Triacylglycerol lipase (EstA)

 

Lipases catalyse the hydrolysis and synthesis of long-chain triglycerols. They have important industrial applications in resolution of racemic mixtures, ester synthesis and transesterification reactions, as well as additives in washing powders.

 

Reference Protein and Structure

Sequence
P37957 UniProt (3.1.1.3) IPR002918 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus subtilis subsp. subtilis str. 168 (Bacteria) Uniprot
PDB
1r4z - Bacillus subtilis lipase A with covalently bound Rc-IPG-phosphonate-inhibitor (1.8 Å) PDBe PDBsum 1r4z
Catalytic CATH Domains
3.40.50.1820 CATHdb (see all for 1r4z)
Click To Show Structure

Enzyme Reaction (EC:3.1.1.3)

triglyceride
CHEBI:17855ChEBI
+
water
CHEBI:15377ChEBI
→
fatty acid anion
CHEBI:28868ChEBI
+
hydron
CHEBI:15378ChEBI
+
diacylglycerol
CHEBI:18900ChEBI
Alternative enzyme names: GA 56, GEH, Meito MY 30, PPL, Takedo 1969-4-9, Tween hydrolase, Tweenase, Tweenesterase, Amano AP, Amano B, Amano CE, Amano CES, Amano P, Amno N-AP, Butyrinase, Cacordase, Capalase L, Glycerol ester hydrolase, Glycerol-ester hydrolase, Heparin releasable hepatic lipase, Hepatic lipase, Hepatic monoacylglycerol acyltransferase, Lipase, Lipazin, Liver lipase, Meito Sangyo OF lipase, Post-heparin plasma protamine-resistant lipase, Salt-resistant post-heparin lipase, Steapsin, Triacetinase, Triacylglycerol ester hydrolase, Tributyrase, Tributyrin esterase, Tributyrinase, Triglyceridase, Triglyceride hydrolase, Triglyceride lipase, Triolein hydrolase, Tween-hydrolyzing esterase,

Enzyme Mechanism

Introduction

His 156 activates Ser 77 through general base catalysis to deprotonate Ser 77 allowing it to make a nucleophilic attack on the ester bond. His 156 donates a proton to the leaving group and then activates a water molecule to allow the hydrolysis of the acyl-enzyme intermediate, again by general base catalysis. Asp 133 alters the pKa of the His 156 to activate it and allow it to act as an effective base in the reaction. The Ile 12 and Met 78 backbone amides form the oxyanion hole to stabilise the transition state.

Catalytic Residues Roles

UniProt PDB* (1r4z)
Ile43 (main-N) Ile12A (main-N) Forms the oxyanion hole to stabilise the transition state. electrostatic stabiliser
Ser108 Ser77A Acts as the active site nucleophile in attack of the substrate, to form the acyl-enzyme intermediate. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor
Asp164 Asp133A Alters the pKa of His 156 to allow it to act as a more efficient general base catalyst. increase basicity, modifies pKa, electrostatic stabiliser
His187 His156A Acts as a general acid/base catalyst to activate Ser 77 and water for nucleophilic attack by proton abstraction, and to facilitate collapse of intermediates and formation of products by proton donation. proton acceptor, proton donor
Met109 (main-N) Met78A (main-N) Stabilises the transition state by formation of the oxyanion hole. 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

bimolecular nucleophilic addition, intermediate formation, overall reactant used, proton transfer, overall product formed, unimolecular elimination by the conjugate base, native state of enzyme regenerated, intermediate terminated

References

  1. van Pouderoyen G et al. (2001), J Mol Biol, 309, 215-226. The crystal structure of Bacillus subtilis lipase: a minimal alpha/beta hydrolase fold enzyme. DOI:10.2210/pdb1i6w/pdb. PMID:11491291.
  2. Kawasaki K et al. (2002), Acta Crystallogr D Biol Crystallogr, 58, 1168-1174. Alternate conformations observed in catalytic serine ofBacillus subtilislipase determined at 1.3 Å resolution. DOI:10.1107/s090744490200714x. PMID:12077437.

Step 1. His156 acts as a general base activating the Ser77 hydroxyl group for nucleophilic attack on the carbonyl carbon of the ester bond. The third component of this catalytic triad- Asp133 acts to increase the basicity of the histidine. The oxyanion intermediate formed is stabilized by the amide groups of Met78 and Ile12.

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Catalytic Residues Roles

Residue Roles
Asp133A modifies pKa, electrostatic stabiliser
Met78A (main-N) electrostatic stabiliser
Ile12A (main-N) electrostatic stabiliser
Asp133A increase basicity
Ser77A covalently attached, proton donor
His156A proton acceptor
Ser77A nucleophile

Chemical Components

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

Step 2. The tetrahedral intermediate collapses and diacylglycerol is eliminated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser77A covalently attached
Ile12A (main-N) electrostatic stabiliser
Met78A (main-N) electrostatic stabiliser
Asp133A electrostatic stabiliser
His156A proton donor

Chemical Components

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

Step 3. His156 activates water for nucleophilic attack and another oxyanion intermediate is formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser77A covalently attached
Asp133A increase basicity, modifies pKa
Ile12A (main-N) electrostatic stabiliser
Met78A (main-N) electrostatic stabiliser
Asp133A electrostatic stabiliser
His156A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 4. The tetrahedral intermediate collapses and Ser77 is eliminated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ile12A (main-N) electrostatic stabiliser
Met78A (main-N) electrostatic stabiliser
Asp133A electrostatic stabiliser
Ser77A proton acceptor
His156A proton donor
Ser77A nucleofuge

Chemical Components

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

Step 1. His156 acts as a general base activating the Ser77 hydroxyl group for nucleophilic attack on the carbonyl carbon of the ester bond. The third component of this catalytic triad- Asp133 acts to increase the basicity of the histidine. The oxyanion intermediate formed is stabilized by the amide groups of Met78 and Ile12.

Step 2. The tetrahedral intermediate collapses and diacylglycerol is eliminated.

Step 3. His156 activates water for nucleophilic attack and another oxyanion intermediate is formed.

Step 4. The tetrahedral intermediate collapses and Ser77 is eliminated.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, James Willey