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

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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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