The Role of General\nAcid Catalysis in the Mechanism\nof an Alkyl Transferase Ribozyme

MTR1 is an in vitro-selected alkyl transferase ribozyme\nthat transfers\nan alkyl group from <i>O</i><sup>6</sup>-alkylguanine to\nN1 of the target adenine in the RNA substrate (A63). The structure\nof the ribozyme suggested a mechanism in which a cytosine (C10) acts\nas a general acid to protonate <i>O</i><sup>6</sup>-alkylguanine\nN1. Here, we have analyzed the role of the C10 general acid and the\nA63 nucleophile by atomic mutagenesis and computation. C10 was substituted\nby n1c and n1c, c5n variants. The n1c variant has an elevated p<i>K</i><sub>a</sub> (11.4 as the free nucleotide) and leads to\na 10<sup>4</sup>-fold lower activity that is pH-independent. Addition\nof the second c5n substitution with a lower p<i>K</i><sub>a</sub> restored both the rate and pH dependence of alkyl transfer.\nQuantum mechanical calculations indicate that protonation of <i>O</i><sup>6</sup>-alkylguanine lowers the barrier to alkyl transfer\nand that there is a significantly elevated barrier to proton transfer\nfor the n1c single substitution. The calculated p<i>K</i><sub>a</sub> values are in good agreement with the apparent values\nfrom measured rates. Increasing the p<i>K</i><sub>a</sub> of the nucleophile by A63 n7c substitution led to a 6-fold higher\nrate. The increased reactivity of the nucleophile corresponds to a\nβ<sub>nuc</sub> of ∼0.5, indicating significant C–N\nbond formation in the transition state. Taken together, these results\nare consistent with a two-step mechanism comprising protonation of\nthe <i>O</i><sup>6</sup>-alkylguanine followed by alkyl\ntransfer.