Differentiation of the slow-binding mechanism for magnesium ion activationand zinc ion inhibition of human placental alkaline phosphatase

The binding mechanism of Mg2+ at the M3 site of human placental alkaline ph osphatase was found to be a slow-binding process with a low binding affinit y (K-Mg(app.) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg 2+-free and Mg2+-containing enzymes by acrylamide showed almost identical d ynamic 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-Mg2enzymes. However, Zn2+ 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 activit y to lower activity (k(obs)) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational ch ange of the enzyme, which locks the enzyme in a conformation (M3'-Zn) havin g an extremely high affinity for the Zn2+ (K*(Zn(app.)) = 0.33 muM). The co nformation of the M3'-Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg2+ activation and Zn2+ inhibition of the enzyme are r eversible processes. Structural information indicates that the M3 site, whi ch is octahedrally coordinated to Mg2+, has been converted to a distorted t etrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching cons tant for acrylamide (K-sv = 3.86 +/- 0.04 M-1), suggesting a conformational change. Both Mg2+ and phosphate prevent the enzyme from reaching this inac tive structure. GTP plays an important role in reactivating the Zn-inhibite d enzyme activity. We propose that, under physiological conditions, magnesi um 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 cat alysis.