Binding of magnesium in a mutant Escherichia coli alkaline phosphatase changes the rate-determining step in the reaction mechanism.

To investigate the role of magnesium at the M3 site in Escherichia coli alkaline phosphatase, site-specific mutagenesis was used to substitute Glu-322, a ligand of the Mg2+ with either aspartic acid (E322D) or alanine (E322A). The residual Mg2+ content of the E322D enzyme is about 16-fold lower than that of the wild-type enzyme, and both mutant enzymes exhibit extremely poor catalytic activity compared to the wild-type enzyme. Mg2+ is a strong activator of the E322D enzyme. The hydrolysis activity of the E322D enzyme maximally stimulated by Mg2+ is 60% of that of the wild-type enzyme. Under conditions that measure the sum of hydrolysis and transphosphorylation activities, the kcat of the E322D enzyme in the presence of 500 mM Mg2+ is 2.6-fold higher than the kcat observed for the wild-type enzyme. Zn2+ also activates the E322D enzyme, although it is not as strong an activator as Mg2+. Competition experiments suggest that the activation of the E322D enzyme by Mg2+ and Zn2+ results from binding of either of these metals to the same site on the enzyme. High concentrations of the substrate p-nitrophenyl phosphate inhibit the activity of the E322D enzyme; however, high concentrations of Mg2+ can overcome this inhibition. Stopped-flow experiments indicate that the rate-limiting step of the nonstimulated E322D enzyme at pH 8.0 differs from that of the wild-type enzyme and involves the breaking of the covalent bond between the enzyme and phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)