Refereed journal article or data article (A1)

Reciprocal effects of substitutions at the subunit interfaces in hexameric pyrophosphatase of Escherichia coli - Dimeric and monomeric forms of the enzyme




List of AuthorsSalminen A, Efimova IS, Parfenyev AN, Magretova NN, Mikalahti K, Goldman A, Baykov AA, Lahti R

PublisherAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Publication year1999

JournalJournal of Biological Chemistry

Journal name in sourceJOURNAL OF BIOLOGICAL CHEMISTRY

Journal acronymJ BIOL CHEM

Volume number274

Issue number48

Start page33898

End page33904

Number of pages7

ISSN0021-9258

DOIhttp://dx.doi.org/10.1074/jbc.274.48.33898


Abstract
A homohexameric molecule of Escherichia coli pyrophosphatase is arranged as a dimer of trimers, with an active site present in each of its six monomers. Earlier we reported that substitution of His(136) and His(140) in the intertrimeric subunit interface splits the molecule into active trimers (Velichko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyytia, T., Goldman, A., Cooperman, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemistry 37, 734-740). Here we demonstrate that additional substitutions of Tyr(77) and Gln(80) in the intratrimeric interface give rise to moderately active dimers or virtually inactive monomers, depending on pH, temperature, and Mg2+ concentration. Successive dissociation of the hexamer into trimers, dimers, and monomers progressively decreases the catalytic efficiency (by 10(6)-fold in total), and conversion of a trimer into dimer decreases the affinity of one of the essential Mg2+-binding sites/monomer. Disruptive substitutions predominantly in the intratrimeric interface stabilize the intertrimeric interface and vice versa, suggesting that the optimal intratrimeric interaction is not compatible with the optimal intertrimeric interaction. Because of the resulting "conformational strain," hexameric wild-type structure appears to be preformed to bind substrate. A hexameric triple variant substituted at Tyr(77), Gln(80), and His(136) exhibits positive cooperativity in catalysis, consistent with this model.


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