Abstract: Ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of ubiquitin (Ub) to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for generating diverse substrate-Ub structures, which provides versatility to this pathway in targeting proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that ubiquitination site/s are selected by E2/E3-mediated positioning of lysine/s toward the E2/E3 active site. By studying polyubiquitination of Sic1 by the E2, Cdc34, and the RING E3, Skp1/Cul1/F box protein (SCF), we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified if this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination.