Ubiquitination occurs by a three-enzyme cascade. (a) Ubiquitin (Ub, yellow) is first activated by an E1, or ubiquitin-activating protein (purple square), which couples ATP hydrolysis to the formation of a thioester bond between the active-site cysteine of the E1 and the carboxyl terminus of ubiquitin. The E1 then transfers the activated ubiquitin to the active-site cysteine of an E2, or ubiquitin-conjugating enzyme (blue). Finally, the E3, or ubiquitin-protein ligase (green), facilitates the transfer of the ubiquitin from the E2 to a lysine on the target protein (substrate, magenta). In the case of the APC and many other E3s, this final step is repeated several times with the same substrate, resulting in ubiquitination of multiple lysines. In addition, specific lysines on ubiquitin itself can be modified, resulting in the assembly of polyubiquitin chains. (b) The APC is a member of the RING-domain family of E3s. These proteins facilitate the final step in ubiquitination by positioning the E2-ubiquitin conjugate next to the substrate, allowing the ε-amino group of a lysine on the substrate to nucleophilically attack the E2-ubiquitin thioester bond, resulting in direct transfer of ubiquitin as shown here. Members of the other major family of E3 proteins, called the HECT-domain E3s, employ an indirect, two-step mechanism (not shown here): ubiquitin is first transferred from the E2 to an active-site cysteine in the E3, after which a lysine in the substrate attacks the E3-ubiquitin thioester bond.
Matyskiela et al. Journal of Biology 2009 8:92 doi:10.1186/jbiol184