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Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Russia.
Department of Chemistry, University of Pennsylvania, USA.
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Russia.
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Russia.
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1999 (Engelska)Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 260, nr 2, s. 308-317Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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.

Ort, förlag, år, upplaga, sidor
Wiley-Blackwell Publishing Inc., 1999. Vol. 260, nr 2, s. 308-317
Nyckelord [en]
pyrophosphatase, magnesium, Tris, quaternary structure, structural modeling
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:liu:diva-132131DOI: 10.1046/j.1432-1327.1999.00181.xPubMedID: 10095764OAI: oai:DiVA.org:liu-132131DiVA, id: diva2:1045923
Tillgänglig från: 2016-11-11 Skapad: 2016-10-18 Senast uppdaterad: 2017-11-29Bibliografiskt granskad

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Turkina, Maria V.
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