Bleomycin hydrolase

 

Bleomycin hydrolase is an aminopeptidase identified in eukaryotes ranging from yeast to mammals. It was originally discovered by its ability to hydrolyse the anti-cancer drug bleomycin, thus limiting the usefulness of this drug in the cell. The yeast enzyme (Gal6p) has been shown to negatively regulate the galactose metabolism system and to bind single-stranded DNA and RNA with high affinity; this binding coupled with the hydrolase activity has a role in the detoxification of bleomycin. Generation of the mature form of Gal6p involves the enzyme initially acting as a carboxypeptidase on its own C terminus to convert itself to an aminopeptidase.

 

Reference Protein and Structure

Sequence
Q01532 UniProt (3.4.22.40) IPR004134 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
1gcb - GAL6, YEAST BLEOMYCIN HYDROLASE DNA-BINDING PROTEASE (THIOL) (2.2 Å) PDBe PDBsum 1gcb
Catalytic CATH Domains
3.90.70.10 CATHdb (see all for 1gcb)
Click To Show Structure

Enzyme Reaction (EC:3.4.22.40)

dipeptide zwitterion
CHEBI:90799ChEBI
+
water
CHEBI:15377ChEBI
→
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
Alternative enzyme names: Aminopeptidase C (Lactococcus lactis),

Enzyme Mechanism

Introduction

Bleomycin hydrolase is a cysteine protease. The resting state contains a thioloate-imidazolium pair (Cys 102 and His 398) that is stabilised by Asn 421. Cys 102 attacks the peptide carbonyl to form an acyl enzyme intermediate, with His 398 protonating the departing amine. Negative charge that accumulates on the substrate carbonyl oxygen during the reaction is stabilised by an 'oxyanion-hole' involving the side chain of Gln 96 and the backbone NH of Cys 102. The thioester intermediate is then hydrolysed by a water molecule that is deprotonated by His 398 acting as a general base.

Catalytic Residues Roles

UniProt PDB* (1gcb)
Cys102 Cys73A Attacks the peptide carbonyl to form an acyl-enzyme intermediate. Backbone NH forms part of the oxyanion hole and stabilises the negative charge that accumulates on the carbonyl oxygen in during the reaction. nucleofuge, nucleophile, proton acceptor, proton donor, electrostatic stabiliser
Cys102 (main-N), Gln96 Cys73A (main-N), Gln67A Forms the oxyanion hole which functions to stabilise the negative charge that accumulates on the carbonyl oxygen of the substrate during the reaction. electrostatic stabiliser
His398 His369A Deprotonates Cys 102 to give the thiolate which acts as a nucleophile to attack the peptide bond. Protonates the departing amine group of the substrate. Later acts as a general base to deprotonate a water molecule that attacks the thioester intermediate. proton acceptor, proton donor
Asn421 Asn392A Stabilises the thiolate-imidazolium pair by interacting with His 398. 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

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

References

  1. Joshua-Tor L et al. (1995), Science, 269, 945-950. Crystal structure of a conserved protease that binds DNA: the bleomycin hydrolase, Gal6. DOI:10.1126/science.7638617. PMID:7638617.
  2. O'Farrell PA et al. (2007), Biochem J, 401, 421-428. Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure. DOI:10.1042/BJ20060641. PMID:17007609.
  3. Lefterov IM et al. (2001), Biochem Biophys Res Commun, 283, 994-999. Cysteine 73 in bleomycin hydrolase is critical for amyloid precursor protein processing. DOI:10.1006/bbrc.2001.4860. PMID:11350084.
  4. O’Farrell PA et al. (1999), Structure, 7, 619-627. Crystal structure of human bleomycin hydrolase, a self-compartmentalizing cysteine protease. DOI:10.1016/S0969-2126(99)80083-5.
  5. Zheng W et al. (1998), Cell, 93, 103-109. The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase. DOI:10.1016/s0092-8674(00)81150-2. PMID:9546396.

Step 1. His398 deprotonates Cys102 which activates Cys102 to attack the carbon of the carbonyl in a nucleophilic addition.

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Catalytic Residues Roles

Residue Roles
Asn392A electrostatic stabiliser
Cys73A electrostatic stabiliser
Gln67A electrostatic stabiliser
Cys73A proton donor, nucleophile
His369A proton acceptor

Chemical Components

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

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The amino group of the N-terminal product accepts a proton from His398.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln67A electrostatic stabiliser
Cys73A (main-N) electrostatic stabiliser
Asn392A electrostatic stabiliser
His369A proton donor

Chemical Components

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

Step 3. His398 deprotonates a water which activates it so it can nucleophilically attack the carbon of the thioester bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln67A electrostatic stabiliser
Cys73A (main-N) electrostatic stabiliser
Asn392A electrostatic stabiliser
His369A proton acceptor

Chemical Components

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

Step 4. The oxyanion initiates another elimination which results in the cleavage of the thioester bond. The released Cys102 now accepts a proton from His398 which returns the enzyme back to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln67A electrostatic stabiliser
Cys73A (main-N) electrostatic stabiliser
Asn392A electrostatic stabiliser
Cys73A proton acceptor, nucleofuge
His369A proton donor

Chemical Components

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

Step 1. His398 deprotonates Cys102 which activates Cys102 to attack the carbon of the carbonyl in a nucleophilic addition.

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The amino group of the N-terminal product accepts a proton from His398.

Step 3. His398 deprotonates a water which activates it so it can nucleophilically attack the carbon of the thioester bond.

Step 4. The oxyanion initiates another elimination which results in the cleavage of the thioester bond. The released Cys102 now accepts a proton from His398 which returns the enzyme back to its native state.

Products.

Contributors

Hannah Gilbert, Steven Smith, Gemma L. Holliday, Charity Hornby