FIGURE 2.

Acidic loop residues Glu-108 and Glu-112 in human Cdc34 function in SCF binding. A, schematic for the assembly and order of addition of the multi-turnover ubiquitination reaction. Reactions were initiated by the addition of either ³²P-labeled β-Cat peptide or Ub-β-catenin peptide (β-Cat-(Ub)₁) substrate followed by quenching and SDS-PAGE. B, increasing amounts of WT Cdc34 were titrated into ubiquitination reactions followed by phosphorimaging and quantitation of products and reactants. Note that each lane corresponds to a single reaction and quantity of Cdc34. The initial velocities of the ubiquitination reaction were plotted against WT Cdc34 concentration and used to estimate the K_m and k_cat of WT Cdc34 for SCF using nonlinear regression (GraphPad Prism software). C, same as B with E108A Cdc34. D, same as B except with E108A/E112A Cdc34 and Ub-β-catenin peptide. The asterisk corresponds to a contaminant of the substrate preparation. E, graph showing the ratio of K_m values for all mutant Cdc34 proteins used in this investigation divided by the K_m for WT Cdc34. Note that all experiments were done in duplicate, and the error bars represent the standard error of measurement. Reaction conditions are summarized in Table 1.