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[摘要]：Protein and RNA enzymes that catalyze phosphoryl transfer reactions frequently contain active site metal ions that interact with the nucleophile and leaving group. Mechanistic models generally hinge upon the assumption that the metal ions stabilize negative charge buildup along the reaction coordinate. However, experimental data that test this assumption directly remain difficult to acquire. We have used an RNA substrate bearing a 3'-thiol group to investigate the energetics of a metal ion interaction directly relevant to transition state stabilization in the Tetrahymena group I ribozyme reaction. Our results show that this interaction lowers the pK(a) of the 3'-thiol by 2.6 units, stabilizing the bound 3'-thiolate by 3.6 kcal/mol. These data, combined with prior studies, provide strong evidence that this metal ion interaction facilitates the forward reaction by stabilization of negative charge buildup on the leaving group 3'-oxygen and facilitates the reverse reaction by deprotonation and activation of the nucleophilic 3'-hydroxyl group. |
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