Revised mechanism of carboxylation of ribulose-1,5-biphosphate by rubisco from large scale quantum chemical calculations

Here, we describe a computational approach for studying enzymes that catalyze complex multi-step reactions and apply it to Ribulose 1,5-bisphosphate carboxylase–oxygenase (Rubisco), the enzyme that fixes atmospheric carbon dioxide within photosynthesis. In the 5-step carboxylase reaction, the substrate Ribulose-1,5-bisphosphate (RuBP) first binds Rubisco and undergoes enolization before binding the second substrate, CO₂. Hydration of the RuBP.CO₂ complex is followed by CC bond scission and stereospecific protonation. However, details of the roles and protonation states of active-site residues, and sources of protons and water, remain highly speculative. Large-scale computations on active-site models provide a means to better understand this complex chemical mechanism. The computational protocol comprises a combination of hybrid semi-empirical quantum mechanics and molecular mechanics within constrained molecular dynamics simulations, together with constrained gradient minimization calculations using density functional theory. Alternative pathways for hydration of the RuBP.CO₂ complex and associated active-site protonation networks and proton and water sources were investigated. The main findings from analysis of the resulting energetics advocate major revision to existing mechanisms such that: hydration takes place anti to the CO₂; both hydration and CC bond scission require early protonation of CO₂ in the RuBP.CO₂ complex; CC bond scission and stereospecific protonation reactions are concerted and, effectively, there is only one stable intermediate, the C3-gemdiolate complex. Our main conclusions for interpreting enzyme kinetic results are that the gemdiolate may represent the elusive Michaelis–Menten-like complex corresponding to the empirical K_m (=K_c) with turnover to product via bond scission concerted with stereospecific protonation consistent with the observed catalytic rate.