Yeast methionyl-tRNA synthetase: analysis of the N-terminal extension and the putative tRNA anticodon binding region by site-directed mutagenesis.
Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA amino-acylation steps than on the adenylate formation step. The Km for ATP and methionine are unaltered, indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3, which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the 3-D structure of the homologous E coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration of the catalytic residues. The tRNA(Met) anticodon binding region of the E coli enzyme has recently been characterized. By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA. Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.
Walter
P
P
Institut de Biologie, Moléculaire et Cellulaire du CNRS, Laboratoire de Biochimie, Strasbourg, France.
Despons
L
L
Laforet
M
M
Ebel
J P
JP
Fasiolo
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000926:Anticodon / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008718:Methionine-tRNA Ligase / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D015003:Yeasts / Q000201:enzymology
D000926:Anticodon; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D008715:Methionine; D008718:Methionine-tRNA Ligase