Affinity labeling of the two species of Escherichia coli lysyl-tRNA synthetase with adenosine di- and triphosphopyridoxals

Lysyl-tRNA synthetase (LysRS), a representative of the class 2 aminoacyl-tRNA synthe-tases, occurs as two species in Escherichia coli: LysRSs and LysRSu. To identify the ATP-binding site in this enzyme, we have applied affinity labeling with reactive adenine nucleotide analogs. Incubation of either enzyme species with adenosine di- or triphos-phopyridoxal, followed by borohydride reduction, resulted in a time-dependent incorpora-tion of the reagent, accompanied with the loss of both tRNALys aminoacylation, and lysine-dependent isotopic ATP-PP, exchange activities. LysRSu appeared less sensitive to adenosine triphosphopyridoxal than LysRSs. Complete inactivation with either reagent corresponded to the incorporation of about 2 mol of reagent per mol of dimeric enzyme. MgATP and ATP protected both enzyme species against the inactivation, suggesting that the modification occurs at the ATP-binding site. Sequence analysis of the labeled peptide isolated from the inactivated LysRSs and LysRSu revealed that bulk of the label wa