E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer.

During ubiquitin ligation, an E2 conjugating enzyme receives ubiquitin from an E1 enzyme and then interacts with an E3 ligase to modify substrates. Competitive binding experiments with three human E2-E3 protein pairs show that the binding of E1s and of E3s to E2s are mutually exclusive. These results imply that polyubiquitination requires recycling of E2 for addition of successive ubiquitins to substrate.