Assembled F1-(αβ) and hybrid F1-α 3β3γ-ATPases from Rhodospirillum rubrum α, wild type or mutant β, and chloroplast γ subunits. Demonstration of Mg2+ versus Ca2+-induced differences in catalytic site structure and function

Ziyun Du, Ward C. Tucker, Mark L. Richter, Zippora Gromet-Elhanan

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Refolding together the expressed α and β subunits of the Rhodospirillum rubrum F1(RF1)-ATPase led to assembly of only α1β1 dimers, showing a stable low MgATPase activity. When incubated in the presence of AlCl3, NaF and either MgAD(T)P or CaAD(T)P, all dimers associated into closed α 3β3 hexamers, which also gained a low CaATPase activity. Both hexamer ATPase activities exhibited identical rates and properties to the open dimer MgATPase. These results indicate that: a) the hexamer, as the dimer, has no catalytic cooperativity; b) aluminium fluoride does not inhibit their MgATPase activity; and c) it does enable the assembly of RrF13β3 hexamers by stabilizing their noncatalytic α/β interfaces. Refolding of the RrF 1-α and β subunits together with the spinach chloroplast F1 (CF1)-γ enabled a simple one-step assembly of two different hybrid RrF13β 3/CF1γ complexes, containing either wild type RrF1-β or the catalytic site mutant RrF1β-T159S. They exhibited over 100-fold higher CaATPase and MgATPase activities than the stabilized hexamers and showed very different catalytic properties. The hybrid wild type MgATPase activity was, as that of RrF1 and CF1 and unlike its higher CaATPase activity, regulated by excess free Mg 2+ ions, stimulated by sulfite, and inhibited by azide. The hybrid mutant had on the other hand a low CaATPase but an exceptionally high MgATPase activity, which was much less sensitive to the specific MgATPase effectors. All these very different ATPase activities were regulated by thiol modulation of the hybrid unique CF1-γ disulfide bond. These hybrid complexes can provide information on the as yet unknown factors that couple ATP binding and hydrolysis to both thiol modulation and rotational motion of their CF 1-γ subunit.

Original languageEnglish (US)
Pages (from-to)11517-11523
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number15
DOIs
StatePublished - Apr 13 2001

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Rhodospirillum rubrum
Proton-Translocating ATPases
Chloroplasts
Dimers
Adenosine Triphosphatases
Catalytic Domain
Demonstrations
Sulfhydryl Compounds
Modulation
Sulfites
Azides
Disulfides
Spinacia oleracea
Hydrolysis
Adenosine Triphosphate
Ions

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Assembled F1-(αβ) and hybrid F1-α 3β3γ-ATPases from Rhodospirillum rubrum α, wild type or mutant β, and chloroplast γ subunits. Demonstration of Mg2+ versus Ca2+-induced differences in catalytic site structure and function. / Du, Ziyun; Tucker, Ward C.; Richter, Mark L.; Gromet-Elhanan, Zippora.

In: Journal of Biological Chemistry, Vol. 276, No. 15, 13.04.2001, p. 11517-11523.

Research output: Contribution to journalArticle

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abstract = "Refolding together the expressed α and β subunits of the Rhodospirillum rubrum F1(RF1)-ATPase led to assembly of only α1β1 dimers, showing a stable low MgATPase activity. When incubated in the presence of AlCl3, NaF and either MgAD(T)P or CaAD(T)P, all dimers associated into closed α 3β3 hexamers, which also gained a low CaATPase activity. Both hexamer ATPase activities exhibited identical rates and properties to the open dimer MgATPase. These results indicate that: a) the hexamer, as the dimer, has no catalytic cooperativity; b) aluminium fluoride does not inhibit their MgATPase activity; and c) it does enable the assembly of RrF1-α3β3 hexamers by stabilizing their noncatalytic α/β interfaces. Refolding of the RrF 1-α and β subunits together with the spinach chloroplast F1 (CF1)-γ enabled a simple one-step assembly of two different hybrid RrF1-α3β 3/CF1γ complexes, containing either wild type RrF1-β or the catalytic site mutant RrF1β-T159S. They exhibited over 100-fold higher CaATPase and MgATPase activities than the stabilized hexamers and showed very different catalytic properties. The hybrid wild type MgATPase activity was, as that of RrF1 and CF1 and unlike its higher CaATPase activity, regulated by excess free Mg 2+ ions, stimulated by sulfite, and inhibited by azide. The hybrid mutant had on the other hand a low CaATPase but an exceptionally high MgATPase activity, which was much less sensitive to the specific MgATPase effectors. All these very different ATPase activities were regulated by thiol modulation of the hybrid unique CF1-γ disulfide bond. These hybrid complexes can provide information on the as yet unknown factors that couple ATP binding and hydrolysis to both thiol modulation and rotational motion of their CF 1-γ subunit.",
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