Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers
1999 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 260, no 2, 308-317 p.Article in journal (Refereed) Published
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 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 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.
Place, publisher, year, edition, pages
Wiley-Blackwell Publishing Inc., 1999. Vol. 260, no 2, 308-317 p.
pyrophosphatase, magnesium, Tris, quaternary structure, structural modeling
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:liu:diva-132131DOI: 10.1046/j.1432-1327.1999.00181.xPubMedID: 10095764OAI: oai:DiVA.org:liu-132131DiVA: diva2:1045923