Isotope effect analyses provide evidence for an altered transition state for RNA 2'-O-transphosphorylation catalyzed by Zn(2+)

Abstract

Solvent D2O and (18)O kinetic isotope effects on RNA 2'-O-transphosphorylation catalyzed by Zn(2+) demonstrate an altered transition state relative to specific base catalysis. A recent model from DFT calculations involving inner sphere coordination to the non-bridging and leaving group oxygens is consistent with the data.

Fig. 1

(A) Mechanisms of RNA 2′-O-transphosphorylation. Specific base catalysis involves equilibrium deprotonation of the 2′O resulting in a 2′ oxyanion that acts as a nucleophile attacking the adjacent phosphoryl group. Experimental and computational data support a mechanism that involves a late TS (similar to TS2). Acid catalysis proceeds via a stepwise mechanism shown in the top pathway resulting in the formation of a stable phosphorane intermediate. The intermediate shown here is anionic for simplicity, however, in the acid mechanism this intermediate is protonated on one or more of the non-bridging oxygens. The potential formation of 2′,5′ diester products resulting from isomerization are also omitted for clarity. (B) Proposed metal ion catalytic modes involving interactions with anionic TSs for RNA cleavage. Potential modes include interactions with the 2′O nucleophile (1), 5′O leaving group (2), and non-bridging oxygen (3). These interactions can involve direct coordination (1–3), H-bonding or transfer (4).

Fig. 2

Proton inventory of specific base (A) and Zn²⁺-catalyzed (B) RNA 2′-O-transphosphorylation. The data are fit to a linear function or to the Gross–Butler equation (eqn S3, ESl). The red dashed line represents a model for one normal fractionation factor of (φ^T = 0.14). The blue dashed line a simulation using eqn S3 (ESI) for a model involving acid catalysis in which there is a modest inverse fractionation factor (φ^R = 2) due to an increase in the protonated form of the catalyst at constant pL that necessitates a large offsetting normal fractionation factor (φ^T = 0.05). The solid black line assumes two normal fractionation factors: one reflecting the change in pK_a of the 2′OL (1/φ^R = 0.2) and a second normal contribution of φ^T = 0.4.

Fig. 3

Summary of KIE measurements for catalysis by specific base (OH−) and Zn(II). (A) Determination of ¹⁸k values by fitting the ln(¹⁸O/¹⁶O) ratio in the unreacted substrate as a function of reaction progress (f) to eqn S4 (ESI). (B) Summary of observed ¹⁸k_NUC, ¹⁸k_NPO and ¹⁸k_LG values. Standard errors in the last numeral are shown in parentheses. The KIEs predicted from the TS models in part C are shown in italics. (C) TS models from DFT calculations for specific base catalysis and a model Zn²⁺-catalyzed mechanism from Chen et al. Distances along the reaction coordinate for the 2′O–P bond formation and 5′O–P bond cleavage are indicated in angstroms. A two metal ion model for non-enzymatic catalysis by Zn²⁺. As described in the text a two metal ion mechanism in which a metal ion interacts with the 5′O leaving group (M_A) and a second metal ion interacts via coordination to a non-bridging oxygen and via H-bonding to the 2′O (M_B).