NanoESI Mass Spectrometry of Rubisco and Rubisco Activase Structures and Their Interactions with Nucleotides and Sugar Phosphates |
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Authors: | Michelle J Blayney Spencer M Whitney Jennifer L Beck |
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Institution: | (1) School of Chemistry, University of Wollongong, New South Wales, 2522, Australia;(2) Plant Science Division, Research School of Biology, Australian National University, Canberra, Australia; |
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Abstract: | Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the protein that is responsible for the fixation of carbon dioxide
in photosynthesis. Inhibitory sugar phosphate molecules, which can include its substrate ribulose-1,5-bisphosphate (RuBP),
can bind to Rubisco catalytic sites and inhibit catalysis. These are removed by interaction with Rubisco activase (RA) via
an ATP hydrolytic reaction. Here we show the first nanoESI mass spectra of the hexadecameric Rubisco and of RA from a higher
plant (tobacco). The spectra of recombinant, purified RA revealed polydispersity in its oligomeric forms (up to hexamer) and
that ADP was bound. ADP was removed by dialysis against a high ionic strength solution and nucleotide binding experiments
showed that ADP bound more tightly to RA than AMP-PNP (a non-hydrolysable ATP analog). There was evidence that there may be
two nucleotide binding sites per RA monomer. The oligomerization capacity of mutant and wild-type tobacco RA up to hexamers
is analogous to the subunit stoichiometry for other AAA+ enzymes. This suggests assembly of RA into hexamers is likely the
most active conformation for removing inhibitory sugar phosphate molecules from Rubisco to enable its catalytic competency.
Stoichiometric binding of RuBP or carboxyarabinitol bisphosphate (CABP) to each of the eight catalytic sites of Rubisco was
observed. |
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Keywords: | |
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