Two-Metal-Ion Mechanism for Hammerhead-Ribozyme Catalysis

Abstract

The hammerhead ribozyme is one of the best studied ribozymes, but it still presents challenges for our understanding of RNA catalysis. It catalyzes a transesterification reaction that converts a 5',3' diester to a 2',3' cyclic phosphate diester via an S(N)2 mechanism. Thus, the overall reaction corresponds to that catalyzed by bovine pancreatic ribonuclease. However, an essential distinguishing aspect is that metal ions are not involved in RNase catalysis but appear to be important in ribozymes. Although various techniques have been used to assign specific functions to metals in the hammerhead ribozyme, their number and roles in catalysis is not clear. Two recent theoretical studies on RNA catalysis examined the reaction mechanism of a single-metal-ion model. A two-metal-ion model, which is supported by experiment and based on ab initio and density functional theory calculations, is described here. The proposed mechanism of the reaction has four chemical steps with three intermediates and four transition states along the reaction pathway. Reaction profiles are calculated in the gas phase and in solution. The early steps of the reaction are found to be fast (with low activation barriers), and the last step, corresponding to the departure of the leaving group, is rate limiting. This two-metal-ion model differs from the models proposed previously in that the two metal ions function not only as Lewis acids but also as general acids/bases. Comparison with experiment shows good agreement with thermodynamic and kinetic data. A detailed analysis based on natural bond orbitals (NBOs) and natural energy decomposition (NEDA) provides insights into the role of metal ions and other factors important for catalysis.

Role of Mg2+ in Hammerhead Ribozyme Catalysis from Molecular Simulation

“…[74][75][76] In the single cation mechanism, the Mg 2+ preserves the integrity of the active site structure, and may serve as an epicenter in the transition state that coordinates the A9 and scissile phosphates, G8 2′OH general acid and O 5′ leaving group. The present work underscores the need for further investigation of the chemical reaction profile using combined QM/MM models.…”

The associative nature of adenylyl transfer catalyzed by T4 DNA ligase

“…In the first case, the phosphorane intermediate is stabilized by a Ϸ12 kcal/mol potential well and its energy is only 2.69 kcal/mol higher compared with RS (17). In the second case, the energy of the phosphorane intermediate is 8.38 kcal/mol lower than that of RS (20). The intermediate has a reverse activation energy of 8.7 kcal/mol and fractional bond numbers of 0.51 for the nucleophile and 0.67 for the leaving group, calculated by using the bond lengths in RS/RI/PS from ref.…”

“…To surpass these experimental limitations, ab initio density functional theory (DFT) quantum mechanical (QM)/molecular mechanical (MM) methods are used as an approach for modeling the free-energy landscapes of chemical reactions and characterizing the corresponding TSs in silico (14). Substitutions at pentavalent phosphorus have been extensively studied, including enzyme catalysis and assistance of metal cations (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). For the associative reactions, calculations in the gas phase show triple-well energy landscapes, with the phosphorane intermediate located in a saddle point between two TSs (22).…”

“…For the associative reactions, calculations in the gas phase show triple-well energy landscapes, with the phosphorane intermediate located in a saddle point between two TSs (22). In the case of the water exchange reaction at the phosphate monoanion (17) or RNA isomerization from a 5Ј,3Ј diester to a 2Ј,3Ј cyclic phosphate diester in hammerheadribozyme (20), the assistance of two Mg 2ϩ ions favors the associative mechanism and decreases the activation barriers of the reaction. In the first case, the phosphorane intermediate is stabilized by a Ϸ12 kcal/mol potential well and its energy is only 2.69 kcal/mol higher compared with RS (17).…”

“…The intermediate has a reverse activation energy of 8.7 kcal/mol and fractional bond numbers of 0.51 for the nucleophile and 0.67 for the leaving group, calculated by using the bond lengths in RS/RI/PS from ref. 20 …”