Mechanism and Catalytic Site Atlas

Introduction

Rubisco has to be activated for reaction. Activation is achieved by carbamylation of a lysine residue(Lys191). This reaction creates a binding site for a Mg(II) ion that stabilises the carbamate by monodentate and completes the active site of the enzyme. After activation, RuBP binds to the enzyme, O2 and O3 become coordinated to Mg(II) ions, displacing 2 water molecules. The coodination of O2 and O3 by Mg(II) ion increases acidity of the protons attached to C3 and O3, thus assisting their subsequent removal. The first step of the reaction is the removal of the proton at the C3 carbon atom of RUBP by carbamate. The combined effect of the Mg2+ polarising the C2 carbonyl and the proximity of a carbamino oxygen positioned to accept the proton favours the formation of the C2-C3 enediolate. The enediol intermediate is further stabilised by protonation of the C2 oxygen by the protonated carbamate. The fact that C3 proton is not conserved in upper 3-P-lgycerate is explained by a proton transfer from C3 to the carbamate and then to O2 which is then abstracted by Lys166. An enediolate is created in which O2 is unprotonated and O3 is protonated and again, carbamate facilitates the transfer of proton from O3 to O2. The next step is the addition of CO2 to the enediolate intermediate. The precise details of the reaction of the enediolate with CO2 and water are unclear. Both a sequential, two step reaction and a concerted mechanism are plausible. In the two step sequential mechanism, the electrons from the enediolate oxygen at C3 forms a double bond, permitting attack of C2 on CO2 to form an intermediate with a keto group at C3. Then, a water molecule, activated by His287, nucleophilically attack the C3 keto group. The alternative mechanism is a concerted one in which attack of water at C3 occurs in the same transition state as attack on CO2. In both case, Lys329 facilitates the addition of CO2 by polarising the 2 oxygen of CO2 and in the former case, it also stabilises the transition state of the carboxylate intermediate. Once the six-carbon hydrated ketone intermediate is formed, C2-C3 bond is cleaved. Cleavage of C2-C3 bond is facilitated by proton removal from OH group at C3. Carbamate on Lys191, again, acts as a base here. Following cleavage of the bond between C2 and C3, one molecule of 3-PGA is immediately yielded from carbon atoms C3 to C5, whereas the second molecule of D-3-PGA is produced by stereospecific protonation of the carbanion derived from C1 and C2.Protonated Lys166 is ideally positioned to act as the acid for this step. The rotation of C1 and C2 leading to aci-carboxylate formation brings C2 in contact with Lys166 and facilitate the protonation. Side chain carbonyl oxygen of Asn192 forms a hydrogen bond to carbamate NH to increase the positive charge of the nitrogen, enhancing the effectiveness of carbamate to act as a base. Based on site mutagenesis, His321 is also found to be stabilising the transition states of the reaction.