A1 Refereed original research article in a scientific journal
Directed mutagenesis studies of the metal binding site at the subunit interface of Escherichia coli inorganic pyrophosphatase
Authors: Efimova IS, Salminen A, Pohjanjoki P, Lapinniemi J, Magretova NN, Cooperman BS, Goldman A, Lahti R, Baykov AA
Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Publication year: 1999
Journal: Journal of Biological Chemistry
Journal name in source: JOURNAL OF BIOLOGICAL CHEMISTRY
Journal acronym: J BIOL CHEM
Volume: 274
Issue: 6
First page : 3294
Last page: 3299
Number of pages: 6
ISSN: 0021-9258
DOI: https://doi.org/10.1074/jbc.274.6.3294
Abstract
Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (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 interface (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 hexamer 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 D26S and D26N variants, when little or no effect is seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the same magnitude for wild type E-PPase and the three variants. The affinity of the intertrimer site for Mg2+ at pH 7.2 is intermediate between those of two Mg2+ binding sites found in the E-PPase active 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.
Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (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 interface (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 hexamer 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 D26S and D26N variants, when little or no effect is seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the same magnitude for wild type E-PPase and the three variants. The affinity of the intertrimer site for Mg2+ at pH 7.2 is intermediate between those of two Mg2+ binding sites found in the E-PPase active 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.
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