FIGURE 13.

Mutations favoring the open state of the NRL and enhancing uncoupled 2OG decarboxylation accelerate the release of DNA substrate. A–C, fluorescence anisotropy assays of the rate of release of 10 nm 5′-CAmCAT-3′ substrate from 4 μm WT (A), W89Y (B), or M61L (C) AlkB-ΔN11 in the presence of 2 mm Suc. Release was initiated by addition of 20 μm unlabeled DNA substrate under the conditions used for the anisotropy assays in Fig. 3. D and E, fluorescence anisotropy assays were used to measure the release rate of 5′-CAmCAT-FAM-3′ (D, k_off = 1.8 min⁻¹) or 5′-TmAT-FAM-3′ (E, k_off ≥108 min⁻¹) from WT AlkB-ΔN11 at 10 °C in standard buffer containing 20 μm Mn(II) and 2 mm Suc. F shows Michaelis-Menten kinetic assays performed for 5′-CAmCAT-3′ (black, k_cat = 21.2 min⁻¹) and 5′-TmAT-3′ (red, k_cat = 5.2 min⁻¹) in the standard assay buffer at 10 °C as described previously (23) with minor modifications described under “Experimental Procedures.” Error bars represent the standard error of the mean.