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

Suratna Hazra, J. Nathan Henderson, Kevin Liles, Matthew T. Hilton, Rebekka Wachter

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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 [Mg2+] in response to changes in available light.

Original languageEnglish (US)
Pages (from-to)24222-24236
Number of pages15
JournalJournal of Biological Chemistry
Volume290
Issue number40
DOIs
Publication statusPublished - Oct 2 2015

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ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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