期刊
JOURNAL OF COMPUTATIONAL CHEMISTRY
卷 39, 期 21, 页码 1656-1665出版社
WILEY
DOI: 10.1002/jcc.25343
关键词
Rubisco carboxylation reaction; large-scale QM/MM; QM and MD calculations; multi-step enzyme mechanism; active-site fragment models; carbon fixation in photosynthesis; constrained gradient minimization; thermodynamic integration; protonation states; molecular dynamics
资金
- Australian Government
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 CC 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 CC bond scission require early protonation of CO2 in the RuBP.CO2 complex; CC 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. (c) 2018 Wiley Periodicals, Inc.
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