Abstract
The carbon-fixing activity of enzyme ribulose-1,5-bisphosphate 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 μm 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.
Highlights
The carbon-fixing activity of enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is regulated by Rubisco activase (Rca), a ring-forming ATPase that catalyzes inhibitor release
To elucidate the relationship between ATPase activity and assembly, we have used fluorescence correlation spectroscopy (FCS) methods to monitor the self-association of tobacco Rca from monomeric species to large supramolecular complexes
To label tobacco Rca with a fluorescent label, the S379C substitution was introduced near the C terminus and covalently labeled with AlexaFluor-546 by means of maleimide chemistry
Summary
The carbon-fixing activity of enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is regulated by Rubisco activase (Rca), a ring-forming ATPase that catalyzes inhibitor release. 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. CO2 fixation reactions on earth and is extensively regulated One such mechanism involves competitive inhibition by phosphorylated metabolites and inhibitory binding of the substrate ribulose-1,5-bisphosphate (RuBP) in the absence of active site carbamylation (1–3). The AAAϩ protein Rubisco activase (Rca) uses ATP hydrolysis to catalyze the release of inhibitors from Rubisco sites (4 –7). In X-ray structures of the nucleotide-free AAAϩ domains of tobacco and Arabidopsis Rca, helical assemblies were observed that contained six subunits per turn, but with differing pitch (Fig. 1) (21, 22). Regardless, conserved central pore residues of Rca have been shown to be critical in Rubisco reactivation (21)
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