protein ubiquitination protein ubiquitination (ubiquitin activation) The first step of the ubiquitination process. Ubiquitin is activated in a two-step reaction by an E1 ubiquitin-activating enzyme in a process requiring ATP as an energy source. The initial step involves production of a ubiquitin-adenylate intermediate. The second step transfers ubiquitin to the E1 active site cysteine residue, with release of AMP. This step results in a thioester linkage between the C-terminal carboxyl group of ubiquitin and the E1 cysteine sulfhydryl group. 2. Transfer of ubiquitin from E1 to the active site cysteine of a ubiquitin-conjugating enzyme E2 via a trans(thio)esterification reaction. 3. The final step of the ubiquitinylation cascade generally requires the activity of one of the hundreds of E3 ubiquitin-protein ligases (often termed simply ubiquitin ligase). E3 enzymes function as the substrate recognition modules of the system and are capable of interaction with both E2 and substrate. E3 enzymes possess one of two domains: * The HECT (Homologous to the E6-AP Carboxyl Terminus) domain * The RING domain (or the closely related U-box domain). pathway UPa:UPA00831