PMID:1847216
| Citation |
Mechulam, Y, Dardel, F, Le Corre, D, Blanquet, S and Fayat, G (1991) Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli. J. Mol. Biol. 217:465-75 |
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| Abstract |
The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre. |
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| Keywords |
Adenosine Triphosphate/metabolism; Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA Mutational Analysis; Diphosphates/metabolism; Escherichia coli/enzymology; Kinetics; Methionine/metabolism; Methionine-tRNA Ligase/chemistry; Methionine-tRNA Ligase/metabolism; Molecular Sequence Data; RNA, Transfer, Met/metabolism; Structure-Activity Relationship; Thermodynamics; Transfer RNA Aminoacylation |
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