AFFINITY LABELING OF THE 2 SPECIES OF ESCHERICHIA-COLI LYSYL-TRANSFER-RNA SYNTHETASE WITH ADENOSINE DIPHOSPHOPYRIDOXALS AND TRIPHOSPHOPYRIDOXALS

Lysyl-tRNA synthetase (LysRS), a representative of the class 2 aminoac yl-tRNA synthetases, 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. In cubation of either enzyme species with adenosine di- or triphosphopyri doxal, followed by borohydride reduction, resulted in a time-dependent incorporation of the reagent, accompanied with the loss of both tRNA( Lys) aminoacylation, and lysine-dependent isotopic ATP-PPi exchange ac tivities. LysRSu appeared less sensitive to adenosine triphosphopyrido xal than LysRSs. Complete inactivation with either reagent corresponde d to the incorporation of about 2 mol of reagent per mol of dimeric en zyme. MgATP and ATP protected both enzyme species against the inactiva tion, suggesting that the modification occurs at the ATP-binding site. Sequence analysis of the labeled peptide isolated from the inactivate d LysRSs and LysRSu revealed that bulk of the label was distributed am ong six lysyl residues at positions 25, 82, 114, 156, 364, and 505, wi th preference for Lys-114 and Lys-156. In LysRSs, Lys-132 and Lys-185 were also modified by both reagents, although these residues are not c onserved in LysRSu. It is concluded that the folding of the LysRSs and LysRSu polypeptides and the relative locations of the identified lysy l residues with respect to the binding site for the two labels are ver y similar.