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.
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.
Mechulam
Y
Y
Laboratoire de Biochimie, URA 240 CNRS, Ecole Polytechnique, Palaiseau, France.
Dardel
F
F
Le Corre
D
D
Blanquet
S
S
Fayat
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008718:Methionine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D012358:RNA, Transfer, Met / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D046249:Transfer RNA Aminoacylation
D011756:Diphosphates; D012358:RNA, Transfer, Met; D000255:Adenosine Triphosphate; D008715:Methionine; D008718:Methionine-tRNA Ligase