Fig. 4.

Initial rate kinetic study of a GST-E6APF849Y point mutant. Incubations containing 100 nM Uba1, 30 nM GST-E6APF849Y, and the indicated concentrations of recombinant UbcH7 were incubated for 10 min at 37°C as outlined in Subheading 3.3. Conjugated ¹²⁵I-ubiquitin was quantitated by excising each lane above the 25-kDa relative molecular weight marker and used to calculate the initial velocity as described in the text. (a) Autoradiogram of the resulting SDS-PAGE resolution of the incubations. (b) Dependence of initial rate (v_o) on (UbcH7)_o (solid line represents the nonlinear regression fit for K_m = 52 ± 8 nM and k_cat = 1.4 ± 0.1 × 10^–3 s^–1). Inset – Double-reciprocal plot of the rate data.