Figure 2.

Nedd4-2 exhibits hyperbolic Michaelis–Menten kinetics. 15-min ¹²⁵I-polyubiquitin conjugation reactions containing 1 nm wild-type or optimized GST-Nedd4-2 or 860 nm GST-Nedd4-2HECT were carried out at 37 °C in the presence of increasing Ubc5B concentrations. A, kinetic analysis of GST-Nedd4-2 under E3-limiting initial velocity conditions. The solid line represents the theoretical non-linear regression fit of the data for K_m = 44 ± 6 nm and k_cat = 0.020 ± 0.007 s⁻¹. Inset, double reciprocal plot of the data in A. B, kinetic analysis of GST-opt.Nedd4-2 (closed circles) and opt.Nedd4-2 (open circles), as described for A. The solid and dashed lines correspond to the theoretical non-linear regression fit for GST-opt.Nedd4-2 (K_m = 41 ± 10 nm; k_cat = 0.1 ± 0.01 s⁻¹) and opt.Nedd4-2 (K_m = 32 ± 5 nm; k_cat = 0.1 ± 0.01 s⁻¹), respectively. The double reciprocal plot of the corresponding data in B is shown in the inset. C, kinetic analysis of GST-Nedd4-2HECT free ¹²⁵I-polyubiquitin chain assembly. The solid line corresponds to the theoretical non-linear sigmoidal regression fit for [S]_½ = 105 ± 20 nm and k_cat = 1.9 ± 0.1 × 10⁻⁶ s⁻¹. The corresponding Hill plot is shown in the inset. The dashed line corresponds to a hyperbolic non-linear regression fit for K_m = 183 ± 123 nm and k_cat = 2.5 ± 0.7 × 10⁻⁶ s⁻¹. Statistical F-test analysis of the data fit to the Hill equation (solid line) compared with the Michaelis–Menten equation (dashed line) indicates that the data fit to the sigmoidal model is statistically significant (p = 0.034).