Figure 4. All E2 partners lower the K_M and K_d of OTUB1 for K48 diUbiquitin.

(a) Kinetic assay of OTUB1 (50 nM) DUB activity in the presence and absence of 10 μM E2, which is above the K_d of all the E2 partners. Reactions were performed in triplicate. The full substrate titration up to 500 μM K48 diUbiquitin can be found in Supplemental Figure S3. (b) Summary table of all determined kinetic parameters: K_M, k_cat, and the calculated k_cat/K_M. (c) Correlation between changes in k_cat/K_M measurements to the increase in fold stimulation for isopeptidase activity (from Figure 2a) in the absence and presence of certain E2 enzymes. Dotted line denotes the linear regression with y = 179.3 M⁻¹ s⁻¹ and R² = 0.95