Significant activity of a modified ribozyme with N7‐deazaguanine at G<sub>10.1</sub>: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Nakamatsu, Yuka; Warashina, Masaki; Kuwabara, Tomoko; Tanaka, Yoshiyuki; Yoshinari, Koichi; Taira, Kazunari

doi:10.1046/j.1365-2443.2000.00352.x

Cited by 22 publications

(32 citation statements)

References 52 publications

(108 reference statements)

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“…26 Since then, additional experimental evidence has accumulated in favor of a two-metal-ion mechanism. [27][28][29][30] The two-metal-ion mechanism is strongly supported by the differential metal-ion effects on the cleavage rate observed for the natural substrate (cleavage activation observed in the presence of Mg 2+ and La 3+ ) 28,30 and for a 5′ thio modified substrate. 27 The metal ions usually involved in chemical catalysis are divalent cations such as Mg 2+ or Mn 2+ .…”

Section: Introductionmentioning

confidence: 91%

Two-Metal-Ion Mechanism for Hammerhead-Ribozyme Catalysis

Leclerc

Karplus

2006

J. Phys. Chem. B

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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.

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Section: Introductionmentioning

confidence: 91%

Two-Metal-Ion Mechanism for Hammerhead-Ribozyme Catalysis

Leclerc

Karplus

2006

J. Phys. Chem. B

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“…The most recent structural and functional studies also identify a Mn 2+ ion with the same G33:N7 direct coordination, and report that G33(7cG) mutation leads to a 1.3 × 10 4 -fold decrease in rate (Wilson et al 2019). This differs considerably from the 30-fold effect of G10.1(7cG) mutation in HHr (Nakamatsu et al 2000). While this binding mode is likely important for Mg 2+ , it is noteworthy that Psr retains modest activity in the presence of the exchange-inert cobalt hexammine as the sole multivalent cation (Harris et al 2015).…”

Section: The Role Of Mg 2+ Ions In β and δ Catalysis Subtly Differs In Psr And Hhrmentioning

confidence: 97%

Molecular simulations of the pistol ribozyme: unifying the interpretation of experimental data and establishing functional links with the hammerhead ribozyme

Kostenbader

York

2019

RNA

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The pistol ribozyme (Psr) is among the most recently discovered RNA enzymes and has been the subject of experiments aimed at elucidating the mechanism. Recent biochemical studies have revealed exciting clues about catalytic interactions in the active site not apparent from available crystallographic data. The present work unifies the interpretation of the existing body of structural and functional data on Psr by providing a dynamical model for the catalytically active state in solution from molecular simulation. Our results suggest that a catalytic Mg 2+ ion makes inner-sphere contact with G33:N7 and outer-sphere coordination to the pro-R P of the scissile phosphate, promoting electrostatic stabilization of the dianionic transition state and neutralization of the developing charge of the leaving group through a metal-coordinated water molecule that is made more acidic by a hydrogen bond donated from the 2 ′ ′ ′ ′ ′ OH of P32. This model is consistent with experimental activity-pH and mutagenesis data, including sensitivity to G33(7cG) and phosphorothioate substitution/metal ion rescue. The model suggests several experimentally testable predictions, including the response of cleavage activity to mutations at G42 and P32 positions in the ribozyme, and thio substitutions of the substrate in the presence of different divalent metal ions. Further, the model identifies striking similarities of Psr to the hammerhead ribozyme (HHr), including similar global fold, organization of secondary structure around an active site three-way junction, catalytic metal ion binding mode, and guanine general base. However, the specific binding mode and role of the Mg 2+ ion, as well as a conserved 2 ′ ′ ′ ′ ′ -OH in the active site, are interrelated but subtly different between the ribozymes.

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“…The transesterification mechanism includes deprotonation of the 2 0 -hydroxyl moiety of a ribose group, nucleophilic attack of the 2 0 -oxygen on the adjacent phosphorus atom, and protonation of the 5 0 -oxyanion leaving group ( Figure 1A). A large body of evidence also indicates that the P9/G 10.1 site binds a metal ion with high affinity, with other metal ion-binding sites being located around the G 5 nucleobase and A 13 phosphate near the site of cleavage (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Thus, the idea that ribozymes are metalloenzymes has been generally accepted.…”

Section: Introductionmentioning

confidence: 99%

“…This finding raises the possibility that hammerhead ribozymes should not be classified as metalloenzymes. Therefore, we decided to investigate ribozyme activity in the presence of Mg 2+ ions and the effects of Mg 2+ ions in the presence of Li + ions on ribozyme activity, using a well-studied model hammerhead ribozyme (R32) and its substrate (S11), both of which are shown in Figure 1B (5)(6)(7)25,28,(32)(33)(34)(38)(39)(40)(41).…”

Section: Introductionmentioning

confidence: 99%

Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

Inoue

Takagi²,

Taira³

2004

Nucleic Acids Research

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Available evidence suggests that Mg2+ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg2+ ions and the effects of Li+ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg2+ ions and did not reach a plateau value even at 1 M Mg2+ ions. Furthermore, this dependence on Mg2+ ions was observed in the presence of a high concentration of Li+ ions. These results indicate that the Mg2+ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg2+ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li+ and Mg2+ ions was more effective than expected, suggesting the existence of a new reaction pathway-a cooperative pathway-in the presence of these multiple ions, and the possibility that a Mg2+ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst.

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Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Cited by 22 publications

References 52 publications

Two-Metal-Ion Mechanism for Hammerhead-Ribozyme Catalysis

Two-Metal-Ion Mechanism for Hammerhead-Ribozyme Catalysis

Molecular simulations of the pistol ribozyme: unifying the interpretation of experimental data and establishing functional links with the hammerhead ribozyme

Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

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Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Cited by 22 publications

References 52 publications

Two-Metal-Ion Mechanism for Hammerhead-Ribozyme Catalysis

Two-Metal-Ion Mechanism for Hammerhead-Ribozyme Catalysis

Molecular simulations of the pistol ribozyme: unifying the interpretation of experimental data and establishing functional links with the hammerhead ribozyme

Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

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Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes