In each subunit of the homodimeric enzyme Escherichia coli alkaline phosphatase, two of the three metal cofactors Zn2+ and Mg2+, are bound by an aspartate side-chain at position 51. Using site-specific mutagenesis, Asp51 was mutated both to alanine and to asparagine to produce the D51A and D51N enzymes, respectively. Over the range of pH values examined, the D51A enzyme did not catalyze phosphate ester hydrolysis above non-enzymic levels and was not activated by the addition of millimolar excess Zn2+ or Mg2+. Replacement of Asp51 by asparagine, however, resulted in a mutant enzyme with reduced activity and a higher pH optimum, compared with the wild-type enzyme. At pH 8.0 the D51N enzyme showed about 1% of the activity of the wild-type enzyme, and as the pH was raised to 9.2, the activity of the D51N enzyme increased to about 10% of the value for the wild-type enzyme. Upon the addition of excess Mg2+ at pH 9.2, the D51N enzyme was activated in a time-dependent fashion to nearly the same level as the wild-type enzyme. The affinity for phosphate of the D51N enzyme decreased tenfold as the concentration of Mg2+ increased. Under optimal conditions, the k(cat)/K(m) ratio for the D51N enzyme indicated that it was 87% as efficient as the wild-type enzyme. To investigate the molecular basis for the observed kinetic differences, X-ray data were collected for the D51N enzyme to 2.3 angstroms resolution at pH 7.5, and then to 2.1 angstroms resolution at pH 9.2 with 20 mM MgCl2. The two structures were then refined. The low magnesium, low pH D51N structure showed that the third metal site was unoccupied, apparently blocked by the amide group of Asn51. At this pH the phosphate anion was bound via one oxygen atom, between the zinc cations at the first and second metal sites, which strongly resembled the arrangement previously determined for the D153H enzyme at pH 7.5. In the high magnesium, high pH D51N structure, the third metal site was also vacant, but the phosphate anion bound closer to the surface of the enzyme, coordinated to the first metal site alone. Electron density difference maps provide evidence that magnesium activates the D51N enzyme by replacing zinc at the second metal site.