## 9 Rubisco Kinetics
Figure 16: (**a**) \(k_{\text{cat,C}}\) vs. S\({}_{\text{C/O}}\) dependence for various Rubiscos measured in vitro at 25 \({}^{\circ}\)C. The organisms from which the Rubiscos were isolated were: Ag, _Artiplex glabiruscalu_ (C3 dicot); Ah, _Amaranthus hybridus_ (C4 dicot); Av, _Anabena uraibilis_ (cyanobacterium); Cr, _Chlamydomonas reinhardtii_ (green alga); Cv, _Chromatium vinosum_ (bacterium); Eg, _Euglena gracilis_ (green alga); Gm, _Griffithsian monilis_ (red alga); Gs, _Galdieria sulfuraria_ (red alga); Nt, _Nicotiana tabacum_ (C3 dicot); Os, _Oryza sativa_ (C3 monocot); Pt, _Pheadecylum tricornutum_ (diatom); Rr, _Rhodospirillum rubrum_ (bacterium); Rs, the bacterial symbiont of the tubeworm _Riftia pachytplitis_; Sb, _Sorghum bicolor_ (C4 monocot); So, _Spinacia olerca_ (C3 dicot); S6301, _Synechococcus_ PCC 6301 (cyanobacterium); 57002, _Synechococcus_ PCC 7002 (cyanobacterium); Ta, _Tritcium aestivum_ (C3 monocot); and Zm, _Zea mays_ (C4 monocot). Dotted line is a linear regression through all the data except for cyanobacteria (S6301 and S7002) which were excluded. (**b**) Energy profile along the Rubisco-catalyzed carboxylation reaction evidencing the structure of Transition States and Intermediates (Figure 16 from ref. [50], adapted and used by permission of PNAS (copyright © 2006)). The major feature of the diagram shown in Figure 16b is the appearance of three energetic barriers (for the CO\({}_{2}\)-addition reaction, carboxylic acid hydration and C2-C3-gemdiolate cleavage, respectively), with the \"C2-C3-gemdiolate cleavage\" barrier being the higher, which identifies the rate determining step. Most important, the authors have proposed that different Rubisco enzymes have evolved by optimizing the energetic profile in response to the CO\({}_{2}\):O\({}_{2}\) ratio available at the subcellular environment, which is characteristic of each species. Moreover, in order to increase the ability of Rubisco to discriminate between CO\({}_{2}\) and O\({}_{2}\) (enhancing thus the CO\({}_{2}\)/O\({}_{2}\) specificity) the authors propose that the TS 1 for the CO\({}_{2}\)-addition reaction (first energetic barrier in Figure 16b) must be product-like. In other words, the enzyme active site must possess a high affinity for the enediolate carboxylate TS 1 and, therefore, the ability to stabilize it in the energetic diagram. Under this hypothesis, the carboxy-ketone intermediate **[I]** and its gem-diol hydrate **[II]**, possessing a carboxylate-moiety, are proposed to bind tightly the active site undergoing energy stabilization. As a consequence, the energy stabilization of intermediate II causes an increase in the activation barrier for the subsequent C2-C3-gemdiolate cleavage-step, which is related to \(k_{cat,C}\). On the whole, according to the author's hypothesis, an increase in CO\({}_{2}\)/O\({}_{2}\) specificity is paid for with a slower rate of C2-C3 bond cleavage and, ultimately, with a slower overall reaction. The occurrence of these two coupled effects is cited in the literature as the \"trade-off\" between CO\({}_{2}\)-specificity and reaction rate: the more the enzyme is specific for CO\({}_{2}\) (vs. O\({}_{2}\)), the slower the overall carboxylation/hydration reaction and vice versa. In 2010 [PERSON] et al. [30] simplified the energetic profile proposed by [PERSON] et al., identifying two energetic \"effective barriers\" along the reaction coordinate corresponding to two irreversible steps which were identified as: (i) \"effective gas binding\" (encompassing RuBP enolization and subsequent CO\({}_{2}\)/O\({}_{2}\) addition) described by the \(k_{an,C}\) kinetic constants; (ii) \"effective catalysis\" (encompassing the C2-carboxylic acid hydrolysis and C2-C3 gemdiolate bond cleavage) described by \(k_{cat,C}\) kinetic constant. Overall, [PERSON]'s study confirmed the negative power low correlation between specificity \(\mathrm{S_{C/O}}\) and \(k_{cat,C}\).