Global Data Fitting Enables Kinetic Dissection of Cullin-RING Ligase-Catalyzed Ubiquitylation: From Substrate Priming to Poly-ubiquitin Chain Formation

The discovery and characterization of the ubiquitin system was founded on classical enzymology approaches that established fundamental principles of protein ubiquitylation. Cullin-RING ligases (CRLs) represent the largest class of human ubiquitin ligases, yet detailed enzymology of these multi-subunit assemblies has lagged behind other systems such as nucleic acid polymerases. Here, we employed pre-steady state kinetics and global data fitting using KinTek Explorer to systematically investigate the kinetic parameters controlling CRL-dependent ubiquitin transfer. Using a well-characterized in vitro reconstituted system exploring CUL2-dependent CRLs, we modeled substrate priming, showing that the system is highly amenable to global fitting of kinetic data. The results show that product dissociation from the CRL is significantly slower than substrate dissociation, explaining previously reported slow steady-state ubiquitylation rates. Extending this framework to poly-ubiquitin chain formation revealed catalytic efficiencies 15-42-fold greater than substrate priming. Comparative analysis of CUL1-dependent and CUL2-dependent systems showed remarkably similar kinetic parameters, including the Km of ubiquitin-carrying enzyme for CRLs, despite differences in substrate receptor and enzyme architecture. We further examined drug-induced neo-substrate ubiquitylation using the pomalidomide-CRBN-SALL4 system, revealing atypical kinetics characterized by rapid ubiquitin transfer paired with an elevated Km, ultimately yielding catalytic efficiencies comparable to natural substrates. The findings establish pre-steady state kinetics and global data fitting as powerful enzymological tools for dissecting CRL-dependent ubiquitylation across different CRL families and substrate contexts, with important implications for understanding both physiological ubiquitylation and therapeutic targeted protein degradation strategies.