Figure 5.

Mg²⁺ dependence of the kinetics for the Schist26 and Schist21 constructs. A plot of k_{obs, cleave} for Schist26 (circles), k_cleave for Schist21 (squares), and calculated k_ligate (diamonds) vs. Mg²⁺ concentration. For k_{obs, cleave} and k_cleave the lines are fits of the Mg²⁺ dependence of the rate constant, k, to a two-state model for Mg²⁺ binding using the equation k = k_max [Mg²⁺]/([Mg²⁺] + [Mg²⁺]_1/2), where k_max is the rate constant at saturating Mg²⁺, and [Mg²⁺]_1/2 is the Mg²⁺ concentration required to achieve half the maximal cleavage rate constant. This analysis assumes a Hill coefficient of 1 since there was no evidence for cooperative binding of Mg²⁺ for any of the kinetic data. These fits yield [Mg²⁺]_1/2 of 55 ± 10 mM for k_{obs, cleave} for Schist26 and 17 ± 3 mM for k_cleave for Schist21. The k_ligate does not saturate at the Mg²⁺ concentrations employed here making it impossible to determine a [Mg²⁺]_1/2. Thus for k_ligate the solid line is a simple linear fit of k_ligate with [Mg²⁺]. The error bars are based on a 20% error between replicate experiments for the measured rate constants (see Table 1).