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°C the purified P231L enzyme has a more than 50-fold increased K m value for asparagine compared to the wild-type enzyme, while the K m value for ATP is increased 8-fold. In the aminoacylation reaction the mutant enzyme shows also significantly increased K m 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.
