A protein designated for catabolism is bound to a series of ubiquitin (Ub) molecules in a process requiring ATP. Initially, free Ub is bound to the Ub-activating enzyme E1 in an ATP dependent process. Ub is subsequently shuttled from the Ub-activating enzyme E1 to Ub-conjugating enzyme E2 through the formation of a thioester bond between Ub and a cysteine residue of the E2 enzyme. The Ub monomer is then conjugated to the target protein through a peptide bond between the ε-amino group of a lysine residue in the target protein and the carboxy-terminal glycine residue in Ub via the action of a Ub-ligase enzyme E3. At least 4 Ub monomers must be attached to the protein before the target protein can be recognized and degraded by the 26S proteasome. In the degradation process, peptides are formed and the ubiquitin is released where it can be recycled again.