Acetylated Ubiquitin Modulates the Catalytic Activity of the E1 Enzyme Uba1

Ubiquitin (Ub) signaling requires the covalent passage of Ub among E1, E2, and E3 enzymes. The choice E2 enzymes combined with multiple rounds cascade leads to formation polyubiquitin chains linked through any one seven lysines on Ub. linkage type length act as a signal trigger important cellular processes such protein degradation or DNA damage response. Recently, proteomics studies have identified that can be acetylated at six its lysine residues under various cell stress conditions. To understand potential differences in caused by acetylation, we synthesized all possible ubiquitin (acUb) variants examined E1-mediated corresponding E2∼acUb conjugates vitro using kinetic methods. A Förster resonance energy transfer assay was optimized which constructs were labeled CyPet fluorophore UBE2D1 YPet monitor E2∼Ub conjugates. Our methods enable detection small may otherwise concealed steady-state ubiquitination experiments. We determined Ub, K11, K27, K33, K48, K63, has altered turnover numbers for conjugate E1 enzyme Uba1. This work provides evidence acetylation alter catalysis early pathway.