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CSA entry for 1gcb
Original Entry
Title:
Dna-binding protein
Compound:
Gal6 hg (emts) derivative
Mutant:
No
UniProt/Swiss-Prot:
Q01532-BLH1_YEAST
EC Class:
3.4.22.40
Other CSA Entries:
Overview of all sites for 1gcb
Homologues of 1gcb
Entries for UniProt/Swiss-Prot: Q01532
Entries for EC: 3.4.22.40
Other Databases:
PDB entry: 1gcb
PDBsum entry: 1gcb
UniProt/Swiss-Prot: Q01532
IntEnz entry: 3.4.22.40
Literature Report:
Introduction:
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.
Mechanism:
Bleomycin hydrolase is a cysteine protease. The resting state contains a thioloate-imidazolium pair (Cys 73 and His 369) that is stabilised by Asn 392. Cys 73 attacks the peptide carbonyl to form an acyl enzyme intermediate, with His 369 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 67 and the backbone NH of Cys 73. The thioester intermediate is then hydrolysed by a water molecule that is deprotonated by His 369 acting as a general base.
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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
GLNA 67 96Sidechain
ElectrostaticTransition state
Side chain forms part of the oxyanion hole which functions to stabilise the negative charge that accumulates on the carbonyl oxygen of the substrate during the reaction.
Evidence from paper Evidence concerns Evidence type
PubMed ID 7638617 Current protein Conservation of residue
PubMed ID 7638617 Current protein Structural similarity to homologue of known mechanism
PubMed ID 9546396 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 73 102Sidechain, Backbone amide
NucleophileSubstrate
ElectrostaticTransition state
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.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9546396 Current protein Structural similarity to homologue of known mechanism
PubMed ID 7638617 Current protein Conservation of residue
PubMed ID 9546396 Current protein Mutagenesis of residue
PubMed ID 9546396 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
HISA 369 398Sidechain
Acid/baseWater
Acid/baseResidue
Acid/baseSubstrate
Deprotonates Cys 73 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.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9546396 Current protein Structural similarity to homologue of known mechanism
PubMed ID 7638617 Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASNA 392 421Sidechain
ElectrostaticResidue
Stabilises the thiolate-imidazolium pair by interacting with His 369.
Evidence from paper Evidence concerns Evidence type
PubMed ID 7638617 Current protein Structural similarity to homologue of known mechanism
PubMed ID 7638617 Current protein Conservation of residue
References:
1
The unusual active site of Gal6/bleomycin hydrolase can act as a carboxypeptidase, aminopeptidase, and peptide ligase.
W. Zheng and S. A. Johnston and L. Joshua-Tor
Cell 93, (1) 103-9, (1998).
9546396
2
Crystal structure of a conserved protease that binds DNA: the bleomycin hydrolase, Gal6.
L. Joshua-Tor and H. E. Xu and S. A. Johnston and D. C. Rees
Science 269, (5226) 945-50, (1995).
7638617
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