Differentiation of the slow‐binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase

Abstract

The binding mechanism of Mg 2+ at the M3 site of human placental alkaline phosphatase was found to be a slow‐binding process with a low binding affinity ( K Mg(app.) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg 2+ ‐free and Mg 2+ ‐containing enzymes by acrylamide showed almost identical dynamic quenching constant ( K sv = 4.44 ± 0.09 M −1 ), indicating that there is no gross conformational difference between the M3‐free and the M3‐Mg 2+ enzymes. However, Zn 2+ was found to have a high affinity with the M3 site ( K Zn(app.) = 0.11 mM) and was observed as a time‐dependent inhibitor of the enzyme. The dependence of the observed transition rate from higher activity to lower activity ( k obs ) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational change of the enzyme, which locks the enzyme in a conformation (M3′‐Zn) having an extremely high affinity for the Zn 2+ ( K * Zn(app.) = 0.33 μM). The conformation of the M3′‐Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg 2+ activation and Zn 2+ inhibition of the enzyme are reversible processes. Structural information indicates that the M3 site, which is octahedrally coordinated to Mg 2+ , has been converted to a distorted tetrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching constant for acrylamide ( K sv = 3.86 ± 0.04 M −1 ), suggesting a conformational change. Both Mg 2+ and phosphate prevent the enzyme from reaching this inactive structure. GTP plays an important role in reactivating the Zn‐inhibited enzyme activity. We propose that, under physiological conditions, magnesium ion may play an important modulatory role in the cell for protecting the enzyme by retaining a favorable geometry of the active site needed for catalysis.