Inactivation of the E6AP E3 ubiquitin ligase (UBE3A gene) causes Angelman syndrome, while aberrant degradation of p53 by E6AP is implicated in cervical cancers. Herein, we describe the development of photo-cross-linkers to discover catalytic residues of E6AP. Using these cross-linkers, we identified covalent modifications of the E6AP catalytic cysteine and two lysines: Lys847 and Lys799. Lys847 is required for the formation of Lys48-linked polyubiquitin chains, while the K799A E6AP mutant was more active at producing Lys48-linked polyubiquitin chains. Thus, opposing roles of Lys799 and Lys847 pave the path forward to pharmacological inhibitors or activators of E6AP for therapeutic purposes.