Uncovering the Enzymatic pKa of the Ribosomal Peptidyl Transferase Reaction Utilizing a Fluorinated Puromycin Derivative

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Abstract
The ribosome-catalyzed peptidyl transferase reaction displays a complex pH profile resulting from two functional groups whose deprotonation is important for the reaction, one within the A-site substrate and a second unidentified group thought to reside in the rRNA peptidyl transferase center. Here we report the synthesis and activity of the β,β-difluorophenylalanyl derivative of puromycin, an A-site substrate. The fluorine atoms reduce the pKa of the nucleophilic α-amino group (<5.0) such that it is deprotonated at all pHs amenable to ribosomal analysis (pH 5.2−9.5). In the 50S modified fragment assay, this substrate reacts substantially faster than puromycin at neutral or acidic pH. The reaction follows a simplified pH profile that is dependent only upon deprotonation of a titratable group within the ribosomal active site. This feature will simplify characterization of the peptidyl transferase reaction mechanism. On the basis of the reaction efficiency of the doubly fluorinated substrate compared to the unfluorinated derivative, the Brönsted coefficient for the nucleophile is estimated to be substantially smaller than that reported for uncatalyzed aminolysis reactions, which has important mechanistic implications for the peptidyl transferase reaction.