The covalent bond between ubiquitin and substrate proteins requires the activation of the C-terminal carboxyl group of Ub to be activated. In this reaction, ubiquitin is linked to the Ubiquitin Activating Enzyme (E1) through a thioester bond between its C-terminal glycine and the active site cysteine residue in the enzyme (E1-S-Ub). E1 hydrolyzes ATP to AMP and PPi with the intermediate formation of an E1-bound ubiquitin adenylate. The activated Ub is then transferred from E1 to any of<br>a family of Ubiquitin Carrier Protein Enzymes (E2s) in a similar thioester linkage (E2-S-Ub). Ubiquitin can then be transferred directly to the ε-Lys of a substrate from E2 enzymes. Other conjugation reactions require the involvement of a third enzyme, Ubiquitin Ligase (E3). E3 binds to substrate proteins and also to an E2 to form an E2-E3-substrate complex. It is the recognition and formation of said complex that has the highest level of substrate specificity for the conjugation<br>cascade.