pK_a Perturbation in Genomic Hepatitis Delta Virus Ribozyme Catalysis Evidenced by Nucleotide Analogue Interference Mapping

Abstract

The Hepatitis Delta Virus (HDV) ribozyme was the first RNA enzyme proposed to use a proton-transfer mechanism for catalysis. Previous biochemical evidence suggested that the genomic HDV ribozyme promotes cis-cleavage using cytosine 75 whose pK(a) is perturbed within the active site. Here we present further biochemical evidence for the involvement of C75 in proton transfer, as well as evidence to support a plausible mechanism for C75 pK(a) perturbation. Nucleotide analogue interference mapping (NAIM) experiments with C analogues having altered N3 pK(a)s demonstrate the importance of C75 ionization in the HDV cis-cleavage reaction. pH-dependent interference rescue with C analogues having enhanced N3 acidity indicates that C75 is the only cytidine residue that must be protonated for ribozyme activity. Furthermore, interference analysis with pseudoisocytidine, a charge-neutral mimic of a C with a protonated N3, shows a pattern consistent with proton transfer, possibly from the C75 N3 to the 5'-oxyanion leaving group during the cis-cleavage reaction. Strong pH-independent inhibition of ribozyme function also occurs at C75 with a C analogue that lacks the N4 amino group, implicating the exocyclic amine in critical interactions in the active site. Interactions with the amino group may play an important role in perturbing the C75 N3 pK(a). Protonation of C41 has been proposed to be important for ribozyme activity; however, no interference at C41 was observed in this analogue series, which argues against a functional role for C41 protonation. These data support a model wherein C75 of the genomic HDV ribozyme acts as a general acid during its cis-cleavage reaction, and provide a glimpse into how RNAs, in a manner similar to protein enzymes, might employ local environmental electronic modulation to catalyze reactions.

Mechanistic characterization of the HDV genomic ribozyme: The cleavage site base pair plays a structural role in facilitating catalysis

“…Kinetic and mutagenesis studies on the HDV ribozyme support a cleavage mechanism involving general acid-base catalysis by C75 (Perrotta et al 1999;Nakano et al 2000;Oyelere et al 2002;Das and Piccirilli 2005;Perrotta et al 2006). Two models have been advanced for how C75 participates in general acid-base catalysis, as recently summarized by Nakano and Bevilacqua (2007).…”

Formamide and the origin of life

“…2 and Scheme 1 pathway "G") [27,87]. Isocytosine is a structural isomer of cytosine, recognizing both guanine (iCG) and cytosine (iCC) in reversed and normal WatsonCrick interactions, respectively [88].…”

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

“…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). Phosphorothioate interference studies have revealed sites of potential site-bound metal ion interactions via coordination to one or more nonbridging oxygens, including the pro-R P position of the scissile phosphate (Jeoung et al 1994;Fauzi et al 1997;Prabhu et al 1997;Raines and Gottlieb 1998;Oyelere et al 2002;Das and Piccirilli 2005;Thaplyal et al 2013). Thiophilic metal ion rescue experiments have been interpreted as supporting a catalytic metal ion interacting with this position.…”