KINETICS AND CRYSTAL-STRUCTURE OF A MUTANT ESCHERICHIA-COLI ALKALINE-PHOSPHATASE (ASP-369-]ASN) - A MECHANISM INVOLVING ONE ZINC PER ACTIVE-SITE

Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (k(cat)) and increased Michaelis constants (K-m). The k(cat) for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the k(cat), measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the k(cat)/K-m ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 Angstrom resolution with an R-factor of 0.189, showed Vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.