Lysophospholipase
Escherichia coli thioesterase I (TAP) is a multifunctional enzyme possessing the activities of thioesterase, esterase, arylesterase, protease and lysophospholipase. This enzyme is also known as protease I and lysophospholipase L1, and is responsible for the degradation of thioester, amide and ester bonds. In particular, TAP catalyses the hydrolytic cleavage/deacylation of fatty acyl-CoA thioesters from fatty acyl-acyl carrier proteins, especially those with long acyl groups. TAP has stereoselectivity for amino acid derivative substrates, and hence is useful for the kinetic resolution of racemic mixtures of industrial chemicals.
Reference Protein and Structure
- Sequence
-
P0ADA1
(3.1.1.2, 3.1.1.5, 3.1.2.2, 3.1.2.14, 3.4.21.-)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Escherichia coli K-12 (Bacteria)
- PDB
-
1j00
- E. coli Thioesterase I/Protease I/Lysophospholipase L1 in complexed with diethyl phosphono moiety
(2.0 Å)
- Catalytic CATH Domains
-
3.40.50.1110
(see all for 1j00)
Enzyme Reaction (EC:3.1.1.5)
Enzyme Mechanism
Introduction
The catalytic triad (Ser 10 - Asp 154 - His 157) and the oxyanion hole (Ser 10 - Gly 44 - Asn 73) are involved in the deacylation catalytic mechanism. The His 157 imidazole ring becomes deprotonated, and its Ne2 lone pair becomes available for hydrogen bonding to the Ser 10 hydroxyl. Asp 154 is positioned close to the entrance of the substrate tunnel and the O-delta-2 atom attracts the proton of N-delta-1-His 157. This draws the His backwards to provide a larger space during substrate binding. Ser 10 OH acts as a nucleophile towards the carbonyl C of the ester substrate, forming an oxyanion intermediate. The oxyanion is stabilised by the oxyanion hole, formed from Ser 10, Gly 44 and Asn 73. The amide protons of ser 10 and Gly 44 acts as proton donors, stabilising the tetrahedral adduct. The thioester bond is cleaved, resulting in product formation and release.
Catalytic Residues Roles
| UniProt | PDB* (1j00) | ||
| Ser36 | Ser10A | Ser 10 is the catalytic nucleophile. Hydrogen bonding to His 157 activates the serine towards nucleophilic attack of the carbonyl C in the substrate ester. Ser 10 is also involved in formation of the oxyanion hole, which stabilises the oxyanion intermediate. The Ser 10 amide proton is devoted to hydrogen bonding with the oxyanion. | covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, electrostatic stabiliser |
| Asn99 | Asn73A | Asn 73 stabilises the oxyanion by contributing to the formation of the oxyanion hole. The asparagine residue offers its gamma-H for hydrogen bonding to the oxyanion. Asn 73 is also involved in the loop 75-80 switch-move motion, essential for accommodating longer acyl-chain-length substrates. | electrostatic stabiliser |
| Gly70 (main-N) | Gly44A (main-N) | Gly 44 forms part of the oxyanion hole, to stabilise the oxyanion, and additionally plays a role in acyl-enzyme intermediate transformation. The Gly 44 amide proton is devoted to hydrogen bonding to the oxyanion, thereby acting as a proton donor. The minimally restrained movement of Gly 44, due to its small H side chain, is important in conformational fine-tuning during oxyanion formation. | electrostatic stabiliser |
| Asp180 | Asp154A | Asp 154 forms part of the catalytic triad. Asp 154 is positioned close to the entrance of the substrate tunnel and the O-delta-2 atom attracts the proton of N-delta-1-His 157. This draws the His backwards to provide a larger space during substrate binding. | increase basicity, electrostatic stabiliser |
| His183 | His157A | The His 157 imidazole ring becomes deprotonated, and its Ne2 lone pair becomes available for hydrogen bonding to the Ser 10 hydroxyl (side chain H-bonding). Thus, His 157 is responsible for the activation of the catalytic serine. Hydrogen bonding between the amide-N of His 157 and the O-gamma-1 of Asp 154 strengthens co-ordination of the catalytic triad and acts as a connector between Ser 10 and Asp 154. His 157 also participates in hydrogen bonding of the acyl-enzyme intermediate. | proton acceptor, electrostatic stabiliser, proton donor |
Chemical Components
bimolecular nucleophilic addition, intermediate formation, overall reactant used, proton transfer, overall product formed, unimolecular elimination by the conjugate base, intermediate terminated, native state of enzyme regeneratedReferences
- Lee LC et al. (2006), Biochem J, 397, 69-76. Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity ofEscherichia colithioesterase I/protease I/phospholipase L1. DOI:10.1042/bj20051645. PMID:16515533.
- Lo YC et al. (2003), J Mol Biol, 330, 539-551. Crystal Structure of Escherichia coli Thioesterase I/Protease I/Lysophospholipase L1: Consensus Sequence Blocks Constitute the Catalytic Center of SGNH-hydrolases through a Conserved Hydrogen Bond Network. DOI:10.1016/s0022-2836(03)00637-5. PMID:12842470.
- Holtkamp K et al. (2003), J Psychiatr Res, 37, 165-169. The effect of therapeutically induced weight gain on plasma leptin levels in patients with anorexia nervosa. DOI:10.1016/s0022-3956(02)00100-0. PMID:12842170.
Step 1. His157 acts as a general base activating the Ser10 hydroxyl group for nucleophilic attack on the carbonyl carbon of the ester bond. The third component of this catalytic triad- Asp154 acts to increase the basicity of the histidine. The oxyanion intermediate formed is stabilized by the amide group of Gly44 and the side chain of Asn73.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Gly44A (main-N) | electrostatic stabiliser |
| Asn73A | electrostatic stabiliser |
| Asp154A | electrostatic stabiliser |
| His157A | electrostatic stabiliser |
| Asp154A | increase basicity |
| Ser10A | covalently attached, electrostatic stabiliser |
| His157A | proton acceptor |
| Ser10A | proton donor, nucleophile |
Chemical Components
ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used, proton transfer
Step 2. The tetrahedral intermediate collapses and choline alfoscerate is eliminated.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Gly44A (main-N) | electrostatic stabiliser |
| Asn73A | electrostatic stabiliser |
| Asp154A | electrostatic stabiliser |
| His157A | electrostatic stabiliser |
| Ser10A | covalently attached, electrostatic stabiliser |
| His157A | proton donor |
Chemical Components
overall product formed, proton transfer, ingold: unimolecular elimination by the conjugate base
Step 3. His157 activates water for nucleophilic attack and another oxyanion intermediate is formed.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Ser10A | covalently attached |
| Asp154A | increase basicity |
| Gly44A (main-N) | electrostatic stabiliser |
| Asn73A | electrostatic stabiliser |
| Asp154A | electrostatic stabiliser |
| His157A | electrostatic stabiliser |
| Ser10A | electrostatic stabiliser |
| His157A | proton acceptor |
Chemical Components
ingold: bimolecular nucleophilic addition, proton transfer
Step 4. The tetrahedral intermediate collapses and Ser10 is eliminated.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Gly44A (main-N) | electrostatic stabiliser |
| Asn73A | electrostatic stabiliser |
| Asp154A | electrostatic stabiliser |
| His157A | electrostatic stabiliser |
| Ser10A | electrostatic stabiliser, proton acceptor |
| His157A | proton donor |
| Ser10A | nucleofuge |
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