A WATER-MEDIATED SALT LINK IN THE CATALYTIC SITE OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE MAY INFLUENCE ACTIVITY

Escherichia coli alkaline phosphatase catalyzes the hydrolysis of a wide variety of phosphomonoesters at similar rates, and the reaction proceeds through a phosphoenzyme intermediate. The active site region is highly conserved between the E. coli and mammalian alkaline phosphatases. The three-dimensional structure of the E. coli enzyme indicates that Lys-328, which is replaced by histidine in all mammalian alkaline phosphatases, is bridged to the phosphate through a water molecule. This water molecule is also hydrogen bonded to Asp-327, a bidendate ligand of the one of the two zinc atoms. Here we report the use of site-specific mutagenesis to convert Lys-328 to both histidine and alanine. Steady-state kinetic studies above pH 7.0 indicate that both mutant enzymes have altered pH versus activity profiles compared to the profile for the wild-type enzyme. At pH 10.3, in the presence of Tris, the Lys-328 --> Ala enzyme is approximately 14-fold more active than the wild-type enzyme. At the same pH in the absence of Tris the Lys-328 --> Ala enzyme is still 6-fold more active than the wild-type enzyme. Both mutant enzymes have lower phosphate affinities than the wild-type enzyme at all pH values investigated. Pre-steady-state kinetics at pH 5.5 reveal that the Lys-328 --> Ala enzyme behaves very similar to the phosphate-free wild-type enzyme. However, at pH 8.0, as opposed to the wild-type enzyme that does not exhibit a transient phase, the mutant enzyme shows a small transient phase in the pre steady state, suggesting a possible change in the rate-limiting step for this mutant enzyme at this pH. The properties of the mutant enzymes can be rationalized on the basis of a reduction in phosphate affinity. These data also suggest a possible alteration of the pK(a) of the zinc-coordinated hydroxyl group.