Mechanistic Characterization of the HDV Genomic Ribozyme: Solvent Isotope Effects and Proton Inventories in the Absence of Divalent Metal Ions Support C75 as the General Acid

Abstract

The hepatitis delta virus (HDV) ribozyme uses the nucleobase C75 and a hydrated Mg(2+) ion as the general acid-base catalysts in phosphodiester bond cleavage at physiological salt. A mechanistic framework has been advanced that involves one Mg(2+)-independent and two Mg(2+)-dependent channels. The rate-pH profile for wild-type (WT) ribozyme in the Mg(2+)-free channel is inverted relative to the fully Mg(2+)-dependent channel, with each having a near-neutral pKa. Inversion of the rate-pH profile was used as the crux of a mechanistic argument that C75 serves as general acid both in the presence and absence of Mg(2+). However, subsequent studies on a double mutant (DM) ribozyme suggested that the pKa observed for WT in the absence of Mg(2+) arises from ionization of C41, a structural nucleobase. To investigate this further, we acquired rate-pH/pD profiles and proton inventories for WT and DM in the absence of Mg(2+). Corrections were made for effects of ionic strength on hydrogen ion activity and pH meter readings. Results are accommodated by a model wherein the Mg(2+)-free pKa observed for WT arises from ionization of C75, and DM reactivity is compromised by protonation of C41. The Brønsted base appears to be water or hydroxide ion depending on pH. The observed pKa's are related to salt-dependent pH titrations of a model oligonucleotide, as well as electrostatic calculations, which support the local environment for C75 in the absence of Mg(2+) being similar to that in the presence of Mg(2+) and impervious to bulk ions. Accordingly, the catalytic role of C75 as the general acid does not appear to depend on divalent ions or the identity of the Brønsted base.

Driving Forces for Nucleic Acid pKa Shifting in an A+·C Wobble: Effects of Helix Position, Temperature, and Ionic Strength

“…pH titrations were conducted in the NMR tube, and the pH of the solution was measured using an ISFET micro pH probe (IQ Scientific Instruments) at the temperature of the titration. We previously reported that ion-dependent effects for this probe result in errors in measurement of pH for certain solutions (32). The effects of potassium ions on pH meter readings were therefore examined.…”

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

“…In addition to numerous studies of self-cleavage by the cisacting genomic and antigenomic HDV ribozymes (Been and Wickham 1997;Perrotta et al 1999aPerrotta et al ,b, 2006Wadkins et al 1999;Nakano et al 2000Nakano et al , 2003Shih and Been 2002;Harris et al 2004;Been 2006;Chadalavada et al 2007;Gong et al 2007Gong et al , 2009Sefcikova et al 2007a,b;Cerrone-Szakal et al 2008;Gong et al 2008;Chen et al 2009), significant work has also been performed to investigate trans-acting forms of this ribozyme for enzymology and therapeutic purposes (Been 1994;Luptak et al 2001;Harris et al 2002;Pereira et al 2002;Jeong et al 2003;Tinsley et al 2003Tinsley et al , 2004Das and Piccirilli 2005;Tinsley and Walter 2007;Walter and Perumal 2009). Generally, trans-acting HDV ribozymes exhibit a 1-2 order-of-magnitude slower catalytic rate constant compared to their cis-acting counterparts, likely due to a less tightly knit structure (Tinsley and Walter 2007).…”

HDV Family of Self-Cleaving Ribozymes

“…4.3). 46,70,71 For the majority of studies conducted on the HDV ribozymes, the active-site cytosine could be assigned a proton-transferring role as either an acid or a base. However, three experiments strongly implied that the nucleobase acts as a general acid in the mechanism.…”

“…35,36 Third, the rate of self-scission in the absence of Mg 2þ results in a pH profile and a kinetic solvent-isotope effect that support transfer from a protonated cytosine. 46,49,[71][72][73] Kinetic pH profiles, proton inventory experiments, and Brønsted analysis indicated that at least one proton is transferred during the rate-limiting step of the reaction, most likely the one from a protonated C75/76 to the oxyanion leaving group of G1. 69,71,74 In order for the cytosine to efficiently transfer a proton to the 5 0 oxyanion leaving group, its pK a needs to be shifted from approximately 4.2 toward neutrality.…”

“…46,49,[71][72][73] Kinetic pH profiles, proton inventory experiments, and Brønsted analysis indicated that at least one proton is transferred during the rate-limiting step of the reaction, most likely the one from a protonated C75/76 to the oxyanion leaving group of G1. 69,71,74 In order for the cytosine to efficiently transfer a proton to the 5 0 oxyanion leaving group, its pK a needs to be shifted from approximately 4.2 toward neutrality. Several biophysical experiments have been designed to measure the equilibrium pK a of the active-site cytosine in various ribozyme constructs.…”