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 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. Incubation of either enzyme species with adenosine di- or triphosphopyridoxal, 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 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 was distributed among six lysyl residues at positions 25, 82, 114, 156, 364, and 505, with 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 conserved in LysRSu. It is concluded that the folding of the LysRSs and LysRSu polypeptides and the relative locations of the identified lysyl residues with respect to the binding site for the two labels are very similar.
Hountondji
C
C
Laboratoire de Biochimie (URA CNRS 240), Ecole Polytechnique, Palaiseau, France.
Gillet
S
S
Schmitter
J M
JM
Fukui
T
T
Blanquet
S
S
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008250:Lysine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives*
D000345:Affinity Labels; D007527:Isoenzymes; C049517:adenosine triphosphopyridoxal; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008250:Lysine-tRNA Ligase; C048236:pyridoxal 5'-diphospho-5'-adenosine