Figure 2. Activator Stimulatesk_cat/K_m of Ubc4 and Ubc1.

(A, B) APC/C (~1 nM) reactions were performed as in Figure 1B with ³⁵S-labeled securin-NApc10 fusion substrate, in the absence (left) or presence (right) of wild-type Cdh1. Purified E2 was titrated into the reactions at the indicated concentrations. Reactions with Ubc4 (A) were incubated for 20 min in the absence of Cdh1 and 10 min in the presence of Cdh1, and reactions with Ubc1 (B) were incubated for 30 min. The rate of substrate turnover (k_obs) was determined by dividing the moles of total substrate modified per minute by the moles of APC/C. Values represent the means (+/− SEM) from three independent experiments. Data were analyzed using Prism and fit using Michaelis-Menten parameters to determine the apparent K_m and maximal k_obs (k_cat), which are summarized in Table S1. Insets show close-ups of activity at lower E2 concentrations.

(C) APC/C reactions were performed at various Ubc4 concentrations as in (A), in the absence or presence of a mutant form of Cdh1 in which critical residues of the C-box were mutated (I57A, P58A). Data from three independent experiments (means +/− SEM) are presented, and the apparent K_m and maximal k_obs (k_cat) are summarized in Table S1.