Abstract

The elucidation of the chemical mechanisms whereby biological molecules control, regulate and catalyze phosphoryl transfer reactions has profound implications for processes such as transcription, energy storage and transfer, cell signalling and gene regulation.[1, 2] The catalytic properties of RNA, in particular, have application in the design of new biotechnology and implications into the evolutionary origins of life itself.[3] Of primary importance to the understanding of mechanism is the characterization of the transition state for these reactions. Kinetic isotope effects (KIEs) offer one of the most powerful and sensitive experimental probes to interrogate the chemical environment of the transition state.[4-6] However, for complex reactions, theoretical methods are required to interpret the experimental measurements in terms of a detailed mechanistic model that traces the pathway from the reactant state through the transition state and into the product state.[7, 8] This paper presents experimental and computational results to characterize the mechanism of model phosphoryl transfer reactions that mimic RNA cleavage transesterification catalyzed by enzymes such as RNase A[1] as well as endonucleolytic ribozymes such as the hammerhead, hairpin, hepatitis delta virus (HDV), VS and glmS ribozymes.[9-11] Herein, secondary kinetic isotope effects are reported for the cleavage transesterification of a dinucleotide system, which, together with previously reported primary isotope effect measurements,[12, 13] represent a comprehensive characterization of isotope effects for a native (unmodified) RNA system. Scheme 1 illustrates the general mechanism for the reverse dianionic in-line methanolysis of ethylene phosphate, a model for base-catalysed RNA phosphate transesterification, with phosphoryl oxygen positions labelled in accord with their RNA counterparts. In this study, the free energy profiles for Scheme 1 were determined with density-functional quantum mechanical/molecular mechanical (QM/MM) simulations in explicit solvent.[14-17] These simulations are state-of-the-art, and take into account the dynamical fluctuations of the solute and solvent degrees of freedom in determination of the free energy profiles. In addition, adiabatic reaction energy profiles were determined with solvation effects treated implicitly with a polarizable continuum model (PCM)[18] specifically calibrated for the native and 3′ and 5′ thio-substituted compounds (Figure 1). The 3′ and 5′ thio-substituted compounds model the corresponding chemically modified RNAs that serve as valuable experimental probes of phosphoryl transfer mechanisms catalyzed by ribozymes.[19] The S5′ substitution, for example, in the HDV ribozyme serves as an enhanced leaving group that suppresses the deleterious effect of mutation of a critical cytosine residue, which has been interpreted to support its role as a general acid catalyst.[20] The energy values for stationary points of the native and thio-substituted reactions are in Table 1. Using our recently developed ab initio path-integral method based on Kleinert’s variational perturbation theory,[7, 21-23] we also calculated kinetic isotope effects, which are shown along with the most relevant experimental values for comparison in Table 2. The agreement that is achieved between the theoretical and experimental results allows a detailed mechanistic interpretation based on the theoretical models.[7, 8] Figure 1 (Color online) Comparison of density-functional QM/MM free-energy and adiabatic PCM profiles for the native reaction (top), and density-functional adiabatic PCM profiles for native and thio-substituted reactions (bottom) as a function of the difference ... Scheme 1 General reaction scheme for the (associative) reverse of dianionic in-line methanolysis of ethylene phosphate: a model for RNA phosphate transesterification under alkaline conditions. In the present work, the native reaction shown in the scheme is studied ... Table 1 Relative free energy (kcal/mol) and reaction coordinate (Δbond) values (A) computed for stationary points along the coordinate of the native and thio-substituted RNA phosphate transesterification reaction models shown in Scheme 1.[[a] ... Table 2 Primary kinetic isotope effects (KIEs) on 2′ nucleophile (18kNu) and 5′ leaving (18kLg) oxygens, and secondary KIEs on O1P (18kO1P) and O2P (18kO2P) oxygens in aqueous solution for the TS1 and TS2 transition states, along with the most ... All of the profiles calculated in this work correspond to associative (or concerted) mechanisms characterized by initial nucleophilic attack, as is typical of phosphate diesters.[6] The departure of the leaving group can be concerted with nucleophilic attack (as in the S5′ substituted reaction) or can occur in a stepwise fashion resulting in the formation of a stable pentavalent phosphorane intermediate. In the stepwise mechanism, two transition states occur: one in which nucleophilic attack occurs (TS1) and another one in which leaving group departure takes place (TS2) as indicated in Scheme 1. These transition states themselves can be characterized as either “early” or “late”, depending on the degree of P-O2′ and P-O5′ bond formation/cleavage.

Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call