FIGURE 3.

Inhibition of WT T5FEN-catalyzed reactions supported by Mn²⁺ and Mg²⁺ viable cofactors by the addition of calcium ions. All data points represent the average of three experiments, but error bars are omitted for clarity except for panel C. Data at 0.1 mm Mn²⁺ and 2 mm Mg²⁺ are from (3). A, inhibition of 0.1 mm (black circles), 0.2 mm (red squares), and 0.4 mm (blue diamonds) Mn²⁺-supported reactions by calcium ions. Experiments were carried out as described under “Experimental Procedures.” Data are fit to a simple single-site inhibition model (Equation 2) to yield K_I = 1.1 ± 0.11, 2.2 ± 0.32, and 3.8 ± 0.41 mm, respectively. Although data fit well in the range k_obs/k₀ > 0.2, the deviation from this behavior at higher concentrations of Ca²⁺ ions is evident. B, the same data as in A fit to a more complex inhibition model where Ca²⁺ displaces either one or two Mn²⁺ ions (Equation 3) yielding K_I1 = 5.3 mm (0.1 mm Mn²⁺) and 8.4 mm (0.2 mm Mn²⁺). In the case of data obtained at 0.4 mm Mn²⁺, where there is limited deviation from the single inhibition model (Equation 2), the value of K_I is outside the data range. C, the dependence of log k_obs/k₀ (where k_obs/k₀ < 0.2) on log [Mn²⁺] at 20 mm Ca²⁺ (circles), 17.5 mm Ca²⁺ (squares), 15 mm Ca²⁺ (diamonds), 12.5 mm Ca²⁺ (crosses), and 10 mm Ca²⁺ (triangles). The slopes of the straight line fits are 1.8 ± 0.1 at 20 mm Ca²⁺, 1.8 ± 0.1 at 17.5 mm Ca²⁺, and 1.8 ± 0.1 at 15 mm Ca²⁺. Although there are only two available data points at 12.5–10 mm Ca²⁺, the same trend is observed in these data. The intercept in each case is a function of 1/[Ca²⁺]². D, inhibition of 0.5 mm (black circles) and 2 mm (red squares) Mg²⁺-supported reactions by calcium ions. Experiments were carried out as described under “Experimental Procedures.” Data are fit to a simple single-site inhibition model (Equation 2) to yield K_I = 0.1 ± 0.01 mm (0.5 mm Mg²⁺) and 0.2 ± 0.02 mm (2 mm Mg²⁺). Although data fit well in the range k_obs/k₀ > 0.2, the deviation from this behavior at higher concentrations of Ca²⁺ ions is evident. E, the same data as in D fit to a more complex inhibition model where Ca²⁺ displaces either one or two Mg²⁺ ions (Equation 3), yielding K_I1 = 8.7 ± 0.1 mm (0.5 mm Mg²⁺) and 9.0 ± 1.3 mm (2 mm Mg²⁺).