Attachment of Ub to Target Protein

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• 1. -NH of Lys residue on protein is covalently attached to the COO- terminus of Ub 

• 2. Another Ub attaches to the Lys 48 -NH of the first Ub

• 3. Repeat Steps 2 twice until you get four covalently attached Ubs

• 4 Ub's = the DEATH MARK

• 1. E1 activates the Ub by adenylating -COO with AMP (hydrolysis of ATP into AMP and PPi)

• 2. E1 transfers Ub onto its Cys residue to form a high E thioester bond, thereby removing AMP

• 3. A transthiolation reaction transfers an activated Ub from E1 to E2

• E2 is now bonded to Ub via a thioester bond

• 4. E3 recognizes and associates with both the E2-Ub complex and the target protein

• E1 = Ub activating enzyme

• E2 = Ub conjugating enzyme

• 5. E3 catalyzes the transfer of Ub onto amino groups on specific Lys on the target protein

• 6. W/ E3 still bound to the target protein, repeat steps 1-5 until 3 more Ubs are attached

• E3 = scaffold b/c it recognizes both target protein and E2-Ub complex

• E3 = Ub ligating enzyme

• Ubiquitin Targeting and Mechanism