FIGURE 5.

Kinetic measurement of mantATP with excess of RecD2·dT₂₀: binding, hydrolysis, and P_i release. Time course of mant fluorescence (solid line), mantADP formation (circles), and P_i release (dashed line) is shown. All measurements were at 2.5 μm mantATP, 12.5 μm RecD2, 15 μm dT₂₀, and 10 μm MDCC-PBP (for P_i measurement) and were carried out under the conditions of Fig. 3 as described under “Experimental Procedures.” The time courses were simulated (dotted lines), based upon a global model for a single turnover of mantATP based on the scheme in Fig. 1B, as described under “Results.” The simulated mant fluorescence and cleavage time course are in the main panel, and the P_i simulation with the experimental data is in the inset. This gave an observed first order rate constant for mantATP binding ([RecD2] × k_+1a) at 200 s⁻¹ followed by the conformation change (k_+1b) at 54 s⁻¹, hydrolysis (k₊₂) at >300 s⁻¹; P_i release (k₊₃) was fast (>300 s⁻¹).