Electrostatic interactions in the hairpin ribozyme account for the majority of the rate acceleration without chemical participation by nucleobases

Nam, Kwangho; Gao, Jiali; York, Darrin M.

doi:10.1261/rna.863108

Cited by 49 publications

(116 citation statements)

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“…Hydrogen bonding interactions between A38(N1) and the 5Ј-oxygen and between A38(N6) and the pro-R P oxygen might facilitate catalysis by positioning and orientating reactive groups in the trigonal bipyramidal geometry needed for an S N 2(P)-type in-line attack mechanism. Molecular dynamics simulations also support a model in which active site interactions provide electrostatic stabilization of the electronegative transition state without direct participation of RNA functional groups in proton transfer (23). Finally, the decrease in Hp Rz activity at low pH might just reflect the destabilization that is characteristic of all RNA structures at pH extremes that disrupt hydrogen bonds and might not be related to the catalytic mechanism at all.…”

mentioning

confidence: 62%

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

Cottrell

Scott

Fedor

2011

Journal of Biological Chemistry

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Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK a values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK a for ionization of 8azaA at position 38 with the apparent pK a for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK a values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5-oxygen-phosphorus bond.Hairpin ribozymes (Hp Rz) 2 belong to one of several families of small self-cleaving RNAs that serve as useful models of RNA catalysis because they are relatively simple and amenable to chemogenetic analyses (1). The Hp Rz remains functional in the absence of divalent metals, relying exclusively on nucleotide functional groups for catalytic chemistry (2-5). High resolution structures of the Hp Rz bound to transition state mimics show two active site purines, G8 and A38, positioned in a manner similar to the two histidines in RNase A that mediate general acid-base catalysis of the same reaction ( Fig. 1A) (6 -8), leading to the proposal that the Hp Rz uses the same concerted general acid-base mechanism (Fig. 1B) (9). A38(N1) is near the 5Ј-oxygen of the reactive phosphodiester, and A38(N6) lies within hydrogen bonding distance of the pro-R P non-bridging oxygen. Exogenous nucleobase rescue experiments confirmed that the amidine group of A38 interacts with the transition state, but its precise role remained unclear (10). In a general acid-base model, the cationic protonated form of A38 would act as a general acid during cleavage to donate a proton to the 5Ј-oxygen leaving group as the 5Ј-oxygen-phosphorus bond breaks. During the reverse ligation reaction, unprotonated A38 would accept a proton to activate the nucleophilic 5Ј-oxygen for attack on phosphorus as the 5Ј-oxygen-phosphorus bond forms. Cleavage and ligation rate constants both increase with increasing pH and exhibit the same apparent pK a values near 6.5 (3), consistent with the law of microscopic reversibility (11). The pH dependence of reaction kinetics can provide information about the identity of general acid-base catalyst when an apparent pK a value clearly corresponds to the microscopic pK a value for a particular active site functional group. However, no RNA functional groups exhibit protonation equilibria near pH 6.5, at least as free nucleotides in solu...

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confidence: 62%

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

Cottrell

Scott

Fedor

2011

Journal of Biological Chemistry

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“…some favoring the hypothesis that G8 contributes to positioning the nucleophile and stabilizing the transition state by electrostatics but not to proton transfer (Lebruska et al 2002;Rupert et al 2002;Kuzmin et al 2004;Salter et al 2006;Nam et al 2008). These doubts have arisen from three observations.…”

Section: Does the Hairpin Ribozyme Share The Same Catalytic Mechanism?mentioning

confidence: 99%

“…This observation is consistent with general acid-base catalysis by two nucleobases of high pK a . Isoguanine will be negatively charged when deprotonated, demonstrating that the assumption that a negative charge will not form in the electronegative environment of the active site, sometimes presented as an argument against G8 acting as a general base (Kuzmin et al 2004;Salter et al 2006;Nam et al 2008), is unlikely to be valid. Lastly, in a recent study Strobel and coworkers (Suydam et al 2010) carried out a NAIM study of ligation in the hairpin ribozyme using a series of adenosine analogs of varying pK a .…”

Section: Does the Hairpin Ribozyme Share The Same Catalytic Mechanism?mentioning

confidence: 99%

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

Wilson

Lilley²

2010

RNA

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The active centers of the hairpin and VS ribozymes are both generated by the interaction of two internal loops, and both ribozymes use guanine and adenine nucleobases to accelerate cleavage and ligation reactions. The centers are topologically equivalent and the relative positioning of key elements the same. There is good evidence that the cleavage reaction of the VS ribozyme is catalyzed by the guanine (G638) acting as general base and the adenine (A756) as general acid. We now critically evaluate the experimental mechanistic evidence for the hairpin ribozyme. We conclude that all the available data are fully consistent with a major contribution to catalysis by general acid-base catalysis involving the adenine (A38) and guanine (G8). It appears that the two ribozymes are mechanistically equivalent.

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“…The purpose of the present paper, based on MD simulations performed with quantum mechanics/molecular mechanics (QM/MM) potentials, is to study the reactivity of the Oryza sativa twister ribozyme by means of a different mechanism previously proposed in the hairpin ribozyme [25][26][27]. In particular, an intramolecular mechanism without participation of acid and bases species has been termed as baseline mechanism (see Scheme 2) [25,26].…”

mentioning

confidence: 99%

“…In particular, an intramolecular mechanism without participation of acid and bases species has been termed as baseline mechanism (see Scheme 2) [25,26]. In this mechanism, the proton from O2′ is transferred to O5′ leaving group through a non-bridging oxygen atom [25][26][27]. This mechanism is explored both in aqueous solution and in the active site of the Oryza sativa twister ribozyme using QM/MM techniques with the purpose of establishing the origin of catalysis in this ribozyme.…”

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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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Electrostatic interactions in the hairpin ribozyme account for the majority of the rate acceleration without chemical participation by nucleobases

Cited by 49 publications

References 49 publications

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

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

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