The dissociation rate of mant-GDP was measured by monitoring the reduction in the fluorescence intensity due to the dissociation of mant-GDP preloaded in Rac1 (10). The purified Rac1 protein (20 μM) was incubated at 30°C for 30 min in buffer containing 50 mM tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM MgCl2, 1 mM DTT, and 50 μM mant-GDP (Invitrogen). The dissociation reaction was initiated by a 100-fold dilution in buffer containing 200 μM GTP and various concentrations of MgCl2. The excitation and emission wavelengths were set to 355 and 448 nm, respectively, with slit widths of 5 nm. The measurements were performed at 30°C. The difference in the activation free energy was calculated according to ΔΔGGDP,off = –RTln(koff,WT/koff,N92I), in which R denotes the gas constant and T represents the absolute temperature.