UBE1 catalyzes the first step in ubiquitin conjugation to mark cellular proteins for degradation. It is responsible for the primary ubiquitin-protein isopeptide bond formation and is a critical component for the initiation of in vitro conjugation reactions. UBE1 activates ubiquitin by the ATP dependent adenylation of its C-terminal glycine carboxyl group. This ubiquitin residue is then linked to the sulphydryl side chain moiety of a cysteine residue within UBE1 by forming a high energy thiol ester bond, liberating free AMP. There are two active sites within the UBE1 molecule allowing it to accommodate two ubiquitin molecules at one time, with a new ubiquitin forming an adenylate intermediate as the previous one is transferred to the thiol site. The activated ubiquitin is then transferred to the lysine of target proteins via the E2/E3 conjugation cascade.