Probing the Leucyl/Phenylalanyl tRNA Protein Transferase Active Site with tRNA Substrate Analogues
Angela Wai Shan Fung, H. Alexander Ebhardt, Kollappillil S. Krishnakumar, Jack Moore, Zhizhong Xu, Peter Strazewski and Richard P. Fahlman
Pages 603-614 (12)
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.
Aminoacyl-tRNA protein transferase, L/F transferase, N-end rule, puromycin, quantitative mass spectrometry.
474 Medical Sciences Building. Department of Biochemistry. University of Alberta. Edmonton, Alberta, T6G 2H7, Canada.