The ubiquitination of terminally misfolded proteins is caused by a cascade of enzymatic reactions. The first of these reactions takes place when the ubiquitin-activating enzyme E1 hydrolyses ATP and forms a high-energy thioester linkage between a cysteine residue in its active site and the C-terminus of ubiquitin. The resulting activated ubiquitin is then passed to E2, which is a ubiquitin-conjugating enzyme. Another group of enzymes, more specifically ubiquitin protein ligases called E3, bind to the misfolded protein. Next they align the protein and E2, thus facilitating the attachment of ubiquitin to lysine residues of the misfolded protein. Following successive addition of ubiquitin molecules to lysine residues of the previously attached ubiquitin, a polyubiquitin chain is formed. A polyubiquitinated protein is produced and this is recognized by specific subunits in the 19S capping complexes of the 26S proteasome. Hereafter, the polypeptide chain is fed into the central chamber of the 20S core region that contains the proteolytically active sites. Ubiquitin is cleaved before terminal digestion by deubiquitinating enzymes. This third step is very closely associated with the second one, since ubiquitination takes place during the translocation event. However, the proteasomal degradation takes place in the cytoplasm.
