Functional Significance of Calcium Binding to Tissue-Nonspecific Alkaline Phosphatase

The conserved active site of alkaline phosphatases (AP) contains catalytically important Zn(2+) (M1 and M2) and Mg(2+)-sites (M3) and a fourth peripheral Ca(2+) site (M4) of unknown significance. We have studied Ca(2+) binding to M1-4 of tissue-nonspecific AP (TNAP), an enzyme crucial for skeletal mineralization, using recombinant TNAP and a series of M4 mutants. Ca(2+) could substitute for Mg(2+) at M3, with maximal activity for Ca(2+)/Zn(2+)-TNAP around 40% that of Mg(2+)/Zn(2+)-TNAP at pH 9.8 and 7.4. At pH 7.4, allosteric TNAP-activation at M3 by Ca(2+) occurred faster than by Mg(2+). Several TNAP M4 mutations eradicated TNAP activity, while others mildly influenced the affinity of Ca(2+) and Mg(2+) for M3 similarly, excluding a catalytic role for Ca(2+) in the TNAP M4 site. At pH 9.8, Ca(2+) competed with soluble Zn(2+) for binding to M1 and M2 up to 1 mM and at higher concentrations, it even displaced M1- and M2-bound Zn(2+), forming Ca(2+)/Ca(2+)-TNAP with a catalytic activity only 4–6% that of Mg(2+)/Zn(2+)-TNAP. At pH 7.4, competition with Zn(2+) and its displacement from M1 and M2 required >10-fold higher Ca(2+) concentrations, to generate weakly active Ca(2+)/Ca(2+)-TNAP. Thus, in a Ca(2+)-rich environment, such as during skeletal mineralization at pH 7.4, Ca(2+) adequately activates Zn(2+)-TNAP at M3, but very high Ca(2+) concentrations compete with available Zn(2+) for binding to M1 and M2 and ultimately displace Zn(2+) from the active site, virtually inactivating TNAP. Those ALPL mutations that substitute critical TNAP amino acids involved in coordinating Ca(2+) to M4 cause hypophosphatasia because of their 3D-structural impact, but M4-bound Ca(2+) is catalytically inactive. In conclusion, during skeletal mineralization, the building Ca(2+) gradient first activates TNAP, but gradually inactivates it at high Ca(2+) concentrations, toward completion of mineralization.