The p Ka and diminished relative reactivity of the α-amino group of phenylalanyl-tRNA from escherichia coli

Abstract

Immense progress has been made in the characterization of the tRNA structure and its function of carrying a particular amino acid to the growing peptide chain (reviewed [I]). Peptide bond formation is a condensation reaction in which the cu-amino group of an aminoacyl-tRNA is brought into contact with the active ester linkage between the preceding tRNA and the nascent peptide. The reaction takes place close to the 3’- and S’ends of the tRNA, which have a special role in it 121. Until now no data have been available concerning the ply, and reactivity of the o-amino group of aminoacyl-tRNA. The major problem for conventional physical techniques is the instability of the macromolecule in the physiological pH range and the resulting contamination of the sample with hydrolysis products. We solved this problem by application of the technique of competitive labelling as used for our studies of the elongation factor Tu [3]. The essential feature of the method is that single-hit tracelabelling is performed under non-denaturing conditions. This is realized here with pmole amounts of sample and by the use of short labelling times (60 s). Our results show that the plea (O*C) for the o-amino group of Phe-tRNA is -8.1, a similar value as for the reference compound Phe-Gly. This indicates that the salt bridge between the protonated amino group and the 5’-phosphate of the tRNA moiety suggested in [7,8] does not exist under physiological conditions. The relative reactivity of the a-amino group, however, is somewhat lower than expected.