title: Functional significance of calcium binding to tissue-nonspecific alkaline phosphatase.
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.
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
authors.forename: Marc F
authors.initials: MF
authors.lastname: Hoylaerts
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
authors.forename: Soetkin
authors.initials: S
authors.lastname: Van Kerckhoven
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, United States of America.
authors.forename: Tina
authors.initials: T
authors.lastname: Kiffer-Moreira
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, United States of America.
authors.forename: Campbell
authors.initials: C
authors.lastname: Sheen
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, United States of America.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Narisawa
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, United States of America.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Millán
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002113:Calcification, Physiologic / Q000502:physiology; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D020125:Mutation, Missense; D011485:Protein Binding
chemical_list: C548267:ALPL protein, human; D000469:Alkaline Phosphatase; D002118:Calcium