## 4 Mechanism of Enzyme Activation by Lys201-Carbamoylation  
The information drawn from X-ray crystal structures and kinetic studies has allowed the proposal of a model describing the Rubisco physiological function as a sequence of \"enzyme activation\" and \"enzyme catalysis\" [29]. According to the model, the \"enzyme activation\" step consists in carbamylation of Lys201 affording an adduct stabilized by Mg\({}^{2+}\) cation (ECM complex).  
In particular, as depicted in Figure 4, the binding pocket of the unbound enzyme (represented as E) binds CO\({}_{2}\) (C) on Lys201 and the carbamylated-Lys201 is stabilized by the binding of Mg\({}^{2+}\) (M) to give the ternary ECM complex Lys-201-NH-COO\({}^{-}\) Mg\({}^{2+}\). This reaction allows the transition from a \"catalytic inactive\" to a \"catalytic active\" enzyme prone to tightly binds RuBP to give the ECMR complex. The ECMR complex may begin the \"enzyme catalysis\", reacting with CO\({}_{2}\) or O\({}_{2}\) according to the RuBP \"carboxylation\" or \"oxygenation\" reactions. Interestingly, it has been demonstrated that the \"activating CO\({}_{2}\) molecule\" (i.e., the CO\({}_{2}\) molecule binding Lys201) is not the CO\({}_{2}\) molecule involved in the RuBP carboxylation reaction (Scheme 1a).  
The apparently \"simple\" Lys201-carbamylation reaction underlines a quite complex reaction mechanism recently disclosed by [PERSON] [15]. The author succeeded in obtaining a high resolution structure (1.9 A) of Rubisco from _Galdieria sulphuraria_ bearing O\({}_{2}\) at the active site (in the center of the TIM barrel fold). Subsequently O2 was replaced by CO\({}_{2}\) by incubation of crystals at 40 \({}^{\circ}\)C in the presence of CO\({}_{2}\) and Mg\({}^{2+}\). After incubation, harvested crystals showed evidence of ECM complex formation. Comparison of the two crystal structures bearing O\({}_{2}\) or the Lys201-CO\({}_{2}\)-Mg\({}^{2+}\) complex allowed describing the structure of the \"activation binding site\" (Figure 5a) evidencing its different location with respect to the \"catalytic binding site\" described in the next section.  
The proposed mechanism for the Lys201 carbamoylation reaction starts with a conformation change of His335 (comparing Figure 5a,b, note the conformational flexibility of the His335 side chain) allowing Mg\({}^{2+}\) to locate in the transient binding site shown in Figure 5b. Interestingly, the metal ion was modelled to coordinate two OH\({}^{-}\) groups, one water molecule and CO\({}_{2}\) (the latter in the end-on mode) displaying a pseudo-tetrahedral geometry with the linear CO\({}_{2}\) molecule pointing towards the \(\epsilon\)-NH\({}_{3}\)\({}^{+}\) group of Lys201. Subsequently, one metal-bound hydroxide promotes the proton abstraction of the \(\epsilon\)-NH\({}_{3}\)\({}^{+}\) group of Lys201 (see Figure 5c), producing the \(\epsilon\)-NH\({}_{2}\) functionality prone to nucleophilic attack of the CO\({}_{2}\) molecule to give the \(\epsilon\)-NH\({}_{2}\)(\({}^{+}\))-CO\({}_{2}\)(\({}^{-}\)) moiety. Finally, further proton abstraction from the \(\epsilon\)-NH\({}_{2}\)(\({}^{+}\))-CO\({}_{2}\)(\({}^{-}\)) moiety produces the Mg\({}^{2+}\)-stabilized acid-carbamate complex that relocates in a new binding site (shown in Figure 5d) where an octahedral  
Figure 4: Schematic representation of the Rubisco “activation” and “catalytic” processes. E (representing the unbound enzyme) react with CO\({}_{2}\) (C) and Mg\({}^{2+}\) (M) to form an active ternary complex (ECM) subsequently binding RuBP to give a quaternary ECMR complex. These events precede the carboxylation or oxygenation reaction (catalytic process) [29]. Previously unpublished figure.  
coordination sphere around Mg\({}^{2+}\) is completed with Glu213 and Asp212 (not shown in Figure 5d). Relocation of the Mg\({}^{2+}\)-complex allows His335-side chain return to its original conformation.