Crystallographic observations of the metal ion triple in the active site region of alkaline phosphatase.

Diffraction analysis reveals three metal ion binding sites, M1, M2 and M3, in each of two symmetric active centers 32 A apart in alkaline phosphatase from Escherichia coli with intermediate distances within the center of 4, 5 and 7 A for M1-M2, M2-M3 and M1-M3, respectively. A fourth site, M4, has been reported 25 A away. Arsenate, a product analog, binds adjacent to M1 and M2. The active serine residue, 102, which is phosphorylated during normal enzymatic turnover, is also adjacent to M1 and M2 and arginine 166 is adjacent to the arsenate. The implication with respect to the mechanism is that M1, M2 and Arg 166 neutralize and redistribute charges within the phosphate group, activate the serine hydroxyl, and stabilize transition states during bond formation and breakage. Three sites, A, B and C, have been deduced from solution studies and defined specifically on the basis of nuclear magnetic resonance data, binding studies and activity data. The evidence suggests correspondence of A to M1, B to M2, and C to M3. Strong antagonism between binding at M1 and M2 is evidenced crystallographically by a pseudo-saturation, which is relieved by phosphate binding. Local destabilization of the protein, particularly residues 323 through 333, is produced by removal of metals from the crystal.