Probing the Leucyl/Phenylalanyl tRNA Protein Transferase Active Site with tRNA Substrate Analogues

ISSN: 1875-5305 (Online)
ISSN: 0929-8665 (Print)


Volume 21, 12 Issues, 2014


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Protein & Peptide Letters

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Probing the Leucyl/Phenylalanyl tRNA Protein Transferase Active Site with tRNA Substrate Analogues

Author(s): Angela Wai Shan Fung, H. Alexander Ebhardt, Kollappillil S. Krishnakumar, Jack Moore, Zhizhong Xu, Peter Strazewski and Richard P. Fahlman

Affiliation: 474 Medical Sciences Building. Department of Biochemistry. University of Alberta. Edmonton, Alberta, T6G 2H7, Canada.

Abstract

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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Article Details

Volume: 21
Issue Number: 7
First Page: 603
Last Page: 614
Page Count: 12
DOI: 10.2174/0929866521666140212110639
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