Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA
onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes
both leucyl-tRNALeu and phenylalanyl-tRNAPhe as substrates. X-ray crystal structures with substrate analogues, the minimal
substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition
by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding.
Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate
analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various
chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively
enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.
Keywords: Aminoacyl-tRNA protein transferase, L/F transferase, N-end rule, puromycin, quantitative mass spectrometry.
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