Asparaginyl-tRNA synthetase from the Escherichia coli temperature-sensitive strain HO202. A proline replacement in motif 2 is responsible for a large increase in Km for asparagine and ATP.
The Escherichia coli K12 mutant gene, asnS40, coding for asparaginyl-tRNA synthetase (AsnRS) in the temperature-sensitive strain HO202, was isolated from genomic DNA using the Polymerase Chain Reaction. DNA sequencing revealed that the mutant enzyme differs from the wild-type AsnRS by two amino acids, but only the P231L replacement leads to a change in aminoacylation activity. In the ATP-PPi exchange reaction at 37 degrees C the purified P231L enzyme has a more than 50-fold increased Km value for asparagine compared to the wild-type enzyme, while the Km value for ATP is increased 8-fold. In the aminoacylation reaction the mutant enzyme shows also significantly increased Km values for asparagine and ATP. Interestingly Pro-231 is part of the conserved motif 2 in class II aminoacyl-tRNA synthetases (Eriani, G., Delarue, M., Poch, O., Gangloff, J. and Moras, D. (1990) Nature 347, 203-206), indicating that this motif might be involved in all class II enzymes in amino acid activation.
Madern
D
D
European Molecular Biology Laboratory, Grenoble, France.
Anselme
J
J
Härtlein
M
M
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism*; D001226:Aspartate-tRNA Ligase; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011392:Proline / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl; D016415:Sequence Alignment; D013696:Temperature
D012346:RNA, Transfer, Amino Acyl; D001216:Asparagine; D000255:Adenosine Triphosphate; D011392:Proline; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase; C029194:asparaginyl-tRNA synthetase