Fig. 6. Kinetic characterization of Rsp5/ByS. (A) Native chemical ligation between Tagless-Ub–MES and FCys. (B) Reaction mixtures were quenched at different time points and the remaining Tagless-Ub–MES was labeled with FCys. Tagless-Ub–MES (250 μM) was incubated for indicated times without Rsp5ΔWW (upper panel) or with Rsp5ΔWW (lower panel, 1.5 μM). (C) Consumption of Tagless-Ub–MES (250 μM) by Rsp5ΔWW (1.5 μM). (D) The initial reaction rates at different Ub–MES concentrations were plotted, and k _cat and K _m values estimated. (E) Tagless-Ub–MES (250 μM) was incubated with the indicated mutant of Rsp5ΔWW (5 μM) and quenched with FCys at five time points starting at 30 seconds. After linear fitting, the y-intercept of each line was adjusted to equal zero. (F) Comparison of gross consumption rates of Tagless-Ub–MES by Rsp5ΔWW mutants.