Fig. 7. pH dependence of the logarithmic single turnover rate (k_c) of T5 FEN with Mg²⁺ added to enzyme prior to mixing enzyme and substrate in a quench-flow apparatus. Experimental details are provided in Materials and methods. Curve fitting (Equation 4) yields pK_a = 8.0 ± 0.11, (k_c)₀ = 256 ± 1/min. The similarity of these results to those obtained when Mg²⁺ is pre-mixed with substrate (Figure 5) indicates that an Mg²⁺-dependent conformational change does not limit the rate of reaction catalysed by T5 FEN.