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Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence

Academic Article
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Overview

authors

  • Burbaum, J. J.
  • Schimmel, Paul

publication date

  • May 1992

journal

  • Protein Science  Journal

abstract

  • Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

subject areas

  • Amino Acid Sequence
  • Cloning, Molecular
  • Consensus Sequence
  • Escherichia coli
  • Kinetics
  • Methionine
  • Methionine-tRNA Ligase
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phenotype
  • Polymerase Chain Reaction
  • Proline
  • Sequence Homology, Amino Acid
  • Serine
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Research

keywords

  • ACTIVE SITE
  • AMINO ACID BINDING
  • AMINOACYL-TRANSFER RNA SYNTHETASE
  • BETA-SHEET
  • PROLINE
  • WEDGE
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Identity

PubMed Central ID

  • PMC2142228

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1002/pro.5560010503

PubMed ID

  • 1304356
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Additional Document Info

start page

  • 575

end page

  • 581

volume

  • 1

issue

  • 5

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