Nucleotide Dependence of Subunit Rearrangements in Short-Form Rubisco Activase from Spinach

Dayna S. Peterson-Forbrook, Matthew T. Hilton, Laura Tichacek, J. Nathan Henderson, Hoang Q. Bui, Rebekka Wachter

    Research output: Contribution to journalArticle

    2 Citations (Scopus)

    Abstract

    Higher-plant Rubisco activase (Rca) plays a critical role in regulating the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In vitro, Rca is known to undergo dynamic assembly-disassembly processes, with several oligomer stoichiometries coexisting over a broad concentration range. Although the hexamer appears to be the active form, changes in quaternary structure could play a role in Rubisco regulation. Therefore, fluorescent labels were attached to the C-termini of spinach β-Rca, and the rate of subunit mixing was monitored by measuring energy transfer as a function of nucleotide and divalent cation. Only dimeric units appeared to exchange. Poorly hydrolyzable substrate analogues provided locked complexes with high thermal stabilities (apparent Tm = 60 °C) and an estimated t1/2 of at least 7 h, whereas ATP-Mg provided tight assemblies with t1/2 values of 30-40 min and ADP-Mg loose assemblies with t1/2 values of <15 min. Accumulation of ADP to 20% of the total level of adenine nucleotide substantially accelerated equilibration. An initial lag period was observed with ATP·Mg, indicating inhibition of subunit exchange at low ADP concentrations. The ADP Ki value was estimated to exceed the Km for ATP (0.772 ± 96 mM), suggesting that the equilibration rate is a function of the relative contributions of high- and low-affinity states. C-Terminal cross-linking generated covalent dimers, required the N-terminal extension to the AAA+ domain, and provided evidence of different classes of sites. We propose that oligomer reorganization may be stalled during periods of high Rubisco reactivation activity, whereas changes in quaternary structure are stimulated by the accumulation of ADP at low light levels.

    Original languageEnglish (US)
    Pages (from-to)4906-4921
    Number of pages16
    JournalBiochemistry
    Volume56
    Issue number36
    DOIs
    StatePublished - Sep 12 2017

    Fingerprint

    Oxygenases
    Spinacia oleracea
    Tissue Plasminogen Activator
    Adenosine Diphosphate
    Nucleotides
    Ribulose-Bisphosphate Carboxylase
    Oligomers
    Adenosine Triphosphate
    Adenine Nucleotides
    Energy Transfer
    Divalent Cations
    Stoichiometry
    Dimers
    Energy transfer
    Labels
    Thermodynamic stability
    Hot Temperature
    ribulose-1,5 diphosphate
    Light
    Substrates

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Peterson-Forbrook, D. S., Hilton, M. T., Tichacek, L., Nathan Henderson, J., Bui, H. Q., & Wachter, R. (2017). Nucleotide Dependence of Subunit Rearrangements in Short-Form Rubisco Activase from Spinach. Biochemistry, 56(36), 4906-4921. https://doi.org/10.1021/acs.biochem.7b00574

    Nucleotide Dependence of Subunit Rearrangements in Short-Form Rubisco Activase from Spinach. / Peterson-Forbrook, Dayna S.; Hilton, Matthew T.; Tichacek, Laura; Nathan Henderson, J.; Bui, Hoang Q.; Wachter, Rebekka.

    In: Biochemistry, Vol. 56, No. 36, 12.09.2017, p. 4906-4921.

    Research output: Contribution to journalArticle

    Peterson-Forbrook, DS, Hilton, MT, Tichacek, L, Nathan Henderson, J, Bui, HQ & Wachter, R 2017, 'Nucleotide Dependence of Subunit Rearrangements in Short-Form Rubisco Activase from Spinach' Biochemistry, vol. 56, no. 36, pp. 4906-4921. https://doi.org/10.1021/acs.biochem.7b00574
    Peterson-Forbrook DS, Hilton MT, Tichacek L, Nathan Henderson J, Bui HQ, Wachter R. Nucleotide Dependence of Subunit Rearrangements in Short-Form Rubisco Activase from Spinach. Biochemistry. 2017 Sep 12;56(36):4906-4921. https://doi.org/10.1021/acs.biochem.7b00574
    Peterson-Forbrook, Dayna S. ; Hilton, Matthew T. ; Tichacek, Laura ; Nathan Henderson, J. ; Bui, Hoang Q. ; Wachter, Rebekka. / Nucleotide Dependence of Subunit Rearrangements in Short-Form Rubisco Activase from Spinach. In: Biochemistry. 2017 ; Vol. 56, No. 36. pp. 4906-4921.
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