These enzymes perform the second step in Ub (or UBL) conjugation reactions by forming a thioester linkage between its active site cysteine with the C-terminal glycine of the modifier. E2 enzymes function alone and in conjunction with E3 ligase to catalyze the attachment of Ub (or UBL) proteins to acceptor lysine residues of target proteins to form isopeptide bonds. E2 enzymes act via selective protein–protein interactions with the E1 and E3 enzymes. It transfers the activated Ub/UBL to downstream effectors (E3s) to facilitate substrate modification. While E3s are involved in substrate selection, E2s are the main determinants for the lysine linkage present in Ub/UBL chains or substrates and thus directly influences the cellular fate of the substrate. The target lysine makes a nucleophilic attack on the carbonyl group of the Ub/Ubl-E2 thioester linkage upon deprotonation.