FIGURE 5.

The UBA domains do not form the S1′-S1 sites. A, hydrolysis of Ub-AMC and ubiquitin modulation of activity by WT, M643E/M711E, and D453A IsoT. WT and mutant IsoT, 8 nm, were incubated with 2 μm Ub-AMC for 50 s in the absence (white bars) and presence of 492 nm ubiquitin (gray bars). Error bars correspond to the S.D. of three measurements. B, binding of monoubiquitin to C335A IsoT (filled squares), C335A/M643E/M711E IsoT (open squares), R221A./C335A IsoT (filled circles), and C335A/D435A IsoT (open triangles). The concentration of ubiquitin in the syringe for the C335A IsoT (2 μm in the cell), C335A/M643E/M711E IsoT (1.57 μm in the cell), and C335A/D435A IsoT (2 μm in the cell) was 91 μm and for R221A/C335A IsoT (2 μm) was 154 μm. The titration was done as in Fig. 2. The solid lines correspond to the fitting of the data points to a single site binding model.