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

Abstract

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…