Hello Anna Truong

E1 enzyme activates the C-terminus of ubiquitin by forming a high energy E1∼Ub thioester adduct, while E2 enzyme transfers ubiquitin from E1 enzyme to the catalytic cysteine of E3 ubiquitin ligase. Thus, for ubiquitin to travel from E1 enzyme to E3 enzyme, two trans thiolation reactions are needed. Since E1 enzyme activates the C-terminus of ubiquitin, and then directs the ubiquitin to downstream pathway, it means that ubiquitin has to be activated.

The E1-Ubiquitin complex is recognized by E2 ubiquitin conjugating enzyme, which receives ubiquitin to form a ubiquitin-charged E2. E2 recognizes E3 that is associated with the substrate and transfers ubiquitin to the substrate.

So, E2 cannot react with free ubiquitin. Ubiquitin activation by E1 is necessary.

I hope this is helpful.

Best.

Thanks Malcolm Nobre.

I understand this cascade happens in cells, but why can’t E2~ubiquitin form in situ when using purified E2 and ubiquitin? The mechanism is simply reaction of the ubiquitin C-terminus and the E2 active site Cys. Is there an energetic barrier that the E1 helps overcome?

Hi Anna Truong

The mechanism would not be a simple reaction of the ubiquitin C-terminus and the E2 active site Cys. This is because the mechanism involved with E1 is slightly different.

The E1 enzyme hydrolyses ATP and adenylates the C-terminal glycine residue of ubiquitin to form a ubiquitin-adenylate intermediate, and then ubiquitin is transferred to a Cys in the catalytic domain of E1 to form the activated ubiquitin~E1 complex and free AMP.

I hope this becomes clearer.