Anthrax lethal factor endopeptidase
Lethal Factor is critical in anthrax pathogenesis, by acting as a specific protease cleaving members of the MAPKK (mitogen-activated protein kinase kinase) family at their amino termini with its commonly cleaving at the sequence BBBBxHxH, where B denotes a basic amino acid Arg or Lys and H denotes a hydrophobic amino acid. This results in signalling pathway inhibition. The catalytic domain of this enzyme is distantly related to the zinc metalloprotease family.
The implications of finding targets for inhibition of anthrax lethal factor are obvious and so the catalytic details of this enzyme should be highly important especially due to its use in biological warfare and when inhaled is fatal.
Reference Protein and Structure
- Sequence
-
P15917
(3.4.24.83)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Bacillus anthracis (Bacteria)
- PDB
-
1pwv
- Crystal structure of Anthrax Lethal Factor wild-type protein complexed with an optimised peptide substrate.
(2.85 Å)
- Catalytic CATH Domains
-
3.40.390.10
(see all for 1pwv)
- Cofactors
- Zinc(2+) (1)
Enzyme Mechanism
Introduction
The active site is centred around a structural zinc cation coordinated by a strictly conserved HExxH+E motif. The zinc ion is directly coordinated by the two histidines of the motif (His719 and His723) , by Glu 735 and by a water molecule. Glu 687 acts as a general base to activate the water molecule for nucleophilic attack by deprotonation and also by further polarization by Zn2+. The activated water can then attack the carbonyl of the peptide bond and form the tetrahedral intermediate which is stabilised by hydrogen bonding with Tyr728 and coordination to Zn2+ which together serve as an oxyanion hole. The oxyanion can the initiate an elimination to start the collapse of the intermediate and the cleavage of the peptide bond. 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.
Catalytic Residues Roles
| UniProt | PDB* (1pwv) | ||
| His723, His719, Glu768 | His690A, His686A, Glu735A | Form the Zinc binding site of the enzyme | metal ligand |
| Glu720 | Glu687A | Acts as a general base to activate the zinc-bound water during catalysis | proton acceptor, proton donor |
| Tyr761 | Tyr728A | Stabilises the transition state by forming a hydrogen bond with the oxygen of the carbonyl of the intermediate | electrostatic stabiliser |
Chemical Components
proton transfer, bimolecular nucleophilic addition, coordination, coordination to a metal ion, cofactor used, intermediate formation, overall reactant used, 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, overall product formedReferences
- Tonello F et al. (2004), Biochem Biophys Res Commun, 313, 496-502. Tyrosine-728 and glutamic acid-735 are essential for the metalloproteolytic activity of the lethal factor of Bacillus anthracis. DOI:10.1016/j.bbrc.2003.11.134. PMID:14697216.
- Smith CR et al. (2011), Theor Chem Acc, 128, 83-90. Quantum mechanical/molecular mechanical study of anthrax lethal factor catalysis. DOI:10.1007/s00214-010-0765-z.
- Kim J et al. (2004), Biochem Biophys Res Commun, 313, 217-222. Implication of pH in the catalytic properties of anthrax lethal factor. DOI:10.1016/j.bbrc.2003.11.110. PMID:14672720.
- Pannifer AD et al. (2001), Nature, 414, 229-233. Crystal structure of the anthrax lethal factor. DOI:10.1038/n35101998. PMID:11700563.
- Hammond SE et al. (1998), Infect Immun, 66, 2374-2378. Lethal factor active-site mutations affect catalytic activity in vitro. PMID:9573135.
Step 1. Glu687 deprotonates water which activates water to attack the carbonyl of the peptide bond by nucleophilc addition. This results in the coordination of the oxyanion to Zinc. Tyr728 and Zn interaction with the negatively charged oxygen of the carbonyl serves as an oxyanion hole which stabilises the transition state.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| His686A | metal ligand |
| His690A | metal ligand |
| Glu735A | metal ligand |
| Tyr728A | electrostatic stabiliser |
| Glu687A | proton acceptor |
Chemical Components
proton transfer, ingold: bimolecular nucleophilic addition, coordination, coordination to a metal ion, cofactor used, intermediate formation, overall reactant used, rate-determining step
Step 2. The oxyanion initaites an elimination which results in the cleavage of the peptide bond. The N-terminal product accepts a proton from Glu687.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| His686A | metal ligand |
| His690A | metal ligand |
| Glu735A | metal ligand |
| Tyr728A | electrostatic stabiliser |
| Glu687A | proton donor |
Chemical Components
ingold: unimolecular elimination by the conjugate base, heterolysis, proton transfer, decoordination from a metal ion, intermediate collapse, native state of cofactor regenerated, native state of enzyme regenerated
Step 3. The N-terminal product deprotonates the C-terminal product to produce the kinetically favourable products.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| His686A | metal ligand |
| His690A | metal ligand |
| Glu735A | metal ligand |
| Tyr728A | electrostatic stabiliser |
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