Reaction mechanism underlying protein ubiquitination by the APC and other RING-domain ubiquitin ligases. (a) The E3 positions the substrate and E2-ubiquitin conjugate in close proximity. (b) Deprotonation of the ε-amino group of a lysine on the substrate promotes nucleophilic attack of the thioester bond between the ubiquitin carboxyl terminus and the E2 active-site cysteine. (c) The initial product of nucleophilic attack is an oxyanion intermediate. (d) Completion of the reaction results in an isopeptide bond between the ubiquitin carboxyl terminus and the substrate lysine.