FIG 2.

The double mutant cycle of R54D ubiquitin and D143K or D143R Ube2R2 demonstrates that each mutant is individually defective but that the mutants complement each other in combination during Ube2R2 catalysis. (a) Schematic showing the components and order of addition for the diubiquitin synthesis assay. UB, ubiquitin. (b) Time course (in hours) for the diubiquitin synthesis assay and all four combinations of the R54D ubiquitin and D143K Ube2R2 double mutant cycle. Representative data are shown. (c) Graph plotting the quantitation of the amounts of diubiquitin product shown in panel b versus time. Ub, ubiquitin. (d) Same as in panel b, except D143R Ube2R2 activity has been assessed in the presence of either WT or R54D acceptor ubiquitin. Error bars represent the standard errors of measurements from duplicate data sets.