KINETIC AND STRUCTURAL CONSEQUENCES OF REPLACING THE ASPARTATE BRIDGEBY ASPARAGINE IN THE CATALYTIC METAL TRIAD OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE

In each subunit of the homodimeric enzyme Escherichia coli alkaline ph osphatase, two of the three metal cofactors, Zn2+ and Mg2+, are bound by an aspartate side-chain at position 51. Using site-specific mutagen esis, Asp51 was mutated both to alanine and to asparagine to produce t he D51A and D51N enzymes, respectively. Over the range of pH values ex amined, the D51A enzyme did not catalyze phosphate ester hydrolysis ab ove non-enzymic levels and was not activated by the addition of millim olar excess Zn2+ or Mg2+. Replacement of Asp51 by asparagine, however, resulted in a mutant enzyme with reduced activity and a higher pH opt imum, compared with the wild-type enzyme. At pH 8.0 the D51N enzyme sh owed about 1% of the activity of the wild-type enzyme, and as the pH w as raised to 9.2, the activity of the D51N enzyme increased to about 1 0% 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 fash ion to nearly the same level as the wild type enzyme. The affinity for phosphate of the D51N enzyme decreased tenfold as the concentration o f Mg2+ increased. Under optimal conditions, the k(cat)/K-m ratio for t he D51N enzyme indicated that it was 87% as efficient as the wild-type enzyme. To investigate the molecular basis for the observed kinetic d ifferences, X-ray data were collected for the D51N enzyme to 2.3 Angst rom resolution at pH 7.5, and the to 2.1 Angstrom resolution at pH 9.2 with 20 mM MgCl2. The two structures were then refined. The low magne sium, low pH D51N structure showed that the third metal site was unocc upied, apparently blocked by the amide group of Asn51. At this pH the phosphate anion was bound via one oxygen atom, between the zinc cation s at the first and second metal sites, which strongly resembled the ar rangement 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 close to the surface of the enz yme, coordinated to the first metal site alone. Electron density diffe rence maps provide evidence that magnesium activates the D51N enzyme b y replacing zinc at the second metal site. (C) 1996 Academic Press Lim ited