Theoretical Examination of Mg²⁺-Mediated Hydrolysis of a Phosphodiester Linkage as Proposed for the Hammerhead Ribozyme

Abstract

The hammerhead ribozyme is an RNA molecule capable of self-cleavage at a unique site within its sequence. Hydrolysis of this phosphodiester linkage has been proposed to occur via an in-line attack geometry for nucleophilic displacement by the 2'-hydroxyl on the adjoining phosphorus to generate a 2',3'-cyclic phosphate ester with elimination of the 5'-hydroxyl group, requiring a divalent metal ion under physiological conditions. The proposed S(N)2(P) reaction mechanism was investigated using density functional theory calculations incorporating the hybrid functional B3LYP to study this metal ion-dependent reaction with a tetraaquo magnesium (II)-bound hydroxide ion. For the Mg(2+)-catalyzed reaction, the gas-phase geometry optimized calculations predict two transition states with a kinetically insignificant, yet clearly defined, pentacoordinate intermediate. The first transition state located for the reaction is characterized by internal nucleophilic attack coupled to proton transfer. The second transition state, the rate-determining step, involves breaking of the exocyclic P-O bond where a metal-ligated water molecule assists in the departure of the leaving group. These calculations demonstrate that the reaction mechanism incorporating a single metal ion, serving as a Lewis acid, functions as a general base and can afford the necessary stabilization to the leaving group by orienting a water molecule for catalysis.

Modeling the RNA 2′OH Activation: Possible Roles of Metal Ion and Nucleobase as Catalysts in Self-Cleaving Ribozymes

“…A reaction pathway based on a Me-GB di mode of activation has been proposed in a single-metal-ion model of catalysis for the hammerhead ribozyme. 11 In this model, the activation proceeds without preactivation because the 2 0 -oxygen remains in the outer-coordination sphere of the metal for the activation and the subsequent reaction To analyze the influence of the inner or outer-sphere coordinations on the respective activation modes Me i -GB di or Me o -GB di , the structure Iw was used as reference. A detailed comparison between Iw and the reactant Iw2 proposed in a previous study 11 is given in the Supporting Information (section S4, Table S3 and Figure S3).…”

“…Iw is more stable than Iw2 in the Me o -GB di pathway. 11 Hence, Iw2 is not a global minimum and appears to be already in some "pre-activated" state ( Figure 4). The metal ion is more tightly bound to the RNA-like ligand in Iw and would require a reorganization of its hydrogen bond network to be converted into Iw2 with a Gibbs free energy penalty of about 10 kcal/mol (Table 3).…”

“…The presence of metal catalysts and their influence on the energy barrier has been studied using minimalist QM models in nonenzymatic reactions ( Figure 2B,C). 11,13,15 The presence of a metal ion close to the 2 0 -oxygen contributes to lowering the energy barrier even more when the metal ion is directly coordinated (Me-GB mono : Figure 2B). 12 Furthermore, the presence of a second noncatalyst metal ion, in the Me-GB mono activation mode, has a cooperative effect.…”

“…The blue lines correspond to the 2 0 OH activation, and the red line corresponds to the rate-determining transition state TS-OS ‡ for the final cleavage reaction in the Me i -GB di pathway. The structures Iw2 and TS-Ow ‡ (green line) correspond to the Me o -GB di mechanism of the cleavage reaction 11. See the text for a more detailed description of the stationary points.…”

Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release

“…The similar behavior observed for both k cat and k cat /K M suggests that the temperature-dependent step occurs at or before cleavage, as this is likely the first irreversible step in the mechanism (Schemes 2, 3). Furthermore, the large magnitude of the measured activation energy (z50 kcal/mol) of this step suggests that the temperature-dependent step is neither substrate association (E a z6 kcal/mol) (Berg and von Hippel 1985;Lohman 1986) nor phosphodiester bond cleavage (E a = 13-20 kcal/mol) (Uhlenbeck 1987;Thomson and Lilley 1999;Torres et al 2003). To explain these data, we propose a reaction scheme with an additional step: a temperaturedependent conformational change in the enzyme-substrate complex prior to catalysis (Scheme 3, ES-E9S), or a temperature-dependent interconversion of RNase P between active and inactive states prior to substrate binding and cleavage (Scheme 3, E, E9, respectively).…”