MTR1 is an in vitro-selected alkyl transferase ribozyme that transfers an alkyl group from O6-alkylguanine to N1 of the target adenine in the RNA substrate (A63). The structure of the ribozyme suggested a mechanism in which a cytosine (C10) acts as a general acid to protonate O6-alkylguanine N1. Here, we have analyzed the role of the C10 general acid and the A63 nucleophile by atomic mutagenesis and computation. C10 was substituted by n1c and n1c, c5n variants. The n1c variant has an elevated pKa (11.4 as the free nucleotide) and leads to a 104-fold lower activity that is pH-independent. Addition of the second c5n substitution with a lower pKa restored both the rate and pH dependence of alkyl transfer. Quantum mechanical calculations indicate that protonation of O6-alkylguanine lowers the barrier to alkyl transfer and that there is a significantly elevated barrier to proton transfer for the n1c single substitution. The calculated pKa values are in good agreement with the apparent values from measured rates. Increasing the pKa of the nucleophile by A63 n7c substitution led to a 6-fold higher rate. The increased reactivity of the nucleophile corresponds to a βnuc of ∼0.5, indicating significant C–N bond formation in the transition state. Taken together, these results are consistent with a two-step mechanism comprising protonation of the O6-alkylguanine followed by alkyl transfer.