Abstract
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.
Highlights
Alkaline phosphatases (APs) occur widely in nature, and are found in many organisms from bacteria to man [1, 2]
We have investigated the functional significance of Ca2+ binding to all four metal ion-binding sites in tissue-nonspecific AP (TNAP) to better understand how the activity of TNAP is regulated during skeletal mineralization in an environment with high local Ca2+ gradients, further aiming to understand the pathophysiological basis for hypophosphatasia
The allosteric effect of [Mg2+] on human TNAP is consistent with that reported for bovine kidney AP [19], with a very slow ka but high affinity
Summary
Alkaline phosphatases (APs) occur widely in nature, and are found in many organisms from bacteria to man [1, 2]. An additional metal-binding site M4, that appears to be occupied by Ca2+ and is not present in the bacterial enzymes, was revealed upon solving the PLAP 3D structure [4, 5]. This fourth metal site is conserved in all human and mouse APs [6] and presumably represents a novel feature common to many if not all mammalian APs. the structural and functional significance of this new M4 metal site remains to be established. We have investigated the functional role of this M4 site for TNAP catalysis, an enzyme crucial for skeletal and dental mineralization
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