CO(2) fixation by Rubisco: computational dissection of the key steps of carboxylation, hydration, and C-C bond cleavage.

Despite intensive experimental and computational studies, some important features of the mechanism of the photosynthetic CO(2)-fixing enzyme, Rubisco, are still not understood. To complement our previous investigation of the first catalytic step, the enolization of D-ribulose-1,5-bisphosphate (King et al., Biochemistry 1998, 44, 15414-15422), we present the first complete computational dissection of subsequent steps of the carboxylation reaction that includes the roles of the central magnesium ion and modeled residues of the active site. We investigated carboxylation, hydration, and C-C bond cleavage using the density functional method and the B3LYP/6-31G(d) level to perform geometry optimizations. The energies were determined by B3LYP/6-311+G(2d,p) single-point calculations. We modeled a fragment of the active site and substrate, taking into account experimental findings that the residues coordinated to the Mg ion, especially the carbamylated Lys-201, play critical roles in this reaction sequence. The carbamate appears to act as a general base, not only for enolization but also for hydration of the beta ketoacid formed by addition of CO(2) and, as well, cleavage of the C2-C3 bond of the hydrate. We show that CO(2) is added directly, without assistance of a Michaelis complex, and that hydration of the resultant beta ketoacid occurs in a separate subsequent step with a discrete transition state. We suggest that two conformations of the hydrate (gem-diol), with different metal coordination, are possible. The step with the highest activation energy during the carboxylation cycle is the C-C bond cleavage. Depending on the conformations of the gem-diol, different pathways are possible for this step. In either case, special arrangements of the metal coordination result in bond breaking occurring at remarkably low activation energies (between 28 and 37 kcal mol(-1)) which might be reduced further in the enzyme environment.