiliser, hydrogen bond donor
Ala170A (main-N) electrostatic stabiliser, hydrogen bond donor
Ser169A hydrogen bond donor
His306A hydrogen bond acceptor, hydrogen bond donor
Asp276A hydrogen bond acceptor
Gln282A hydrogen bond donor
Met278A (main-N) hydrogen bond donor
His306A increase nucleophilicity, electrostatic stabiliser
Asp276A electrostatic stabiliser
Ser169A nucleophile

Chemical Components

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

Step 2. Collapse of tetrahedral intermediate to an acyl-enzyme complex and release of alcohol product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly92A (main-N) hydrogen bond donor
Gly93A (main-N) hydrogen bond donor
Ala170A (main-N) hydrogen bond donor
Ser169A hydrogen bond donor
His306A hydrogen bond donor
Gln282A hydrogen bond donor
Met278A (main-N) hydrogen bond donor
Asp276A hydrogen bond acceptor
Ser169A covalent catalysis
His306A promote heterolysis

Chemical Components

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

Step 3. A water molecule enters the active site. Nucleophilic attack of a water molecule on the acyl-enzyme complex facilitated by His306. A second tetrahedral intermediate forms.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His306A hydrogen bond acceptor, hydrogen bond donor
Ser169A hydrogen bond acceptor
Asp276A hydrogen bond acceptor
Gly92A (main-N) hydrogen bond donor
Gly93A (main-N) hydrogen bond donor
Ala170A (main-N) hydrogen bond donor
Gln282A hydrogen bond donor
Met278A (main-N) hydrogen bond donor
His306A increase nucleophilicity
Ser169A covalent catalysis

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 4. Ser169 disconnects from the acyl complex and the active site is regenerated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser169A hydrogen bond acceptor
Gly92A (main-N) hydrogen bond donor
Gly93A (main-N) hydrogen bond donor
Ala170A (main-N) hydrogen bond donor
His306A hydrogen bond donor
Gln282A hydrogen bond donor
Met278A (main-N) hydrogen bond donor
Asp276A hydrogen bond acceptor
His306A promote heterolysis
Ser169A nucleofuge

Chemical Components

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

Step 1. The carbonyl group forms three hydrogen bonds to the backbone nitrogen atoms of Gly92 and Gly93 of the oxyanion hole region and the backbone nitrogen of two of them with the backbone nitrogen atoms of Ala170. His306 facilitates the nucleophilic attack of Ser169 to the substrate. The first tetrahedral intermediate forms.

Step 2. Collapse of tetrahedral intermediate to an acyl-enzyme complex and release of alcohol product.

Step 3. A water molecule enters the active site. Nucleophilic attack of a water molecule on the acyl-enzyme complex facilitated by His306. A second tetrahedral intermediate forms.

Step 4. Ser169 disconnects from the acyl complex and the active site is regenerated.

Products.

Contributors

Noa Marson, Marko Babić, Stuart Lucas, Craig Porter, Gemma L. Holliday