## 7 Conformational Changes during Catalysis: \"Open\" and \"Closed\" Form of the Enzyme
A very important point noted by [PERSON] et al., is that all Rubisco crystallized in the \"closed\" state show 1P and 5P at a distance less than 9.1 A, while all proteins crystallized in the \"open\" state show the two phosphates at a distance higher than 9.4 A, pointing out that decrease of the 1P-5P distance triggers important conformational changes. Figure 8b evidences in red the 1P- and 5P-group binding sites of RuBP in Rubisco from the cyanobacterium _Synechocystis_ sp. ([PERSON] et al., 2016). In the \"open\" state, 1P binds the main-chain NH-groups of Gly381, Gly404 and Gly403 (located at \(\beta\)-strand-7 and loop-8 of the TIM barrel) and engages in hydrogen bonding with Trp66 located at the N-terminal domain of the antiparallel L-subunit (not shown in Figure 8b). The 5P-anion engages in hydrogen bonding with Arg295 and His298-H\({}^{+}\) occupying the so called \"upper subsite\" located at \(\beta\)-strand-5 of the TIM barrel. The third phosphate group shown in Figure 8b interacts with the latch-site which is assigned a particular role in regulation of catalysis. During the open-to-closed transition the 5P-group displaces, occupying the so called \"lower subsite\" upon interaction with Arg295 and His327 (Figure 8b). Moreover, the 1P-anion reaches a position closer to the 5P-group engaging in hydrogen bonding with Thr65 of the N-terminal domain of the adjacent L2 subunit (not shown in Figure 8b). A change of the backbone \(\psi\) torsion angle of Thr65 moving towards the 1P-group seems to be the event triggering the rotation of the N-domain to cover the C-terminal domain TIM barrel. It is important to note that amino acid residues from Trp66 to Thr75 are located in the middle of the loop connecting \(\alpha\)-helix-B to \(\beta\)-strand-C of the N-terminal domain (see Figure S2). This loop constitutes the major interface between the N-terminal domain of one L subunit and the TIM-barrel of the antiparallel L subunit and does not alter in position relative to the TIM barrel during open-to-closed transition. In contrast, the remainder of the N-terminal subunit is triggered to rotate. Conformational changes occurring at loop 6 enable loop mobility between two well defined states i.e., \"retracted\" (while the enzyme is \"open state\") and \"extended\" (while the enzyme is \"closed state\"): extension of loop 6 over the active site (to reach the enzyme closed state) is due to dramatic changes in the \(\varphi\) and \(\psi\) angles of Gly333 and Gly337. These residues provide two flexible elements favoring the rearrangement. Moreover, the 5P-anion binding to the \"upper sub-site\" exerts steric hindrance toward Leu335 of loop 6 favoring the \"retracted\" vs. \"extended\" conformation of the loop. As the enzyme needs to reach the \"closed state\" before initiating the carboxylation/oxygenation reactions, conformational changes significantly affect the \"entropic cost\" of enzyme functioning. [PERSON] points out that the required conformational changes may explain, in part, the Rubisco's low catalytic turnover rates. Moreover, [PERSON] et al. (2016) point out the low \(k_{cat,\text{C}}\) of Rubisco enzymes may be due to the complexity of the reaction mechanism with all reaction steps taking place at a single active site. The following section will focus on the description of the reaction mechanism as recently proposed by [PERSON]'s research group.