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Increased rates of tRNA charging through modification of the enzyme‐aminoacyl‐adenylate complex of phenylalanyl‐tRNA synthetase
Author(s) -
Ibba Michael,
Johnson Christopher M.,
Hennecke Hauke,
Fersht Alan R.
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)01454-9
Subject(s) - adenylate kinase , transfer rna , amino acid , aminoacyl trna synthetase , enzyme , chemistry , biochemistry , amino acyl trna synthetases , escherichia coli , mutant , biology , stereochemistry , rna , gene
The transfer of amino acid to tRNA by Escherichia coli phenylalanyl‐tRNA synthetase (PheRS) was studied using replacements of Ala 294 in the α subunit previously shown to have modified amino acid specificity. Steady‐state analysis of tRNA charging showed little difference between wild‐type and mutants, whereas pre‐steady‐state analysis revealed higher rates of tRNA charging by both the A294S PheRS‐phenylalanyl adenylate and the A294G PheRS‐ p ‐Cl‐phenylalanyl adenylate. The decrease in energy required for the formation of the transition state of amino acid transfer in these mutants could be related to a weaker binding of the amino acid in the aminoacyl adenylate complex. Thus a compromise appears to exist between amino acid activation and tRNA charging, because slowing down the first step increases the rate of the second step, possibly as a result of decreased stability of the PheRS·amino acid‐AMP complex.