Assembly-disassembly is coupled to the ATPase cycle of tobacco Rubisco activase.

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.

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

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 ß-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.