MAGNESIUM IN THE ACTIVE-SITE OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE IS IMPORTANT FOR BOTH STRUCTURAL STABILIZATION AND CATALYSIS

Site-specific mutagenesis was used to explore the roles of the side chains of residues Lys-328 and Asp-153 in Escherichia coli alkaline phosphatase. The D153H enzyme exhibits a 3.5-fold decrease in activity at pH 8.0 compared to that of the wild-type enzyme, while a double mutant D153H/K328H exhibits a 16-fold decrease in activity under these conditions. However, the K(m) values for both enzymes, employing the substrate p-nitrophenyl phosphate, are lower than the value for the wild-type enzyme. The K(i) for phosphate, which is pH- and Mg2+-dependent, is decreased for the D153H enzyme and increased for the D153H/K328H enzyme. Relative to the wild-type enzyme, both mutant enzymes bind Mg2+ more weakly and undergo a time-dependent activation induced by Mg2+. The half-time of the activation process is independent of the Mg2+ concentration, indicating that the activation most probably involves a conformational change. The pH versus activity profiles of both enzymes are altered relative to that of the wild-type enzyme and exhibit greatly enhanced activity, relative to that of the wild-type enzyme, at high pH values. The pre-steady-state kinetics for the D153H and D153H/K328H enzymes exhibit a transient burst of product formation at pH 8.0, under conditions at which the wild-type enzyme exhibits no transient burst, indicating that at pH 8.0 the hydrolysis of the covalent enzyme-phosphate complex is rate-determining and not the release of phosphate from the noncovalent enzyme-phosphate complex as is observed for the wild-type enzyme. Therefore, these mutations are directly influencing catalysis. The introduction of either the D153H or the D153H/K328H mutations reduces the heat stability of the enzyme, whereas the K328H mutation alone exhibits the same heat stability as the wild-type enzyme. Energy minimization and Langevin molecular dynamics calculations suggest that the enhanced phosphate affinity of the D153H enzyme is due to the repositioning of Lys-328 so that it can directly interact with the phosphate and thereby stabilize its binding to the enzyme. These data also indicate that although Asp-153 is not a direct ligand to Mg2+ in the wild-type enzyme, it plays a role in stabilizing the binding of Mg2+ in the M3 site and indicates that the Mg2+ at the M3 site is important for catalysis by stabilizing the active conformation of the enzyme. The introduction of histidine residues at both position 153 and 328 results in an enzyme that is remarkably similar to mammalian alkaline phosphatases in terms of pH versus activity profile, heat stability, maximal activity, and stimulation by Mg2+, suggesting that these two mutations are responsible for many of the differences in properties between the E. coli and mammalian alkaline phosphatases.