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

Abstract

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.