Ubiquitin carboxyl-terminal hydrolase family 1, cysteine active-site (UCH_AS)
Type: Conserved_site

This entry represents the Ubiquitin carboxyl-terminal hydrolase family 1 cysteine active-site.

Ubiquitin carboxyl-terminal hydrolases (EC 3.4.19.12) (UCH) (deubiquitinating enzymes) are thiol proteases that recognise and hydrolyse the peptide bond at the C-terminal glycine of ubiquitin. These enzymes are involved in the processing of poly-ubiquitin precursors as well as that of ubiquinated proteins.

There are two distinct families of UCH. The first class is represented by:

Mammalian isozymes L1 and L3.

Yeast YUH1.

Drosophila Uch.

One of the active site residues of class-I UCH is a cysteine and the region surrounding this residue is highly conserved.

This group of cysteine peptidases belong to the MEROPS peptidase family C12 (ubiquitin C-terminal hydrolase family, clan CA). Families within the CA clan are loosely termed papain-like as protein fold of the peptidase unit resembles that of papain, the type example for clan CA. The type example is the human ubiquitin C-terminal hydrolase UCH-L1.

Ubiquitin is highly conserved, commonly found conjugated to proteins in eukaryotic cells, where it may act as a marker for rapid degradation, or it may have a chaperone function in protein assembly . The ubiquitin is released by cleavage from the bound protein by a protease . A number of deubiquitinising proteases are known: all are activated by thiol compounds , and inhibited by thiol-blocking agents and ubiquitin aldehyde , and as such have the properties of cysteine proteases .

The deubiquitinsing proteases can be split into 2 size ranges: 20-30kDa (this entry) and 100-200kDa (

) . The 20-30kDa group includes the yeast yuh1, which is known to be active only against small ubiquitin conjugates, being inactive against conjugated beta-galactosidase . A mammalian homologue, UCH (ubiquitin conjugate hydrolase), is one of the most abundant proteins in the brain . Only one conserved cysteine can be identified, along with two conserved histidines. The spacing between the cysteine and the second histidine is thought to be more representative of the cysteine/histidine spacing of a cysteine protease catalytic dyad .

A cysteine peptidase is a proteolytic enzyme that hydrolyses a peptide bond using the thiol group of a cysteine residue as a nucleophile. Hydrolysis involves usually a catalytic triad consisting of the thiol group of the cysteine, the imidazolium ring of a histidine, and a third residue, usually asparagine or aspartic acid, to orientate and activate the imidazolium ring. In only one family of cysteine peptidases, is the role of the general base assigned to a residue other than a histidine: in peptidases from family C89 (acid ceramidase) an arginine is the general base. Cysteine peptidases can be grouped into fourteen different clans, with members of each clan possessing a tertiary fold unique to the clan. Four clans of cysteine peptidases share structural similarities with serine and threonine peptidases and asparagine lyases. From sequence similarities, cysteine peptidases can be clustered into over 80 different families . Clans CF, CM, CN, CO, CP and PD contain only one family.

Cysteine peptidases are often active at acidic pH and are therefore confined to acidic environments, such as the animal lysosome or plant vacuole. Cysteine peptidases can be endopeptidases, aminopeptidases, carboxypeptidases, dipeptidyl-peptidases or omega-peptidases. They are inhibited by thiol chelators such as iodoacetate, iodoacetic acid,
N
-ethylmaleimide or
p
-chloromercuribenzoate.

Clan CA includes proteins with a papain-like fold. There is a catalytic triad which occurs in the order: Cys/His/Asn (or Asp). A fourth residue, usually Gln, is important for stabilising the acyl intermediate that forms during catalysis, and this precedes the active site Cys. The fold consists of two subdomains with the active site between them. One subdomain consists of a bundle of helices, with the catalytic Cys at the end of one of them, and the other subdomain is a β-barrel with the active site His and Asn (or Asp). There are over thirty families in the clan, and tertiary structures have been solved for members of most of these. Peptidases in clan CA are usually sensitive to the small molecule inhibitor E64, which is ineffective against peptidases from other clans of cysteine peptidases .

Clan CD includes proteins with a caspase-like fold. Proteins in the clan have an α/β/α sandwich structure. There is a catalytic dyad which occurs in the order His/Cys. The active site His occurs in a His-Gly motif and the active site Cys occurs in an Ala-Cys motif; both motifs are preceded by a block of hydrophobic residues . Specificity is predominantly directed towards residues that occupy the S1 binding pocket, so that caspases cleave aspartyl bonds, legumains cleave asparaginyl bonds, and gingipains cleave lysyl or arginyl bonds.

Clan CE includes proteins with an adenain-like fold. The fold consists of two subdomains with the active site between them. One domain is a bundle of helices, and the other a β-barrel. The subdomains are in the opposite order to those found in peptidases from clan CA, and this is reflected in the order of active site residues: His/Asn/Gln/Cys. This has prompted speculation that proteins in clans CA and CE are related, and that members of one clan are derived from a circular permutation of the structure of the other.

Clan CL includes proteins with a sortase B-like fold. Peptidases in the clan hydrolyse and transfer bacterial cell wall peptides. The fold shows a closed β-barrel decorated with helices with the active site at one end of the barrel . The active site consists of a His/Cys catalytic dyad.