Increased rates of tRNA charging through modification of the enzyme-aminoacyl-adenylate complex of phenylalanyl-tRNA synthetase.
The transfer of amino acid to tRNA by Escherichia coli phenylalanyl-tRNA synthetase (PheRS) was studied using replacements of Ala294 in the alpha 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.
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH Zentrum, Zürich, Switzerland.
M
M
Ibba
C M
CM
Johnson
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H
Hennecke
A R
AR
Fersht
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*
C026617:phenylalanyl adenylate; D000249:Adenosine Monophosphate; D012343:RNA, Transfer; D010652:Phenylalanine-tRNA Ligase