Ubiquitylation is a multistep reaction. (a) E1 enzymes use ATP to activate the carboxyl terminus of ubiquitin (Ubi) as a high-energy anhydride (Ubi-AMP). The E1 active site cysteine then attacks the adenylated ubiquitin to form a thioester intermediate. Subsequently, the active site cysteine of the E2 receives ubiquitin via trans-thioesterification. (b) E3 enzymes catalyze the formation of an isopeptide bond between the ubiquitin carboxyl terminus and a primary amino group of an acceptor. The acceptor can be a target protein (mono-ubiquitylation/ubiquitin chain initiation) or another ubiquitin molecule (ubiquitin chain elongation). Catalysis by HECT- and RBR-type E3 enzymes proceeds through an intermediate, in which the ubiquitin carboxyl terminus is thioester-linked to a cysteine residue at the active site of the E3, followed by aminolysis of the thioester. In contrast, RING-type E3s catalyze direct transfer of ubiquitin from the E2 active site cysteine to amino groups on the acceptor.