Differentiation of the slow-binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase.
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 microM). 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.
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Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China. ibio33@ndmctsgh.edu.tw
Chang
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D000469:Alkaline Phosphatase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D001665:Binding Sites; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006801:Humans; D008274:Magnesium / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D020011:Protective Agents / Q000494:pharmacology; D011487:Protein Conformation; D013997:Time Factors; D015032:Zinc / Q000494:pharmacology*
D010710:Phosphates; D020011:Protective Agents; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc