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

Pre-steady state kinetics with global fitting in a CUL2-dependent CRL system shows product dissociation is significantly slower than substrate dissociation, explaining slow steady-state ubiquitylation rates.

• Why it matters: CRL enzymology lags behind other systems, limiting understanding of which kinetic steps control ubiquitin transfer and chain building in multi-subunit ligases.
• What they did: In vitro reconstituted CUL2-dependent CRLs were analyzed with pre-steady state kinetics and global data fitting in KinTek Explorer, including substrate priming and poly-ubiquitin chain formation.
• The result: Catalytic efficiencies for poly-ubiquitin chain formation were 15–42-fold greater than substrate priming, and CUL1- and CUL2-dependent systems showed remarkably similar kinetic parameters, including the Km of the ubiquitin-carrying enzyme.

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