- 泛素的活化：: 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 活性部位 半胱氨酸 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.
- Transfer of ubiquitin from E1 to the 活性部位 cysteine of a 泛素缀合酶 E2 via a 转硫酯化反应. Mammalian genomes contain 30–40 UBCs.
- The final step of the ubiquitylation cascade creates an isopeptide bond between a lysine of the target protein and the C-terminal glycine of ubiquitin. In general, this step requires the activity of one of the hundreds of E3 ubiquitin-protein ligases (often termed simply 泛素连接酶). E3 enzymes function as the substrate recognition modules of the system and are capable of interaction with both E2 and substrate.
In the ubiquitination cascade, E1 can bind with dozens of E2s, which can bind with hundreds of E3s in a hierarchical way. Other ubiquitin-like proteins (ULPs) are also modified via the E1–E2–E3 cascade.
E3酶具有possess one of two domains:
- The HECT (Homologous to the E6-AP Carboxyl Terminus) domain
- The RING (Really Interesting New Gene) domain (or the closely related U-box domain)
Transfer can occur in two ways:
- Directly from E2, catalysed by RING domain E3s.
- Via an E3 enzyme, catalysed by HECT domain E3s. In this case, a covalent E3-ubiquitin intermediate is formed before transfer of ubiquitin to the substrate protein.
The anaphase-promoting complex (APC) and the SCF complex (for Skp1-Cullin-F-box protein complex) are two examples of multi-subunit E3s involved in recognition and ubiquitination of specific target proteins for degradation by the proteasome.