The Mechanism of RNA Strand Scission: An Experimental Measure of the Brønsted Coefficient, β_nuc

Abstract

A closer look at RNA cleavage: A series of fluorine‐bearing nucleotide analogues (X) were incorporated into RNA to perturb the pKa value of the 2′‐OH group systematically. These analogues enable the use of physical organic approaches to investigate bonding to the nucleophile during the RNA scission reaction. Measurement of the Brønsted coefficient, βnuc, provides new insights into the transition‐state structure for this classic reaction.

Transition State Features in the Hepatitis Delta Virus Ribozyme Reaction Revealed by Atomic Perturbations

“…53 Crudely estimating the effective charge on the leaving group in the ground state as +0.5 gives an effective charge equal to ~ −0.8 (i.e., 0.5 – 1.28) in the transition state in the absence of a catalyst. 54 If the transition state in the HDV ribozyme active site resembles the transition state for the nonenzymatic reaction, the relatively large value of α app , aforementioned caveats notwithstanding, implies that the negative charge on the leaving group would be neutralized entirely. General acid catalysis appears to impart a significant fraction of the rate acceleration provided by the HDV ribozyme, similar to classic models of ribonuclease A catalysis, 48,55,56 in which Brønsted and KIE analysis of the leaving group reveal significant decreases in β lg 55 and 18 O lg , 56 respectively, compared to corresponding nonenzymatic reactions.…”

Integration of kinetic isotope effect analyses to elucidate ribonuclease mechanism

“…Quantum chemical calculations provide a molecular level characterization of the structure and bonding in the transition state that can aid in the interpretation of experimental LFER analysis[32]. For reactions involving phosphoryl transfer reactions of ribose hydroxyls, the β EQ is not available, however a value of −1.35 is measured for equilibrium ionization of substituted phenols[14] and a value of −1.56 has been estimated[15]. A β LG of −1.28 is measured for the reaction of a series of uridine 3’ alkyl phosphodiesters indicating a late transition state with respect to bond cleavage (α = 0.71)[16].…”

“…12.5, and this feature has been attributed to a change in mechanism from a concerted to stepwise mechanism[16]. A β NUC of 0.75 for solution RNA transphosphorylation has been measured using a series of 2’ substituted analogs[15]. For comparison, the β NUC values measured for oxygen and nitrogen nucleophiles attacking monoesters are in the range of 0.1–0.2[18].…”

“…The inactivity of RNase A toward this substrate is not understood, but could be an issue related to reaction geometry, or due to stable non-catalytic interactions that form due to the presence of a full negative charge at the 2’ position in the ground state. A β NUC of 0.75 for solution RNA transphosphorylation has been measured using a series of 2’ substituted analogs[15]. This result suggests that the 2′O nucleophile has an effective charge of only −0.25 in the rate limiting transition state for the spontaneous reaction.…”

Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2′-O-transphosphorylation

“…These data suggest a change in mechanism with increasing leaving group p K a that may involve transition from a concerted to stepwise reaction [16]. Piccirilli and colleagues reported a β NUC of 0.75 for RNA transphosphorylation using a series of 2′-substituted analogs (α ≈ 0.5) [17] demonstrating the TS for RNA reactions is advanced along the 2′O-P bond formation coordinate. Significant β LG and β NUC values support a late, TS2-like transition state in either a concerted or stepwise mechanism.…”