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.

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.