Hen's egg yolk alkaline phosphatase: general characterization and kinetic study with inhibitors.

1. The non-specific hen's egg yolk alkaline phosphatase is a metalloprotein (Zn2+?) composed of two identical inactive subunits. 2. A second metal site preferably binds Mg2+ (15-fold activation). Me(II)(H2O)H+, a charged arginine, and tyrosine in the active site are involved in positioning and binding of the substrate and metal ion. 3. Substrate inhibition differs with pH. This may be related to the presence of two active sites in the enzyme, one in each subunit. 4. Uncompetitive inhibition with l-phenylalanine and analogues suggests a phosphorylated intermediate. 5. Inhibition is weakly competitive with Pi, strong non-competitive with PPi as compared to Mg2+- free PPi, and partially competitive with arsenate. 6. The purified enzyme is stabilized and activated by amines and proteins.