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?

Web Figure 8.5.A   Modulation of rubisco by rubisco-activase. The combination or the interaction of rubisco (E) with modulators distributes the enzyme among different states (yellow boxes). The unprotonated form of a specific lysine residue (E-NH₂) reacts with CO₂ releasing a proton and forming the carbamate (E-NH-CO₂^-) that is stabilized by the cofactor Mg²⁺ (E-NH-CO₂^–. Mg²⁺). At this stage (blue box), rubisco is catalytically competent and thereby binds ribulose-1,5-bisphosphate (RuBP) yielding the ternary complex (E-NH-CO₂^– . Mg²⁺ . RuBP) in which the latter is converted to the enediol form. This entails the incorporation of CO₂ or O₂ in the active site to generate the products of carboxylation or oxygenation, respectively. The high affinity of ribulose-1,5-bisphosphate for free rubisco (E-NH³⁺) generates the dead-end complex (E-NH³⁺ . RuBP) thereby displacing the above equilibria to a catalytically incompetent state. Rubisco-activase (RA) interacts noncovalently with the binary complex and facilitates the release of ribulose-1,5-bisphosphate (red box) freeing the enzyme for the next carbamylation cycle. This process requires the concurrent hydrolysis of ATP.

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.