Effect of magnesium on the properties of zinc alkaline phosphatase

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Abstract
Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 4.0 .+-. 0.3 g-atoms of tightly bound Zn/mol (Kd .mchlt. 1 .mu.M) and 1.3 .+-. 0.2 g-atoms of Mg/mol. The binding of Mg is dependent upon pH and Zn content. The failure to assign the maximal Mg stoichiometry to enzyme isolated by conventional procedures may be due to the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of Mg to alkaline phosphatase. Under the conditions of the present studies, a maximum of 6 metal sites are available to bind Zn and Mg, i.e., 4 for Zn and 2 for Mg. Mg alone does not activate the apoenzyme, but it regulates the nature of the Zn-dependent restoration of catalytic activity to apophosphatase, increasing the activity of enzyme containing 2 g-atoms of Zn 5-fold and that of enzyme containing 4 g-atoms of Zn 1.4-fold. H-3H exchange reveals the stabilizing effects of Mg on the structural properties of phosphatase, but neither the KM for substrate or the phosphate binding stoichiometry and KI are significantly altered by Mg. Mg which is specifically bound to the enzyme, stabilizes the dynamic protein structure and regulates the expression of catalytic activity by Zn in alkaline phosphatase.