Figure 2. UCH37–RPN13^C preferentially binds and deubiquitinates K6/K48-branched Ub₃.

(A) Binding affinities between His-TEV-UCH37(C88S)–RPN13^C and various polyUb chains were measured by microscale thermophoresis. Binding isotherms ( Figure 2—figure supplement 1) were fit with a single-site-binding model; best-fit K_D values are shown with standard errors. (B) Schematic of the free Ub sensor-based deubiquitination assay. (C) Michaelis–Menten kinetics of branched Ub₃ hydrolysis by NS–UCH37–RPN13^C. The table shows best-fit K_M, k_cat, and k_cat/K_M values with standard errors from two independent replicates.

Figure 2—figure supplement 1. UCH37–RPN13^C preferentially binds and deubiquitinates K6/K48-branched Ub₃.

(A) Ub chain-binding isotherms of His-TEV-UCH37(C88S)–RPN13^C were determined by microscale thermophoresis; fluorescent labeling used RED-tris-NTA that binds to the His-tag. Lines show the fitted curves using a single-site-binding model.(B) Each UCH37 preparation was evaluated by Ub-AMC hydrolysis. Specific activities were determined with 1 nM enzyme and 8 µM Ub-AMC; AU, arbitrary fluorescence unit. (C) Gel-based deubiquitinating enzyme (DUB) assays were performed to compare debranching efficiencies by the UCH37 preparations described in (B). Enzymes (1 µM) were incubated with 5 µM substrate as indicated for 2 hr at 37 °C. Reaction products were quantified and plotted in (D). U–R^C and U–R^FL represent UCH37–RPN13^C and UCH37–RPN13^FL, respectively.