Abstract
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.
Highlights
Rubisco activase (Rca) maintains carbon fixation, coordinates photosynthetic pathways, and regulates darklight transitions
We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation
ATPase Activity of Tobacco -Rca Provides a Michaelis Constant in the Mid-micromolar Range—To examine the regulation of Rca turnover by the available ATP and ADP pools, a continuous enzyme-coupled ATPase assay was developed that monitors the kinetics of phosphate (Pi) release, while tolerating moderate amounts of ADP in the reaction (EnzCheck assay) [53]
Summary
Rubisco activase (Rca) maintains carbon fixation, coordinates photosynthetic pathways, and regulates darklight transitions. Results: Positive cooperativity of ATP hydrolysis is afforded by ADP binding to oligomeric assemblies in the Rca-Mg-ATP-Mg state. Conclusion: Magnesium-mediated allosteric regulation involves the coexistence of ATP- and ADP-bound states. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ؎ 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. 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. 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. 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 [Mg2؉] in response to changes in available light
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