Figure 1. The Km values of ARIH1, UBE2D3, or UBE2R2 for β-Catenin and Cyclin E peptide substrates were estimated for comparison with their physiological concentrations.
Multi-turnover ubiquitylation reactions were assembled in the presence of constant amounts of SCFFBW7 and 32P-labeled Cyclin E peptide or SCFβTRCP and 32P-labeled β-Catenin peptide and increasing amounts of (a) ARIH1 protein (along with sufficient UBE2L3 to saturate ARIH1), (b) UBE2D3, or (c) UBE2R2. In the case of UBE2R2, mono-ubiquitylated versions of the peptide substrates were used. The enzyme concentrations have been provided in Supplementary file 2. Reaction velocities were calculated (see Materials and methods) and plotted as a function of the protein concentration. The data were fit to the Michaelis-Menten equation, , where [S] represents the substrate concentration and Km is the Michaelis constant, using nonlinear curve fitting (Prism 8 software). All reactions were performed in duplicate technical replicates. Figure 1—figure supplement 1 shows representative autoradiograms of the ubiquitylation reactions. The R2 values for the fit of the data to the model have been provided in Table 1.
Figure 1—figure supplement 1. Autoradiograms of the ubiquitylation reactions to estimate the Km values of ARIH1, UBE2D3, or UBE2R2 for β-Catenin and Cyclin E peptide substrates.
