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.