Fig 1. Allosteric activation of Zn²⁺-TNAP by MgCl₂.

a. Progressive AbM2-bound Zn²⁺-TNAP activation, visualized as increasing slopes in plots of A405 nm vs. time, for the indicated [Mg²⁺], added to Chelex-pretreated pNPP (10 mM) at pH 9.8 (left panel); repeat of the experiment in (a) at 15-fold lower [TNAP] over a time-interval 0–90 min, for the indicated [Mg²⁺] (right panel); b. Slopes (first derivatives) to the lines in Fig. 1A, right panel vs. time, for the indicated [Mg²⁺], describing formation of Mg²⁺/Zn²⁺-TNAP as a function of time (left panel); plots of k_app (calculated from Fig. 1B left panel) vs. [Mg²⁺] and determination of k_a and k_d for binding of Mg²⁺ to Zn²⁺-TNAP (right panel);. Experiments representative of at least three replicates with variable enzyme and MgCl₂ concentrations.