Regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase: product inhibition, cooperativity, and magnesium activation.

In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mm tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min(-1), the Km for ATP by 0.104 ± 0.024 mm, and the Ki for ADP by 0.037 ± 0.007 mm. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg(2+)] in response to changes in available light.

ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase hexamer formation at low micromolar concentrations.

We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton ß-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 µM). Hexamer fractions peak at 30 µM and dominate at 8-70 µM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 µM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 µM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

Biophysical characterization of higher plant Rubisco activase.

Rubisco activase (Rca) is a chaperone-like protein of the AAA+ family, which uses mechano-chemical energy derived from ATP hydrolysis to release tightly bound inhibitors from the active site of the primary carbon fixing enzyme ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco). Mechanistic and structural investigations of Rca have been hampered by its exceptional thermolability, high degree of size polydispersity and propensity towards subunit aggregation. In this work, we have characterized the thermal stability and self-association behavior of recombinant Rca preparations, and have developed ligand screening methods. Thermal denaturation profiles generated by circular dichroism indicate that creosote and tobacco short-form Rcas are the most stable proteins examined, with an estimated mid-point temperature of 45-47°C for protein denaturation. We demonstrate that ADP provides a higher degree of stabilization than ATP, that magnesium ions have a small stabilizing effect on ATP-bound, but a significant destabilizing effect on ADP-bound Rca, and that phosphate provides weak stabilization of the ADP-bound form of the protein. A dimeric species was identified by size-exclusion chromatography, suggesting that the two-subunit module may comprise the basic building block for larger assemblies. Evidence is provided that chromatographic procedures reflect non-equilibrium multimeric states. Dynamic light scattering experiments performed on nucleotide-bearing Rca support the notion that several larger, highly polydisperse assembly states coexist over a broad concentration range. No significant changes in aggregation are observed upon replacement of ADP with ATP. However, in the absence of nucleotides, the major protein population appears to consist of a monodisperse oligomer smaller than a hexamer.