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

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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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