A single substitution in the motif 1 of Escherichia coli lysyl-tRNA synthetase induces cooperativity toward amino acid binding.
The constitutive lysyl-tRNA synthetase (LysRS) of the Escherichia coli strain OEL134 differs from the wild-type enzyme by the single substitution of threonine 208 with methionine. In vitro study of the isotopic [32P]PPi-ATP exchange reaction catalyzed by purified T208M LysRS revealed specific features that are not observed with the wild-type LysRS: (i) The steady state of the reaction was reached after a approximately 1-min lag when the addition of the enzyme was used to initiate the reaction. This lag disappeared upon preincubation of the enzyme with lysine and ATP. (ii) The variation of the steady state rate as a function of the lysine concentration in the assay was sigmoidal (Hill coefficient of 1.65), suggesting cooperativity of lysine binding to this dimeric enzyme. The allosteric behavior of the mutant enzyme was further established by showing that, at low concentrations of lysine, low amounts of cadaverine stimulated T208M LysRS activity. T208A LysRS, in which threonine 208 had been changed into alanine by site-directed mutagenesis, displayed the same properties as T208M LysRS. Remarkably, Thr 208 makes part of the first signature motif of class II aminoacyl-tRNA synthetases, a motif likely to be involved in the dimerization of the enzyme subunits. Therefore, the behavior of the Thr 208 mutants of LysRS supports the idea that the dimerization of class II aminoacyl-tRNA synthetases is important for an efficient structuration of their active site.
Commans
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Laboratoire de Biochimie, URA 240 CNRS, Ecole Polytechnique, Palaiseau, France.
Blanquet
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Plateau
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008250:Lysine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D010710:Phosphates; D000255:Adenosine Triphosphate; D008250:Lysine-tRNA Ligase; D008239:Lysine