Conversion of a magnesium binding site into a zinc binding site by a single amino acid substitution in Escherichia coli alkaline phosphatase.

Abstract

The replacement of aspartic acid by histidine at position 153 in Escherichia coli alkaline phosphatase results in a mutant enzyme that is remarkably similar to certain mammalian alkaline phosphatases that are activated by magnesium in a time-dependent fashion.These mammalian alkaline phosphatases have histidine at the position corresponding to 153 of the E. coli sequence.Here we report the three-dimensional structure of the mutant E. coli alkaline phosphatase with histidine at position 153.The structure reveals that the octahedral magnesium binding site has been converted to a tetrahedral zinc binding site with an imidazole ring nitrogen of His-153 as one of the ligands to the zinc.The alteration in metal binding caused by the mutation could explain the origin of the magnesium activation observed with the mammalian alkaline phosphatases.The structure also reveals differences in the mode of phosphate binding, explaining the enhanced phosphate affinity and the reduced activity of the mutant enzyme in the presence of zinc.Alkaline phosphatase (EC 3.1.3.1) is a nonspecific phosphomonoesterase that functions through a phosphoseryl intermediate (1) to produce inorganic phosphate and an alcohol.The alkaline phosphatase from Escherichia coli is a dimeric metalloenzyme that contains two atoms of zinc and one atom of magnesium per monomer.The x-ray structure