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Kinetic Isotope Effects for RNA Cleavage by 2′-O- Transphosphorylation: Nucleophilic Activation by Specific Base

2010; American Chemical Society; Volume: 132; Issue: 33 Linguagem: Inglês

10.1021/ja103550e

1943-2984

Michael E. Harris, Qing Dai, Hong Gu, Daniel Kellerman, Joseph A. Piccirilli, Vernon Anderson,

RNA and protein synthesis mechanisms

To better understand the interactions between catalysts and transition states during RNA strand cleavage, primary 18O kinetic isotope effects (KIEs) and solvent D2O isotope effects were measured to probe the mechanism of base-catalyzed 2′-O-transphosphorylation of the RNA dinucleotide 5′-UpG-3′. The observed 18O KIEs for the nucleophilic 2′-O and in the 5′-O leaving group at pH 14 are both large relative to reactions of phosphodiesters with good leaving groups, indicating that the reaction catalyzed by hydroxide has a transition state (TS) with advanced phosphorus−oxygen bond fission to the leaving group (18kLG = 1.034 ± 0.004) and phosphorus−nucleophile bond formation (18kNUC = 0.984 ± 0.004). A breakpoint in the pH dependence of the 2′-O-transphosphorylation rate to a pH independent phase above pH 13 has been attributed to the pKa of the 2′−OH nucleophile. A smaller nucleophile KIE is observed at pH 12 (18kNUC = 0.995 ± 0.004) that is interpreted as the combined effect of the equilibrium isotope effect (ca. 1.02) on deprotonation of the 2′-hydroxyl nucleophile and the intrinsic KIE on the nucleophilic addition step (ca. 0.981). An alternative mechanism in which the hydroxide ion acts as a general base is considered unlikely given the lack of a solvent deuterium isotope effect above the breakpoint in the pH versus rate profile. These results represent the first direct analysis of the transition state for RNA strand cleavage. The primary 18O KIE results and the lack of a kinetic solvent deuterium isotope effect together provide strong evidence for a late transition state and 2′-O nucleophile activation by specific base catalysis.