Figure 5.

Nedd4-2 exhibits two E2∼ubiquitin thioester-binding sites. A, kinetic analysis of 1 nm GST-Nedd4-2 identical to that of Fig. 2 examining the rate of ¹²⁵I-polyubiquitin chain assembly under E3-limiting initial velocity conditions as a function of [Ubc5B]_o in the absence (closed circles) or presence (open circles) of 2 μm Ubc5BC85A. B, double reciprocal plot of initial rates of ¹²⁵I-polyubiquitin chain assembly as a function of [Ubc5B]_o as in A, in the absence (closed circles) or presence (open circles) of 550 nm Ubc5BC85S-¹²⁵I-ubiquitin oxyester. C, double reciprocal plot demonstrating the change in initial velocity (V_max − v_o) as a function of [Ubc5B]_o at substrate inhibition concentrations (0.6–4.8 μm) in the absence (closed circles) or presence (open circles) of 550 nm Ubc5BC85S-¹²⁵I-ubiquitin oxyester.