Kinetic analysis of the leucyl/phenylalanyl-tRNA-protein transferase with acceptor peptides possessing different N-terminal penultimate residues

:
• A systematic kinetic analysis of L/F-transferase with different acceptor peptides.
• L/F-transferase discriminates the N-terminal penultimate residue of substrate peptides.
• The side chain of this residue affects the peptide binding affinity for L/F-transferase.
• Ser or Arg is this position optimizes introduction of non-natural amino acids into peptides
• The N-terminal penultimate residue of a protein may affect its stability in vivo.

The introduction of non-natural amino acids at the N-terminus of peptides/proteins using leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) is a useful technique for protein engineering. To accelerate the chemoenzymatic reaction, here we systematically optimized the N-terminal penultimate residue of the acceptor peptide. Positively charged, small, or hydrophilic amino acids at this position show positive effects for the reaction. Kinetic analysis of peptides possessing different penultimate residues suggests that the side chain of the residue affects peptide-binding affinity towards the L/F-transferase. These findings also provide biological insight into the effect of the penultimate amino acid on substrate specificity of natural proteins to be degraded via the N-end rule pathway.

Graphical AbstractFigure optionsDownload as PowerPoint slide