Dissociative Transition State in Hepatitis Delta Virus Ribozyme Catalysis

Weissman, Benjamin P.; Ekesan, Şölen; Lin, Hsuan-Chun; Gardezi, Shahbaz; Li, Nan‐Sheng; Giese, Timothy J.; McCarthy, Erika; Harris, Michael E.; York, Darrin M.; Piccirilli, Joseph A.

doi:10.1021/jacs.2c10079

Cited by 4 publications

(3 citation statements)

References 58 publications

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“…There are considerable mechanistic similarities between primer extension and the ribozyme-catalyzed cleavage of RNA by transphosphorylation, which involves deprotonation of the 2′-hydroxyl nucleophile, stabilization of the in-line geometry for nucleophilic attack, electrophilic phosphate activation, and stabilization of the leaving group, which provide a helpful context for interpreting the mechanism of metal ion-catalyzed primer extension. As an example, metal ions play a similar role in the HDV ribozyme by coordinating to a nonbridging phosphoryl oxygen and activating the nucleophile . If primer extension proceeded through a fully dissociative mechanism, then the products would exhibit mixed stereochemistry, which we did not observe.…”

Section: Discussioncontrasting

confidence: 57%

“…As an example, metal ions play a similar role in the HDV ribozyme by coordinating to a nonbridging phosphoryl oxygen and activating the nucleophile. 53 If primer extension proceeded through a fully dissociative mechanism, then the products would exhibit mixed stereochemistry, which we did not observe. Assuming an S N 2-like mechanism (Figure S5), reactant substrate chirality can be deduced from that of the products.…”

Section: ■ Discussioncontrasting

confidence: 55%

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Catalytic Metal Ion–Substrate Coordination during Nonenzymatic RNA Primer Extension

Fang,

Pazienza,

Zhang

et al. 2024

J. Am. Chem. Soc.

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Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3′-hydroxyl on the adjacent phosphate of a 5′-5′-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazoliumbridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg 2+ and Cd 2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg 2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-S P position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5′-5′imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg 2+ ion with the pro-S P oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed S N 2(P) reaction.

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Section: Discussioncontrasting

confidence: 57%

Section: ■ Discussioncontrasting

confidence: 55%

Catalytic Metal Ion–Substrate Coordination during Nonenzymatic RNA Primer Extension

Fang,

Pazienza,

Zhang

et al. 2024

J. Am. Chem. Soc.

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“…Both nucleic acid and protein catalyze RNA 2′- O -transphosphorylation providing contrasting examples of active sites with very different physiochemical properties. Recent comparisons of RNA 2′- O -transphosphorylation catalyzed by the protein enzyme ribonuclease A and the HDV ribozyme (HDVrz) using combined quantum mechanical/molecular mechanical (QM/MM) simulations benchmarked by 18 O kinetic isotope effects reveal that they traverse through remarkably different transition states, underscoring the breadth of the mechanistic landscape. , To define this mechanistic landscape, there is a clear need for additional comparisons between RNA and protein catalysts that have similar or distinct active site architectures. Such detailed investigation of different RNA catalysts at a high level of mechanistic detail is essential for defining fundamental aspects of biological catalysis and providing general principles and strategies for engineering novel catalysts or ones with expanded chemical range or that respond to signaling molecules. ,,, …”

Section: Introductionmentioning

confidence: 99%

Rapid Kinetics of Pistol Ribozyme: Insights into Limits to RNA Catalysis

et al. 2023

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Pistol ribozyme (Psr) is a distinct class of small endonucleolytic ribozymes, which are important experimental systems for defining fundamental principles of RNA catalysis and designing valuable tools in biotechnology. High-resolution structures of Psr, extensive structure−function studies, and computation support a mechanism involving one or more catalytic guanosine nucleobases acting as a general base and divalent metal ion-bound water acting as an acid to catalyze RNA 2′-O-transphosphorylation. Yet, for a wide range of pH and metal ion concentrations, the rate of Psr catalysis is too fast to measure manually and the reaction steps that limit catalysis are not well understood. Here, we use stopped-flow fluorescence spectroscopy to evaluate Psr temperature dependence, solvent H/D isotope effects, and divalent metal ion affinity and specificity unconstrained by limitations due to fast kinetics. The results show that Psr catalysis is characterized by small apparent activation enthalpy and entropy changes and minimal transition state H/D fractionation, suggesting that one or more pre-equilibrium steps rather than chemistry is rate limiting. Quantitative analyses of divalent ion dependence confirm that metal aquo ion pK a correlates with higher rates of catalysis independent of differences in ion binding affinity. However, ambiguity regarding the rate-limiting step and similar correlation with related attributes such as ionic radius and hydration free energy complicate a definitive mechanistic interpretation. These new data provide a framework for further interrogation of Psr transition state stabilization and show how thermal instability, metal ion insolubility at optimal pH, and pre-equilibrium steps such as ion binding and folding limit the catalytic power of Psr suggesting potential strategies for further optimization.

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Estimation of frequency factors for the calculation of kinetic isotope effects from classical and path integral free energy simulations

Giese

York

2023

The Journal of Chemical Physics

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We use the modified Bigeleisen–Mayer equation to compute kinetic isotope effect values for non-enzymatic phosphoryl transfer reactions from classical and path integral molecular dynamics umbrella sampling. The modified form of the Bigeleisen–Mayer equation consists of a ratio of imaginary mode vibrational frequencies and a contribution arising from the isotopic substitution’s effect on the activation free energy, which can be computed from path integral simulation. In the present study, we describe a practical method for estimating the frequency ratio correction directly from umbrella sampling in a manner that does not require normal mode analysis of many geometry optimized structures. Instead, the method relates the frequency ratio to the change in the mass weighted coordinate representation of the minimum free energy path at the transition state induced by isotopic substitution. The method is applied to the calculation of 16/18O and 32/34S primary kinetic isotope effect values for six non-enzymatic phosphoryl transfer reactions. We demonstrate that the results are consistent with the analysis of geometry optimized transition state ensembles using the traditional Bigeleisen–Mayer equation. The method thus presents a new practical tool to enable facile calculation of kinetic isotope effect values for complex chemical reactions in the condensed phase.

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Dissociative Transition State in Hepatitis Delta Virus Ribozyme Catalysis

Cited by 4 publications

References 58 publications

Catalytic Metal Ion–Substrate Coordination during Nonenzymatic RNA Primer Extension

Catalytic Metal Ion–Substrate Coordination during Nonenzymatic RNA Primer Extension

Rapid Kinetics of Pistol Ribozyme: Insights into Limits to RNA Catalysis

Estimation of frequency factors for the calculation of kinetic isotope effects from classical and path integral free energy simulations

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