Assembly– disassembly is coupled to the ATPase cycle of tobacco Rubisco activase

Andrew J. Serban, Isabella L. Breen, Hoang Q. Bui, Marcia Levitus, Rebekka Wachter

    Research output: Contribution to journalArticle

    Abstract

    The carbon-fixing activity of enzyme ribulose-1,5-bisphos-phate carboxylase/oxygenase (Rubisco) is regulated by Rubisco activase (Rca), a ring-forming ATPase that catalyzes inhibitor release. For higher plant Rca, the catalytic roles played by different oligomeric species have remained obscure. Here, we utilized fluorescence-correlation spectroscopy to estimate dissociation constants for the dimer–tetramer, tetramer– hexamer, hexamer–12-mer, and higher-order assembly equilibria of tobacco Rca. A comparison of oligomer composition with ATPase activity provided evidence that assemblies larger than hexamers are hydrolytically inactive. Therefore, supramolecular aggregates may serve as storage forms at low-energy charge. We observed that the tetramer accumulates only when both substrate and product nucleotides are bound. During rapid ATP turnover, about one in six active sites was occupied by ADP, and 36% of Rca was tetrameric. The steady-state catalytic rate reached a maximum between 0.5 and 2.5 M Rca. In this range, significant amounts of dimers, tetramers, and hexamers coexisted, although none could fully account for the observed activity profile. Therefore, we propose that dynamic assembly– disassembly partakes in the ATPase cycle. According to this model, the association of dimers with tetramers generates a hexamer that forms a closed ring at high ATP and magnesium levels. Upon hydrolysis and product release, the toroid breaks open and dissociates into a dimer and tetramer, which may be coupled to Rubisco remodeling. Although a variant bearing the R294V substitution assembled in much the same way, highly stabilized states could be generated by binding of a transition-state analog. A tight-binding pre-hydrolysis state appears to become more accessible in thermally labile Rcas.

    Original languageEnglish (US)
    Pages (from-to)19451-19465
    Number of pages15
    JournalJournal of Biological Chemistry
    Volume293
    Issue number50
    DOIs
    StatePublished - Jan 1 2018

    Fingerprint

    Oxygenases
    Tobacco
    Tissue Plasminogen Activator
    Ribulose-Bisphosphate Carboxylase
    Adenosine Triphosphatases
    Dimers
    Hydrolysis
    Bearings (structural)
    Adenosine Triphosphate
    Fluorescence Spectrometry
    Oligomers
    Adenosine Diphosphate
    Magnesium
    Catalytic Domain
    Substitution reactions
    Carbon
    Nucleotides
    Fluorescence
    Association reactions
    Spectroscopy

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Cite this

    Assembly– disassembly is coupled to the ATPase cycle of tobacco Rubisco activase. / Serban, Andrew J.; Breen, Isabella L.; Bui, Hoang Q.; Levitus, Marcia; Wachter, Rebekka.

    In: Journal of Biological Chemistry, Vol. 293, No. 50, 01.01.2018, p. 19451-19465.

    Research output: Contribution to journalArticle

    Serban, Andrew J. ; Breen, Isabella L. ; Bui, Hoang Q. ; Levitus, Marcia ; Wachter, Rebekka. / Assembly– disassembly is coupled to the ATPase cycle of tobacco Rubisco activase. In: Journal of Biological Chemistry. 2018 ; Vol. 293, No. 50. pp. 19451-19465.
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    abstract = "The carbon-fixing activity of enzyme ribulose-1,5-bisphos-phate carboxylase/oxygenase (Rubisco) is regulated by Rubisco activase (Rca), a ring-forming ATPase that catalyzes inhibitor release. For higher plant Rca, the catalytic roles played by different oligomeric species have remained obscure. Here, we utilized fluorescence-correlation spectroscopy to estimate dissociation constants for the dimer–tetramer, tetramer– hexamer, hexamer–12-mer, and higher-order assembly equilibria of tobacco Rca. A comparison of oligomer composition with ATPase activity provided evidence that assemblies larger than hexamers are hydrolytically inactive. Therefore, supramolecular aggregates may serve as storage forms at low-energy charge. We observed that the tetramer accumulates only when both substrate and product nucleotides are bound. During rapid ATP turnover, about one in six active sites was occupied by ADP, and 36{\%} of Rca was tetrameric. The steady-state catalytic rate reached a maximum between 0.5 and 2.5 M Rca. In this range, significant amounts of dimers, tetramers, and hexamers coexisted, although none could fully account for the observed activity profile. Therefore, we propose that dynamic assembly– disassembly partakes in the ATPase cycle. According to this model, the association of dimers with tetramers generates a hexamer that forms a closed ring at high ATP and magnesium levels. Upon hydrolysis and product release, the toroid breaks open and dissociates into a dimer and tetramer, which may be coupled to Rubisco remodeling. Although a variant bearing the R294V substitution assembled in much the same way, highly stabilized states could be generated by binding of a transition-state analog. A tight-binding pre-hydrolysis state appears to become more accessible in thermally labile Rcas.",
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