Inverse Thio Effects in the Hepatitis Delta Virus Ribozyme Reveal that the Reaction Pathway Is Controlled by Metal Ion Charge Density

Abstract

The hepatitis delta virus (HDV) ribozyme self-cleaves in the presence of a wide range of monovalent and divalent ions. Prior theoretical studies provided evidence that self-cleavage proceeds via a concerted or stepwise pathway, with the outcome dictated by the valency of the metal ion. In the present study, we measure stereospecific thio effects at the nonbridging oxygens of the scissile phosphate under a wide range of experimental conditions, including varying concentrations of diverse monovalent and divalent ions, and combine these with quantum mechanical/molecular mechanical (QM/MM) free energy simulations on the stereospecific thio substrates. The RP substrate gives large normal thio effects in the presence of all monovalent ions. The SP substrate also gives normal or no thio effects, but only for smaller monovalent and divalent cations, such as Li+, Mg2+, Ca2+, and Sr2+; in contrast, sizable inverse thio effects are found for larger monovalent and divalent cations, including Na+, K+, NH4+, and Ba2+. Proton inventories are found to be unity in the presence of the larger monovalent and divalent ions, but two in the presence of Mg2+. Additionally, rate–pH profiles are inverted for the low charge density ions, and only imidazole plus ammonium ions rescue an inactive C75Δ variant in the absence of Mg2+. Results from the thio effect experiments, rate–pH profiles, proton inventories, and ammonium/imidazole rescue experiments, combined with QM/MM free energy simulations, support a change in the mechanism of HDV ribozyme self-cleavage from concerted and metal ion-stabilized to stepwise and proton transfer-stabilized as the charge density of the metal ion decreases.

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

“…However, unlike typical results for other metalloribozymes (Frederiksen and Piccirilli 2009), the metal rescue is unconventional in that the substitution decreases the susceptibility of the ribozyme to inhibition by thiophilic metal ions rather than being activated by their presence (Thaplyal et al 2013). Recent studies of stereospecific thio effects over a wide range of ionic conditions reveal inverse thio effects at the pro-S P position with large monovalent and divalent ions (Thaplyal et al 2015). These results, taken together with pH-rate profiles, proton inventories, ammonium/imidazole rescue experiments, and computational simulations, have been interpreted to suggest that the HDVr mechanism can shift from a concerted metal ion-stabilized to a stepwise proton transfer-stabilized pathway with decreasing metal ion charge density (Thaplyal et al 2015).…”

“…Detailed biochemical and kinetic studies have revealed multiple functional divalent metal binding sites and demonstrated that molar concentrations of monovalent ions alone can support catalysis (Nakano et al 2001(Nakano et al , 2003. Sitebound metal ion interactions have been identified and characterized via crystallography (Ke et al 2004(Ke et al , 2007Chen et al 2010), spectroscopy ), chemical probing experiments (Chen et al , 2013Lévesque et al 2012;Thaplyal et al 2015), and molecular simulation (Lee et al 2011;Veeraraghavan et al 2011a,b;Chen et al 2013). Moreover, pH-rate profiles for the reaction in the absence of Mg 2+ and for mutants designed to disrupt binding of the proposed active site ion are inverted relative to the reaction of the native HDVr in Mg 2+ Chen et al 2013).…”

“…Recent studies of stereospecific thio effects over a wide range of ionic conditions reveal inverse thio effects at the pro-S P position with large monovalent and divalent ions (Thaplyal et al 2015). These results, taken together with pH-rate profiles, proton inventories, ammonium/imidazole rescue experiments, and computational simulations, have been interpreted to suggest that the HDVr mechanism can shift from a concerted metal ion-stabilized to a stepwise proton transfer-stabilized pathway with decreasing metal ion charge density (Thaplyal et al 2015). Clearly the evidence for the participation of an active site metal is very strong, but the details of its interactions in the transition state and its role in catalysis are not yet clear.…”

In Vitro Selection of a DNAzyme Cooperatively Binding Two Lanthanide Ions for RNA Cleavage

“…17 Most known RNA-cleaving enzymes bind metal using the pro-Rp oxygen; when this oxygen is replaced by sulfur, the activity is significantly suppressed (typically by 30-200 fold). 32,[48][49][50][51] On the other hand, if the pro-Sp oxygen is replaced by sulfur, the enzyme remains quite active (with only a several fold drop in activity).…”

An Ultrasensitive Light-up Cu2+ Biosensor Using a New DNAzyme Cleaving a Phosphorothioate-Modified Substrate

“…The PS modification has been used for probing ribozyme mechanisms, designing anti-sense oligonucleotides, and assembling nanomaterials. [35][36][37][38][39][40] Since Cu 2+ is a thiophilic soft metal, we herein use a PS containing library for selecting new DNAzymes in the presence of Cu 2+ , and develop a biosensor for Cu 2+ detection.…”