Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme.
MESI, the structural gene for methionyl-tRNA synthetase from Saccharomyces cerevisiae encodes an amino-terminal extension of 193 amino acids, based on the comparison of the encoded protein with the Escherichia coli methionyl-tRNA synthetase. 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 aminoacylation step 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 three-dimension 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 to the catalytic residues.
Walter
P
P
Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Laboratoire de Biochimie 15, Strasbourg, France.
Weygand-Durasevic
I
I
Sanni
A
A
Ebel
J P
JP
Fasiolo
F
F
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D005800:Genes, Fungal / Q000235:genetics*; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012358:RNA, Transfer, Met / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic
D012358:RNA, Transfer, Met; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase