Figure 2.

Activity of genetically tethered pseudohexamers. A, rates of hydrolysis of 5 mm ATP by the genetically tethered W-W₃, W-E₃, and E-W₃ pseudohexamers. Values are averages (n ≥ 5) ± S.D. (error bars). B, rates of unfolding of different concentrations of thrombin-split ^I37AArc-^cp6GFP-β5/β6-st11-ssrA by wild-type HslU or genetically tethered variants. Lines, non-linear least squares fits to the Michaelis-Menten equation. K_m values for all enzymes were 1–3 μm but were not well determined because of the small number of low-concentration data points. Average V_max values ± S.D. calculated from the highest four substrate concentrations were 0.155 ± 0.003 min⁻¹ enz⁻¹ (HslU), 0.143 ± 0.008 min⁻¹ enz⁻¹ (W-W₃), 0.0802 ± 0.007 min⁻¹ enz⁻¹ (E-W₃), and 0.0716 ± 0.006 min⁻¹ enz⁻¹ (W-E₃). Fitted V_max values were 10–15% higher. C, the kinetics of degradation of Arc-st11-ssrA (10 μm) by HslV (10 μm) and HslU (0.3 μm) or W-W₃ (0.3 μm) at 50 °C was monitored by SDS-PAGE. Reactions contained 5 mm ATP and a regeneration system.