Functional Significance of Calcium Binding to Tissue-Nonspecific Alkaline Phosphatase

The conserved active site of alkaline phosphatases (AP) contains catalytically important Zn²⁺ (M1 and M2) and Mg²⁺-sites (M3) and a fourth peripheral Ca²⁺ site (M4) of unknown significance. We have studied Ca²⁺ binding to M1-4 of tissue-nonspecific AP (TNAP), an enzyme crucial for skeletal mineralization, using recombinant TNAP and a series of M4 mutants. Ca²⁺ could substitute for Mg²⁺ at M3, with maximal activity for Ca²⁺/Zn²⁺-TNAP around 40% that of Mg²⁺/Zn²⁺-TNAP at pH 9.8 and 7.4. At pH 7.4, allosteric TNAP-activation at M3 by Ca²⁺ occurred faster than by Mg²⁺. Several TNAP M4 mutations eradicated TNAP activity, while others mildly influenced the affinity of Ca²⁺ and Mg²⁺ for M3 similarly, excluding a catalytic role for Ca²⁺ in the TNAP M4 site. At pH 9.8, Ca²⁺ competed with soluble Zn²⁺ for binding to M1 and M2 up to 1 mM and at higher concentrations, it even displaced M1- and M2-bound Zn²⁺, forming Ca²⁺/Ca²⁺-TNAP with a catalytic activity only 4–6% that of Mg²⁺/Zn²⁺-TNAP. At pH 7.4, competition with Zn²⁺ and its displacement from M1 and M2 required >10-fold higher Ca²⁺ concentrations, to generate weakly active Ca²⁺/Ca²⁺-TNAP. Thus, in a Ca²⁺-rich environment, such as during skeletal mineralization at pH 7.4, Ca²⁺ adequately activates Zn²⁺-TNAP at M3, but very high Ca²⁺ concentrations compete with available Zn²⁺ for binding to M1 and M2 and ultimately displace Zn²⁺ from the active site, virtually inactivating TNAP. Those ALPL mutations that substitute critical TNAP amino acids involved in coordinating Ca²⁺ to M4 cause hypophosphatasia because of their 3D-structural impact, but M4-bound Ca²⁺ is catalytically inactive. In conclusion, during skeletal mineralization, the building Ca²⁺ gradient first activates TNAP, but gradually inactivates it at high Ca²⁺ concentrations, toward completion of mineralization.