Ubiquitinyl hydrolase 1 (peptidase C19 type)

 

Deubiquinylation is a vital process in eukaryotic cells because it protects proteins from degradation. It involves the hydrolysis of the peptide bond that joins the C terminal residue of ubiquitin to the lysine residue in the protein, and is catalysed by DUPs (Deubiquinylating proteins) such as HAUSP, described here. This family of enzymes is one of the largest in the human genome, reflecting their importance in the cell. For example, the tumour suppressing protein p53 relies on HAUSP action in order to prevent it from being degraded; thus malfunctions in the expression or activity of HAUSP can lead to cancer. HAUSP, along with the rest of the homologous family, is a cysteine protease, and displays structural and sequence homology to other cysteine proteases with different roles in the cell.

 

Reference Protein and Structure

Sequences
P0CG48 UniProt
Q93009 UniProt (3.4.19.12) IPR001394 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1nbf - Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde (2.3 Å) PDBe PDBsum 1nbf
Catalytic CATH Domains
(see all for 1nbf)
Click To Show Structure

Enzyme Reaction (EC:3.4.19.12)

Lys-Gly
CHEBI:73604ChEBI
+
water
CHEBI:15377ChEBI
→
L-lysine
CHEBI:18019ChEBI
+
glycine
CHEBI:15428ChEBI
Alternative enzyme names: Ubiquitin C-terminal hydrolase, Yeast ubiquitin hydrolase, Ubiquitin thiolesterase, Ubiquitin carboxyl-terminal hydrolase,

Enzyme Mechanism

Introduction

The scissile peptide bond targeted by HAUSP is between a Lysine residue's epsilon amino group and the glycine residue that makes up the C terminal of ubiquitin. Cys 223 acts as the nucleophile, with deprotonation by His 464, with Asp 481 acting as a primer. This leads to a tetrahedral intermediate which is stabilised by the amide portion of Cys 223 and the side chain of Asn 281. Protonation of the leaving group by His 464 results in the release of the protein and forms a thioacyl intermediate where the ubiquitin remains bound to the enzyme. Hydrolysis of this intermediate by a water molecule activated by His 464 results in the release of the ubiquitin and the completion of the reaction cycle.

Catalytic Residues Roles

UniProt PDB* (1nbf)
Asn218 Asn218(11)A Acts to stabilise the tetrahedral intermediate through hydrogen bonding between its side chain and the oxyanion that forms in the reaction. Subsequent collapse of the tetrahedral intermediate results in product release. electrostatic stabiliser
Cys223 Cys223(16)A Acts as a nucleophile, attacking the scissile peptide bond to form a tetrahedral intermediate which subsequently collapses and is hydrolysed to the products. Also stabilises the tetrahedral intermediate through its NH group's hydrogen bonding capacity. nucleofuge, nucleophile, proton acceptor, proton donor
His464 His464(257)A Acts as acid base to deprotonate Cys 223 so it can act as a nucleophile. Subsequently protonates the leaving group to allow the thioacyl enzyme intermediate to form. Finally deprotonates the attacking water molecule to allow hydrolysis of the thioacyl enzyme intermediate to release ubiquitin. proton acceptor, proton donor
Asp481 Asp481(274)A Acts to modify the pKa of His 464 to increase its ability to act as an acid base during the reaction. 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. Hu M et al. (2002), Cell, 111, 1041-1054. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. DOI:10.2210/pdb1nbf/pdb. PMID:12507430.
  2. Reverdy C et al. (2012), Chem Biol, 19, 467-477. Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating enzyme. DOI:10.1016/j.chembiol.2012.02.007. PMID:22520753.
  3. Zhang W et al. (2011), Biochemistry, 50, 4775-4785. Contribution of active site residues to substrate hydrolysis by USP2: insights into catalysis by ubiquitin specific proteases. DOI:10.1021/bi101958h. PMID:21542621.

Step 1. His464 deprotonates Cys223 which activates it so that it can attack the carbon of the carbonyl in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn218(11)A electrostatic stabiliser
Asp481(274)A electrostatic stabiliser
Cys223(16)A proton donor
His464(257)A proton acceptor
Cys223(16)A nucleophile

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 scissille bond. The amine of the N-terminal group then accepts a proton from His464.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn218(11)A electrostatic stabiliser
Asp481(274)A electrostatic stabiliser
His464(257)A proton donor

Chemical Components

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

Step 3. His464 abstracts a proton from a water which activates it so it can nucleophilically attack the carbon of the thioester bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn218(11)A electrostatic stabiliser
Asp481(274)A electrostatic stabiliser
His464(257)A 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 Cys223 can now accept a proton from His464 which returns the enzyme to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn218(11)A electrostatic stabiliser
Asp481(274)A electrostatic stabiliser
Cys223(16)A proton acceptor, nucleofuge
His464(257)A 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. His464 deprotonates Cys223 which activates it so that it can attack the carbon of the carbonyl in a nucleophilic addition.

Step 2. The oxyanion initiates an elimination which results in the cleavage of the scissille bond. The amine of the N-terminal group then accepts a proton from His464.

Step 3. His464 abstracts a proton from a water which activates it so it can nucleophilically attack the carbon of the thioester bond in a nucleophilic addition.

Step 4. The oxyanion initiates another elimination which results in the cleavage of the thioester bond. The released Cys223 can now accept a proton from His464 which returns the enzyme to its native state.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Charity Hornby