SecYEG reactivates GTP hydrolysis from the RNC–SRP–FtsY complex. (A) Stimulated GTP hydrolysis by SRP and FtsY in the absence (circles) and presence (squares) of RNC, and in the presence of both RNC and SecYEG (triangles). The data were fit to Eq. 3 and gave k_cat values of 80, 22, and 66 min⁻¹ for the SRP–FtsY, RNC–SRP–FtsY, and RNC–SRP–FtsY–SecYEG complexes, respectively. (B and C) Effect of SecYEG (B) or DDM (C) on GTP hydrolysis from the RNC–SRP–FtsY complex. The data in B were fit to Eq. 3 and gave a K_d value of 2 µM and a k_max value of 57 min⁻¹. (D) DDM reduces the GTP hydrolysis rate from the SRP–FtsY complex. k_cat values were determined as in A in the absence and presence of 0.02% DDM. (E) SecYEG does not affect the basal GTPase activity of FtsY. Reactions were performed in the presence of 4 µM FtsY, 100 µM GTP, and the indicated concentrations of SecYEG. (F) In the absence of RNC, SecYEG does not significantly affect GTP hydrolysis from the SRP–FtsY complex. The data were fit to Eq. 3 and gave k_cat values of 60 and 51 min⁻¹ in the absence (closed) and presence (open) of 12 µM SecYEG, respectively. The data in A, B, D, E, and F are the average of two experiments ± SD (error bars).