Rubisco catalyzes the incorporation of CO2 to the C-2 of the enediol form of ribulose-1,5-bisphosphate and the subsequent cleavage of the unstable intermediate to yield two molecules of 3-phosphoglycerate (see Web Topic 8.3 and textbook Figure 8.4) The conversion of the inactive form of rubisco to an active state, which is required for both the carboxylation and oxygenation reactions, requires the carbamylation of a specific lysine residue (in spinach, Lys-201) (Web Figure 8.5.A). As a consequence of the binding of the CO2– molecule to the lysine residue, the enzyme acquires a triad of anionic residues that provide the binding site for the cofactor Mg2+ (in spinach, the other two residues are Asp-203 and Glu-204). At this stage, the incorporation of ribulose-1,5-bisphosphate causes conformational changes in rubisco that allows the enzyme to sequester the sugar bisphosphate from the solvent and, upon the binding of a CO2 or O2 molecule, to start the catalytic cycle. However, the tight binding of ribulose-1,5-bisphosphate to the uncarbamylated enzyme molecules displaces the equilibrium to the dead end complex, rubisco-ribulose-1,5-bisphosphate, so that the rate of reaction declines rapidly until it finally stops. How is this problem solved?
A combination of genetic, physiological and biochemical studies have provided the solution to the long-term riddle of rubisco activation. In vivo, some mutants of Arabidopsis thaliana lack light-mediated activation of rubisco and can only grow at high concentrations of CO2. Surprisingly, the mutation does not impair the response of the enzyme to modulation by CO2 and Mg2+ in vitro. These contradictory results led to the discovery of a protein, rubisco-activase, that uncouples ribulose-1,5-bisphosphate from decarbamylated active sites and, in so doing, promotes the access of CO2 and Mg2+ for the carbamylation of the enzyme.The importance of this process is that the rate at which rubisco-activase enhances the catalytic capacity of rubisco is linked to the hydrolysis of ATP (50 molecules of ATP hydrolyzed / 1 rubisco site activated). Two isoforms of rubisco activase, (42 and 46 kDa) found in several species differ in the C-terminal region as result of the differential splicing of a common pre-mRNA. Both isoforms catalyze the activation of rubisco and the hydrolysis of ATP but the smaller isoform has higher capacity than the larger isoform for both activities. Evidence for the fact that the two processes are independent are provided by studies showing that the digestion of native rubisco-activase with trypsin or the modification of its primary structure by site-directed mutagenesis do not elicit the same responses in the stimulation of rubisco and the hydrolysis of ATP (Esau et al. 1996, Kallis et al. 2000). More importantly, the deletion of some residues at the C-region of rubisco-activase doubles the rate of rubisco activation with only a minor effect on the hydrolysis of ATP.
Rubisco activation by the activase requires a mutual recognition of both proteins. Rubisco-activase from tobacco (Solanaceae) fails to activate rubisco from spinach or Chlamydomonas (non-Solanaceae) and, similarly, the rubisco-activase from spinach or Chlamydomonas fails to stimulate rubisco from tobacco. However, the substitution of some residues on the surface of the large subunit of Chlamydomonas rubisco shifts the specificity from non-Solanaceae to Solanaceae rubisco-activase (Ott et al. 2000). These experiments underscore the contribution of specific amino acids to protein-protein interaction and provide experimental evidence for a recognition step separated from the subsequent activation process.