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

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 beta,beta-difluorophenylalanyl derivative of puromycin, an A-site substrate. The fluorine atoms reduce the pK(a) of the nucleophilic alpha-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 Bronsted 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.

Comparative studies of coordination properties of puromycin and puromycin aminonucleoside towards copper(II) ions

“…The respective pK a values are consistent with the literature on similar amino groups [22,[27][28][29][30][31][32].…”

pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA

“…1), for the chemistry of peptide bond formation (15). In fact, the pH-dependence of the peptidyl transfer reaction has previously been observed only for small aa-tRNAanalogs, like puromycin, C-puromycin, CC-puromycin (23,30,31), or puromycin analogs (32). For these, the pH-dependence is complex, with the effect of two protons on the rate of peptidyl transfer to puromycin at 37°C (23,30) as well as at 20°C (31).…”

The interaction between C75 of tRNA and the A loop of the ribosome stimulates peptidyl transferase activity

“…This apparent pK a value may correspond to a general base involved in the reaction, a pH-dependent conformational change (Bayfield et al 2001;Muth et al 2001) or even to an aggregate pK a value representing a larger number of titratable groups of pK a far removed from this value (Knitt and Herschlag 1996). Subsequent studies have focused on trying to identify which nucleotide(s) might function as a general base or as the pHdependent conformational switch critical for the peptidyl transfer reaction, though no definitive answer has emerged (Katunin et al 2002;Beringer et al 2003;Hesslein et al 2004;Youngman et al 2004;Okuda et al 2005).…”