Thermolysin

 

This entry covers the metalloproteinase family M4, which contains thermolysin (EC3.4.24.27), bacillolysins (EC 3.4.24.28), pseudolysins (EC 3.4.24.26), Vibriolysin (EC3.4.24.25) and several other proteases which do not have EC numbers. Family M4 are the only members of clan MA to have structures within the PDB. The active site lies between the two domains and contains a catalytic zinc ion.

 

Reference Protein and Structure

Sequence
P00800 UniProt (3.4.24.27) IPR023612 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus thermoproteolyticus (Bacteria) Uniprot
PDB
1kei - Thermolysin (substrate-free) (1.6 Å) PDBe PDBsum 1kei
Catalytic CATH Domains
1.10.390.10 CATHdb 3.10.170.10 CATHdb (see all for 1kei)
Cofactors
Zinc(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.4.24.27)

water
CHEBI:15377ChEBI
+
dipeptide zwitterion
CHEBI:90799ChEBI
→
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
+
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
Alternative enzyme names: Bacillus thermoproteolyticus neutral proteinase, TLN, Thermoase, Thermoase Y10,

Enzyme Mechanism

Introduction

His231 is activated by Asp226 to act as a general acid/base. In the first step, His231 deprotonates water, which attacks the peptide bond in a nucleophilic addition, resulting in the coordination of the oxyanion to the zinc cofactor. The oxyanion collapses, eliminating the neutral terminal products. The kinetically favoured products of this reaction are the carboxyanion and the amine cation, thus in a final step the N-terminal amine deprotonates the C-terminal carboxyacid. However, there is no evidence of direct enzyme involvement in this final step [PMID:8652513].

Catalytic Residues Roles

UniProt PDB* (1kei)
Tyr389 Tyr157A Tyr157 stabilises the transition state and orients the catalytic water. hydrogen bond donor, steric role, electrostatic stabiliser
Asp458 Asp226A Asp226 "backs up" the His231 - which acts as the general base - in a manner analogous to the Ser-His-Asp triads. activator, hydrogen bond acceptor, electrostatic stabiliser
His463 His231A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu375 Glu143A Glu143 electrostatically stabilises the cationic intermediates. metal ligand, electrostatic stabiliser
His378, His374, Glu398 His146A, His142A, Glu166A Forms part of the zinc binding site. 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 addition, coordination, overall reactant used, cofactor used, intermediate formation, coordination to a metal ion, rate-determining step, unimolecular elimination by the conjugate base, heterolysis, decoordination from a metal ion, intermediate collapse, native state of cofactor regenerated, native state of enzyme regenerated, intermediate terminated, overall product formed

References

  1. Mock WL et al. (1996), Biochemistry, 35, 7369-7377. Arazoformyl Dipeptide Substrates for Thermolysin. Confirmation of a Reverse Protonation Catalytic Mechanism†. DOI:10.1021/bi952827p. PMID:8652513.
  2. Dedachi K et al. (2009), Chem Phys Lett, 479, 290-295. A combined simulation with ab initio MO and classical vibrational analysis on the specific interactions between thermolysin and dipeptide ligands. DOI:10.1016/j.cplett.2009.08.036.
  3. Blumberger J et al. (2007), J Chem Theory Comput, 3, 1837-1850. Peptide Hydrolysis in Thermolysin:  Ab Initio QM/MM Investigation of the Glu143-Assisted Water Addition Mechanism. DOI:10.1021/ct7000792. PMID:26627626.
  4. Juers DH et al. (2005), Biochemistry, 44, 16524-16528. Structural Analysis of Silanediols as Transition-State-Analogue Inhibitors of the Benchmark Metalloprotease Thermolysin†,‡. DOI:10.1021/bi051346v. PMID:16342943.
  5. Marie-Claire C et al. (1998), FEBS Lett, 438, 215-219. Differences in transition state stabilization between thermolysin (EC 3.4.24.27) and neprilysin (EC 3.4.24.11). DOI:10.1016/s0014-5793(98)01267-8. PMID:9827548.
  6. Rawlings ND et al. (1995), Methods Enzymol, 248, 183-228. [13] Evolutionary families of metallopeptidases. DOI:10.1016/0076-6879(95)48015-3. PMID:7674922.

Step 1. His231 deprotonates water, which attacks the peptide bond in a nucleophilic addition, resulting in the coordination of the oxyanion to the zinc cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His142A metal ligand
His146A metal ligand
Glu166A metal ligand
Glu143A metal ligand
Glu143A electrostatic stabiliser
Tyr157A hydrogen bond donor, steric role, electrostatic stabiliser
His231A hydrogen bond donor, hydrogen bond acceptor
Asp226A hydrogen bond acceptor, activator
His231A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, coordination, overall reactant used, cofactor used, intermediate formation, coordination to a metal ion, rate-determining step

Step 2. The oxyanion initiates an elimination that cleaves the peptide bond. The N-terminal product deprotonates His231.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His142A metal ligand
His146A metal ligand
Glu166A metal ligand
Glu143A metal ligand
Glu143A electrostatic stabiliser
Tyr157A hydrogen bond donor
His231A hydrogen bond donor
Asp226A hydrogen bond acceptor, electrostatic stabiliser
His231A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, heterolysis, decoordination from a metal ion, intermediate collapse, intermediate formation, native state of cofactor regenerated, native state of enzyme regenerated

Step 3. The N-terminal product then deprotonates the C-terminal product to form the kinetically favoured products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His142A metal ligand
His146A metal ligand
Glu166A metal ligand
Glu143A metal ligand
His231A hydrogen bond acceptor, hydrogen bond donor
Asp226A hydrogen bond acceptor

Chemical Components

proton transfer, intermediate terminated, overall product formed
Download: Image, Marvin File

Step 1. His231 deprotonates water, which attacks the peptide bond in a nucleophilic addition, resulting in the coordination of the oxyanion to the zinc cofactor.

Step 2. The oxyanion initiates an elimination that cleaves the peptide bond. The N-terminal product deprotonates His231.

Step 3. The N-terminal product then deprotonates the C-terminal product to form the kinetically favoured products.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Christian Drew, Craig Porter