title: Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues.
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-tRNA(Leu) and phenylalanyl-tRNA(Phe) 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.
authors: Angela Wai Shan
authors: AW
authors: Fung
authors: H Alexander
authors: HA
authors: Ebhardt
authors: Kollappillil S
authors: KS
authors: Krishnakumar
authors: Jack
authors: J
authors: Moore
authors: Zhizhong
authors: Z
authors: Xu
authors: Peter
authors: P
authors: Strazewski
authors: 474 Medical Sciences Building. Department of Biochemistry. University of Alberta. Edmonton, Alberta, T6G 2H7, Canada. rfahlman@ualberta.ca.
authors: Richard P
authors: RP
authors: Fahlman
mesh_terms: D019881:Aminoacyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011691:Puromycin / Q000494:pharmacology; D012356:RNA, Transfer, Leu / Q000737:chemistry* / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins
chemical_list: D029968:Escherichia coli Proteins; D012356:RNA, Transfer, Leu; D012360:RNA, Transfer, Phe; D011993:Recombinant Fusion Proteins; D011691:Puromycin; D019881:Aminoacyltransferases; C021511:leucyltransferase