Fig. 3.

Ubiquitin cleavage assays with CidB^wMel variants. CidB^wMel proteins are truncated to the DUB domain (residues H717 through R1128). (V-L) and (H-Y) are substitutions found in CidB^wYak. (C-A) is catalytic mutant negative control. Silver-stained SDS-PAGE analysis of 1-h digests with K63-linked di-Ubiquitin (Ub₂) (A) and K48 linked Ub₂ (B). The (V-L) mutant has reduced DUB activity compared to wild-type (WT). Markers are kDa. Michaelis–Mentin graphs showing reaction velocity versus substrate concentration from timed 5-min digests for K63 (C) and K48 (D). (V-L) reaction velocities are reduced compared to WT. At substrate concentrations of 2 μM, the difference is statistically significant (P = 0.008, unpaired t test with Welch’s correction). (E) Quantification of kinetic parameters for WT and (V-L) enzymes. Enzyme efficiency (kcat/KM) of (V-L) mutants are reduced 2.4-fold and 1.4-fold from the WT for K63 and K48, respectively.