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Ubiquitin/Ubl Conjugating Enzymes

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Ubiquitin/Ubl conjugating enzymes are responsible for the selective modification of substrates. LifeSensors supplies a wide assortment of these enzymes to aid researchers in their efforts to understand the specificity and selectivity of this complex enzyme pathway.


E1 Enzymes

There is one major ubiquitin activating enzyme (UBE1) utilized in the conjugation of ubiquitin to substrates. UBE1 is the first enzyme in the pathway conjugating ubiquitin to target substrates through a ubiquitin-protein isopeptide bond. The C-terminal glycine of ubiquitin (G76) is initially adenylated in an ATP dependent manner which activates it so it can be transferred to the active site cysteine of UBE1 forming an ubiquitin-UBE1 thioester. With the help of additional enzymes in the ubiquitin conjugation pathway (E2/E3 enzymes) the ubiquitin is transferred to a specific lysine residue on a target substrate.




E2 Enzymes

Ubiquitin conjugating enzymes (E2s) are involved in the second step of the reaction to conjugate ubiquitin to target substrates through a ubiquitin-protein isopeptide bond. After activation of the C-terminus of ubiquitin by the E1 (UBE1), the ubiquitin is transferred from the cysteine of the E1 to the active site cysteine of the E2 enzyme. This thiol-transfer reaction leads to an ubiquitin-E2 thioester intermediate which is primed to either transfer the ubiquitin to an active site cysteine of an E3 ubiquitin ligase (HECT domain containing E3s), or transfer the ubiquitin directly to a lysine residue of a substrate protein with the aid of RING finger domain containing E3s (RING or SCF E3s).

Characterizing E2/E3 Pair Interactions

When initiating studies with a particular E3 ligase, many scientists select UBE2D3 (UbcH5c) as the E2 enzyme, due to reported promiscuity associated with this E2.  However, it should be noted that several E3 ligases (including Parkin) have been reported not to work with UBE2D3.  UBE2L3 (UbcH7) has been shown to function with several E3 ligases, including E6-AP and Parkin.  UBE2N/UBE2V2 function as a heterodimeric E2 enzyme for the production of K63-linked ubiquitin chains, and is a good choice if you know that the E3 of interest polyubiquitylates through this linkage type.  This information can be used as a starting point in matching E2/E3 enzyme pairs for your ligase assays, but comprehensive profiling is still sorely needed.


  • Identify cognate E2/E3 pairs
  • Characterize upregulation/downregulation of E2 enzymes
  • Build poly-ubiquitin chains
  • Characterize ubiquitin chain specificity of E2 enzymes
  • Analyze expression of E2 enzymes


E3 Enzymes

Ubiquitin protein ligases (E3s) are involved in the final step of attaching ubiquitin to target substrates. E3s generally determine substrate recognition in the ubiquitin conjugation pathway, and E3s are the most abundant of the ubiquitin conjugation pathway enzymes with hundreds (>800) being identified so far. There are two main classes of E3s, HECT domain containing E3s, and RING finger domain containing E3s. The HECT domain E3s contain an active site cysteine, and in a thiol-transfer reaction with the ubiquitin-E2 thioester intermediate, ubiquitin is transferred from the E2 to the E3 forming an ubiquitin-E3 thioester intermediate. The HECT E3s also contain a substrate binding domain transferring ubiquitin directly to a specific lysine on the substrate. RING finger domain E3s act as scaffolds containing a RING domain (which binds the ubiquitin-E2 thioester) and containing a substrate binding domain. The RING E3s bring the E2 and substrate together to facilitate transfer of ubiquitin from the E2 to the substrate. Within the RING E3 family there are two major subclasses, simple RING ligases and SCF ligases. Simple RING ligases contain the RING domain and the substrate binding domain in one protein. SCF ligases are modular protein complexes with the RING domain and the substrate binding domain being contained on different proteins.


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