title: Evidence that the 3' end of a tRNA binds to a site in the adenylate synthesis domain of an aminoacyl-tRNA synthetase.
abstract: Aminoacylation requires that an enzyme-bound aminoacyladenylate is brought proximal to the 3' end of a specific transfer RNA. In Escherichia coli alanyl-tRNA synthetase, the first 368 amino acids encode a domain for adenylate synthesis while sequences on the carboxyl-terminal side of this domain are required for much of the enzyme-tRNAAla binding energy. The 3' end of E. coli tRNAAla has been cross-linked to the enzyme, and sequence analysis showed that Lys-73 is the major site of coupling. A mutant enzyme with a Lys-73----Gln replacement has a 50-fold reduced kcat/Km (with respect to tRNAAla) for aminoacylation but has a relatively small alteration of its kinetic parameters for ATP and alanine in the adenylate synthesis reaction. The data provide evidence that the 3' end of tRNAAla binds to a site in the enzyme domain responsible for adenylate synthesis and that a residue (Lys-73) in this domain is important for a tRNAAla-dependent step that is subsequent to the synthesis of the aminoacyladenylate intermediate.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: K
authors.initials: K
authors.lastname: Hill
authors.forename: P
authors.initials: P
authors.lastname: Schimmel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000412:Alanine-tRNA Ligase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D012344:RNA, Transfer, Ala / Q000378:metabolism*; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*
chemical_list: D011756:Diphosphates; D010446:Peptide Fragments; D012344:RNA, Transfer, Ala; D012345:RNA, Transfer, Amino Acid-Specific; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D000412:Alanine-tRNA Ligase