@article{TEXTUAL,
      recid = {13625},
      author = {Wilson, Timothy J. and McCarthy, Erika and Ekesan, Şölen  and Giese, Timothy J. and Li, Nan-Sheng and Huang, Lin and  Piccirilli, Joseph A. and York, Darrin M. and Lilley, David  M. J.},
      title = {The Role of General Acid Catalysis in the Mechanism of an  Alkyl Transferase Ribozyme},
      journal = {ACS Catalysis},
      address = {2024-10-02},
      number = {TEXTUAL},
      abstract = {MTR1 is an in vitro-selected alkyl transferase ribozyme  that transfers an alkyl group from  O&lt;sup&gt;6&lt;/sup&gt;-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 O&lt;sup&gt;6&lt;/sup&gt;-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 pK&lt;sub&gt;a&lt;/sub&gt; (11.4 as the free  nucleotide) and leads to a 10&lt;sup&gt;4&lt;/sup&gt;-fold lower  activity that is pH-independent. Addition of the second c5n  substitution with a lower pK&lt;sub&gt;a&lt;/sub&gt; restored both the  rate and pH dependence of alkyl transfer. Quantum  mechanical calculations indicate that protonation of  O&lt;sup&gt;6&lt;/sup&gt;-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 pK&lt;sub&gt;a&lt;/sub&gt; values are in good agreement with  the apparent values from measured rates. Increasing the  pK&lt;sub&gt;a&lt;/sub&gt; of the nucleophile by A63 n7c substitution  led to a 6-fold higher rate. The increased reactivity of  the nucleophile corresponds to a β&lt;sub&gt;nuc&lt;/sub&gt; 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  O&lt;sup&gt;6&lt;/sup&gt;-alkylguanine followed by alkyl transfer.},
      url = {http://knowledge.uchicago.edu/record/13625},
}