Functional significance of calcium binding to tissue-nonspecific alkaline phosphatase

PloS One
Marc F HoylaertsJosé Luis Millán

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+ adequate...Continue Reading

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Citations

Nov 23, 2015·Calcified Tissue International·José Luis Millán, Michael P Whyte
Jul 18, 2017·Journal of Tissue Engineering and Regenerative Medicine·Hyungjin JungOzan Akkus
Jan 10, 2018·Journal of Biomedical Materials Research. Part B, Applied Biomaterials·Amanda N de FariaAna P Ramos
Mar 5, 2020·Archiv der Pharmazie·Dana M ZaherJamshed Iqbal
May 16, 2017·Nature Reviews. Nephrology·Mathias HaarhausPer Magnusson
Nov 19, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Keun-Yeong JeongHwan Mook Kim
Jan 23, 2021·International Journal of Molecular Sciences·Stephanie GraserFranz Jakob
Sep 25, 2017·Metabolism: Clinical and Experimental·Etienne Mornet
May 1, 2021·International Journal of Molecular Sciences·Juan Miguel Villa-SuárezManuel Muñoz-Torres
Sep 7, 2019·The Science of the Total Environment·Guoqiang ZhaoQunqun Liu

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Methods Mentioned

BETA
glycosylation
Protein Assay
PLAP

Software Mentioned

MODEL
Chimera
SWISS
GraphPad Prism
PdbViewer

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