Transition state stabilization by the 'high' motif of class I aminoacyl-tRNA synthetases: the case of Escherichia coli methionyl-tRNA synthetase.
Methionyl-tRNA synthetase belongs to the class I aminoacyl-tRNA synthetase family characterized both by a catalytic center built around a Rossmann Fold and by the presence of the two peptidic marker sequences HIGH and KMSKS. In this study, the role of the 21HLGH24 motif of Escherichia coli methionyl-tRNA synthetase was studied in a systematic fashion by site-directed mutagenesis. It is shown that the two histidine residues play a crucial role in the catalysis of the methionyl adenylate formation by participating in the stabilisation of the ATP phosphate chain during the transition state. Moreover, the results suggest the involvement of the epsilon-imino group of histidine 21 and of the delta-imino group of histidine 24. Notably, the substitution of either the leucine or the glycine residue of the HLGH motif by alanine had no effect on the catalysis. From the data and from other studies with class I aminoacyl-tRNA synthetases, concomitant positive contributions of the HIGH and KMSKS sequences to reach the transition state of aminoacyl adenylate formation can be envisaged.
Laboratoire de Biochimie, Unité de Recherche Associée no. 1970 du Centre National de la Recherche Scientifique, Ecole Polytechnique, Palaiseau, France.
E
E
Schmitt
M
M
Panvert
S
S
Blanquet
Y
Y
Mechulam
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000255:Adenosine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008715:Methionine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D008718:Methionine-tRNA Ligase / Q000737:chemistry / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; C011501:methioninyl adenylate; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D008715:Methionine; D008718:Methionine-tRNA Ligase