Increased rates of tRNA charging through modification of the enzyme‐aminoacyl‐adenylate complex of phenylalanyl‐tRNA synthetase

FEBS Letters

Hennecke, Hauke²  Johnson, Christopher M.¹  Fersht, Alan R.¹  Ibba, Michael² 

[1] Cambridge Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK;Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH Zentrum, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland

关键词: p-Cl-phenylalanine;    Phenylalanine;    Phenylalanyl-tRNA synthetase;    Pre-steady-state;    tRNA;    Escherichia coli;   

DOI  :  10.1016/0014-5793(94)01454-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The transfer of amino acid to tRNA by Escherichia coli phenylalanyl-tRNA synthetase (PheRS) was studied using replacements of Ala²⁹⁴ 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.

【 授权许可】

Unknown