Ubiquitin activating enzyme E1 is responsible for the first step in ubiquitin-protein isopeptide bond formation and is a critical component for the initiation of in vitro conjugation reactions. E1 activates ubiquitin by first adenylating with ATP the C-terminal glycine carboxyl group of ubiquitin and, thereafter, linking this residue to the sulphydryl side chain moiety of a cysteine residue in E1 by forming a high energy thiol ester bond and liberating free AMP. There are two active sites within the E1 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.