The Role of General Acid Catalysis in the Mechanism of an Alkyl Transferase Ribozyme.

ABSTRACT: MTR1 is an in vitro-selected alkyl transferase ribozyme that transfers an alkyl group from O ⁶-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 ⁶-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 _a (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 pK _a restored both the rate and pH dependence of alkyl transfer. Q

SUBMITTER: Wilson TJ 

PROVIDER: S-EPMC11494507 | biostudies-literature | 2024 Oct

REPOSITORIES: biostudies-literature