Biochemistry, Nottingham University

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3.0 Enzymes of the Ubiquitin Pathway

3.1 Ubiquitin-activating enzyme, E1

The first step in ubiquitinylation involves activation of ubiquitin, followed by transfer to a ubiquitin conjugating enzyme. This first step is carried out by the activating enzyme, or E1. Several genes for E1 from different species have now been cloned and sequenced and code for proteins of 115-125kD. Wheat, and possibly other organisms, contain genes for more that one E1 [10]. The role of these different E1s is not understood. E1s contain a conserved cysteine at the active site and a nuleotide binding site. E1 is found in the nucleus and cytosol and associated with the cytoskeleton [15]. E1 is also phosphorylated in mammalian cells by the kinase cdc2 [16], which presumably reflects the role of the ubiquitin in cell cycle control. E1 is also found to form complexes with ubiquitin conjugating enzymes and E3s [17].

3.2 Ubiquitin-conjugating enzymes

While there may be two or even three species of E1 in cells S. cerevisiae is currently known to contain at least 10 genes coding for ubiquitin-conjugating enzymes (E2). Why are there so many E2s? Because some at least of the specificity of ubiquitinylation appears to be dependent on E2s. New E2 enzymes from a growing variety of species are being discovered all the time. All contain a cysteine in a conserved domain of about 16kD (the UBC domain) which contains the UBC motif. This cysteine accepts ubiquitin from E1 to form a thiol ester. Substitution of this cysteine abolishes E2 activity [18]. A suggested motif rich in basic residues is found at the N-terminus of the UBC domain which is may be involved in E1 binding [18].

E2s (UBC) can be classified on the basis of their structure into three classes.

At least one E2s does not fit into any of the three classes. A large (230kDa) E2 in reticulocytes (the precursor cells of erythrocytes) is expressed during erythroid differentiation and may be involved in remodelling events leading to the mature cell. A multiple alignment of three UBCs, 2 Class I (UBC1 of A. thaliana and UBC4 of yeast) and a Class II (UBC2 of yeast) highlights the conserved regions and shows the C-terminal extension of UBC2/RAD6. The 3D crystal structures of UBC1 of A. thaliana and UBC4 of yeast have been solved and can also be viewed here.

3.3 Ubiquitin ligases - "recognins" or E3s

Some E2s appear to require no other protein factor, at least in vitro, to transfer ubiquitin to a suitable target. Others require an additional protein factor, an E3. The E3 binds the target protein, and presumably therefore selects the target. This implies E3 recognize a motif in the substrate protein that targets it for ubiquitinylation. It has been suggested that such motifs be called "degrons", degrons being any motif that targets proteins for degradation (by the ubiquitin-dependent or and other systems). An E3 can therefore be called a "recognin". Unfortunately these useful terms have not caught on and the term "ubiquitin-ligase" is still used for E3s. Until recently the proximal donor of ubiquitin to target proteins was thought to a ubiquitin charged E2 (UBC), and E3s were not thought to be directly involved in the transfer of ubiquitin to the substrate protein. This is indeed the case for one class of E3s. However, recently E3s have been discovered which form thiol esters with ubiquitin and these charged E3s are the proximal donors to targets. These E3s may also act as "recognins". To date then two classes of E3s have been identified:

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