A revised mechanism for the alkaline phosphatase reaction involving three metal ions

Here, X-ray crystallography has been used to investigate the proposed doubl e in-line displacement mechanism of Escherichia coli alkaline phosphatase i n which two of the three active-site metal ions have a direct role in catal ysis. Two new X-ray crystal structures of the wild-type enzyme in the absen ce and presence of inorganic phosphate have been refined at 1.75 Angstrom, to final working R-factors of 15.4% and 16.4%, respectively. In the refinem ent of both structures, residues in the active sites were treated anisotrop ically. The ellipsoids resulting from the partial anisotropic refinement sh ow a clear route for the binding and release of substrate/ product. In addi tion, a direct comparison of the refined structures with and without phosph ate reveal a strong correlation between the occupancy of the third metal-bi nding site and the conformation of the Ser102 nucleophile. These findings c larify two important and unresolved aspects of the previously proposed cata lytic mechanism, how Ser102 is activated for nucleophilic attack and why a magnesium ion in the third metal site is required for catalysis. Analysis o f these results suggest that three metal-ion assisted catalysis is a more a ccurate description of the mechanism of the alkaline phosphatase reaction. A revised mechanism for the catalytic reaction of alkaline phosphatase is p roposed on the basis of the two new X-ray crystal structures reported. (C) 2000 Academic Press.