Fig. 4.

The rate constant for ADP release by SecA (k_off[ADP]) is equal to the steady-state ATPase rate (k_cat). k_off[ADP] was measured by displacement of ADP bound to SecA by MANT-ADP, resulting in an increase in FRET between the intrinsic tryptophans in SecA and the MANT fluorophore. SecA (0.5 μM) preincubated with 2.5 μM ADP was mixed with 12.5 μM MANT-ADP in a stopped-flow device and the fluorescence change was measured using excitation light at 296 nm and a 399-nm emission cut-off filter. The data were fitted to a single exponential equation (Eq. 4), giving a k_off[ADP] of 0.0073 ± 1.1 × 10⁻⁵ s⁻¹.