PMID:10095764
| Citation |
Baykov, AA, Hyytiä, T, Turkina, MV, Efimova, IS, Kasho, VN, Goldman, A, Cooperman, BS and Lahti, R (1999) Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers. Eur. J. Biochem. 260:308-17 |
|---|---|
| Abstract |
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, we found that the previous data markedly underestimate Mg(2+)-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, Mg(2+)-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 notion 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. |
| Links | |
| Keywords |
Binding Sites; Catalysis; Escherichia coli; Hydrogen-Ion Concentration; Kinetics; Magnesium/metabolism; Models, Chemical; Models, Molecular; Protein Conformation; Pyrophosphatases/metabolism; Tromethamine |
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