Engineering a new magnesium binding site in the subunit contact region of Escherichia coli inorganic pyrophosphatase

Three Gln-80 residues belonging to different subunits of homohexameric Esch erichia coli pyrophosphatase are separated by only one water molecule to wh ich they are hydrogen bonded. Substitution of Glu for Gln-80 stabilizes qua ternary structure of the enzyme but has only a small effect on enzyme activ ity. The substitution stimulates Mg2+ binding and changes the appearance of the Mg2+ concentration dependence of the rate constant for the trimer --> hexamer transition. These data suggest that a new Mg2+ binding site is form ed in the intersubunit contact region as a result of the substitution. Thre e-dimensional modeling of the mutated protein showed that a chelate complex might form involving two of the three Glu-80 residues.