Directed mutagenesis studies of the metal binding site at the subunit interface of Escherichia coli inorganic pyrophosphatase

Recent crystallographic studies on Escherichia coli inorganic pyrophosphata se (E-PPase) have identified three Mg2+ ions/enzyme hexamer in water-filled cavities formed by Asn(24), Ala(25), and Asp(26) at the trimer-trimer inte rface (Kankare, J., Salminen, T., Lahti, R., Cooperman, B., Baykov, A. A., and Goldman, A. (1996) Biochemistry 35, 4670-4677). Here we show that D26S and D26N substitutions decrease the stoichiometry of tight Mg2+ binding to E-PPase by approximately 0.5 mol/mol monomer and increase hexamer stability in acidic medium. Mg2+ markedly decelerates the dissociation of enzyme hex amer into trimers at pH 5.0 and accelerates hexamer formation from trimers at pH 7.2 with wild type E-PPase and the N24D variant, in contrast to the D 26S and D26N variants, when little or no effect is seen. The catalytic para meters describing the dependences of enzyme activity on substrate and Mg2concentrations are of the same magnitude for wild type E-PPase and the thre e variants. The affinity of the intertrimer site for Mg2+ at pH 7.2 is inte rmediate between those of two Mg2+ binding sites found in the E-PPase activ e site. It is concluded that the metal ion binding site found at the trimer -trimer interface of E-PPase is a high affinity site whose occupancy by Mg2 + greatly stabilizes the enzyme hexamer but has little effect on catalysis.