Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers

Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, nle found that the previous data markedly underestimate Mg2+-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg2+-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer, These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed, As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the nation that one of the tightly binding sites is located at the trimer-trimer interface, Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.