Ubiquitination is catalyzed in a stepwise manner by the three enzymes: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating and E3 ubiquitin-protein ligase. First, E1 catalyzes the ATP-dependent conjugation of a sulfhydryl group to ubiquitin. Then, the ubiquitin is then transferred to the sulfhydryl group of E2 ubiquitin-conjugating enzyme. Finally, E3 catalyzes the transfer of ubiquitin from E2 to the target protein. Successive ubiquitin molecules can be linked to lysine 48 of the attached ubiquitin to form poly-ubiquitin chains, amplifying the signal.