Quantum Mechanical/Molecular Mechanical Free Energy Simulations of the Self-Cleavage Reaction in the Hepatitis Delta Virus Ribozyme

Ganguly, Abir; Thaplyal, Pallavi; Rosta, Edina; Bevilacqua, Philip C.; Hammes‐Schiffer, Sharon

doi:10.1021/ja4104217

Cited by 70 publications

(167 citation statements)

References 61 publications

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“…The observed concerted coupled proton transfer appears to be a general mechanism in enzymes that catalyzes phosphate transfer and cleavage reactions. 43,76,[95][96][97][98] Our calculations also provided evidence that the specific metal ion coordination mode may play a prominent role in the catalytic reaction. We developed a new implementation to quantitatively determine the symmetry around the metal ion during the catalytic reaction.…”

Section: Discussionmentioning

confidence: 54%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

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Most enzymes present a catalytic mechanism where one or more proton transfer events occur coupled tightly together with the enzymatic chemical reaction. We show here that inactivating mutations decouple this proton transfer step from the phosphate cleavage reaction in dUTPase. Homotrimeric dUTPase enzymes catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, using largely similar structural and functional groups as most AAA+ enzymes. dUTPases typically use a single Mg 2+ ion as a cofactor in the active site that is formed by direct protein-protein contacts including all three protomers. Here we focus on the C-terminal arm structural motif, which has sequence and functional similarities to P-loop motifs, and is required for catalysis. In this work, we have studied the functional roles of the C-terminal arm in ligand binding and catalysis by using QM/MM (quantum mechanics/molecular mechanics) calculations in conjunction with site-directed mutagenesis experiments. We also present a new method to assess the metal ion coordination symmetry during the catalytic reaction. Using this new implementation, we identified that the coordination symmetry follows a consistent pattern in the three systems studied, reaching the most symmetrical state near the transition states. We found that the phosphate cleavage proceeds with a concerted bimolecular (A N D N ) mechanism with a loose dissociative transition state, and that it is coupled with a proton transfer step involving a unanimously conserved Asp residue. We show that the main mechanistic effect of the lack of the C-terminal arm is to decouple the phosphate cleavage from the subsequent proton transfer step, resulting in a highbarrier altered reaction pathway.

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Section: Discussionmentioning

confidence: 54%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

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“…The calculations have provided a detailed catalytic pathway partially consistent with experimental data as well as some specific mechanistic predictions. These simulations predicted a change from a concerted mechanism when the active site Mg 2+ is present to a stepwise pathway (via a protonated phosphorane intermediate) in the presence of Na + and absence of Mg 2+ (Ganguly et al 2014). The work presented here provides a detailed computational perspective on active site Mg 2+ association and its contribution to catalysis.…”

Section: Introductionmentioning

confidence: 94%

“…As such, we proceed under the assumption that C75 is protonated in all states considered here. In the present work, as in that of Ganguly et al (2014), the deprotonation step is assumed to have already occurred before the reaction proceeds (i.e., during the QM/MM simulation). Instead, preequilibrium assumptions are used to estimate the fraction of active enzyme from the relative free energies (calculated by MM/3D-RISM) of the various unreacted states (i.e., combinations of Mg 2+ bound/unbound and O2…”

Section: Hdvr Ground Statesmentioning

confidence: 99%

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Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM

Radak

Harris

et al. 2015

RNA

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The hepatitis delta virus ribozyme is an efficient catalyst of RNA 2 ′ -O-transphosphorylation and has emerged as a key experimental system for identifying and characterizing fundamental features of RNA catalysis. Recent structural and biochemical data have led to a proposed mechanistic model whereby an active site Mg 2+ ion facilitates deprotonation of the O2 ′ nucleophile, and a protonated cytosine residue (C75) acts as an acid to donate a proton to the O5 ′ leaving group as noted in a previous study. This model assumes that the active site Mg 2+ ion forms an inner-sphere coordination with the O2 ′ nucleophile and a nonbridging oxygen of the scissile phosphate. These contacts, however, are not fully resolved in the crystal structure, and biochemical data are not able to unambiguously exclude other mechanistic models. In order to explore the feasibility of this model, we exhaustively mapped the free energy surfaces with different active site ion occupancies via quantum mechanical/ molecular mechanical (QM/MM) simulations. We further incorporate a three-dimensional reference interaction site model for the solvated ion atmosphere that allows these calculations to consider not only the rate associated with the chemical steps, but also the probability of observing the system in the presumed active state with the Mg 2+ ion bound. The QM/MM results predict that a pathway involving metal-assisted nucleophile activation is feasible based on the rate-controlling transition state barrier departing from the presumed metal-bound active state. However, QM/MM results for a similar pathway in the absence of Mg 2+ are not consistent with experimental data, suggesting that a structural model in which the crystallographically determined Mg 2+ is simply replaced with Na + is likely incorrect. It should be emphasized, however, that these results hinge upon the assumption of the validity of the presumed Mg 2+ -bound starting state, which has not yet been definitively verified experimentally, nor explored in depth computationally. Thus, further experimental and theoretical study is needed such that a consensus view of the catalytic mechanism emerges.

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“…Thus, experimental studies and theoretical calculations have suggested that the nucleophile is activated in the HDV ribozyme through a divalent cation located in the active site, which can be partially hydrated, and would serve either as a Lewis acid, a Brønsted base or both [13,14]. In the case of the hammerhead, experimental and computational studies suggest that an adenine (A38) and a guanine (G8) have an important role in the catalytic mechanism, the later acting as an acid ("AH" in Scheme 1) whose hydroxyl O2′ is activated by the presence of a Mg 2+ cation [15,16].…”

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confidence: 99%

Molecular mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a twister ribozyme

et al. 2017

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isotope effects on the nucleophile and leaving oxygen atoms, in very good agreement with experiments, also support this description. Nevertheless, the free energy profiles in the enzyme and in solution are almost coincident which, despite that the rate-limiting activation free energy is in very good agreement with experimental data of counterpart reactions in solution, rule out this substrate-assisted catalysis mechanism for the twister ribozyme from O. sativa. Catalysis must come from the role of alternative acid-base species not available in aqueous solution, but the rate-limiting transition state must be associated with the P-O5′ bond cleavage.

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Quantum Mechanical/Molecular Mechanical Free Energy Simulations of the Self-Cleavage Reaction in the Hepatitis Delta Virus Ribozyme

Cited by 70 publications

References 61 publications

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM

Molecular mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a twister ribozyme

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