Affinity labeling of aminoacyl-tRNA synthetases with adenosine triphosphopyridoxal: probing the Lys-Met-Ser-Lys-Ser signature sequence as the ATP-binding site in Escherichia coli methionyl-and valyl-tRNA synthetases.
Pyridoxal 5'-triphospho-5'-adenosine (AP3-PL), the affinity labeling reagent specific for lysine residues in the nucleotide-binding site of several enzymes [Tagaya, M., & Fukui, T. (1986) Biochemistry 25, 2958-2964; Yagami, T., Tagaya, M., & Fukui, T. (1988) FEBS Lett. 229, 261-264], was used to identify the ATP-binding site of Escherichia coli methionyl-tRNA synthetase (MetRS). Incubation of this enzyme with AP3-PL followed by reduction with sodium borohydride resulted in a rapid inactivation of both the tRNA(Met) aminoacylation and the methionine-dependent ATP-PPi exchange activities. Complete inactivation corresponded to the incorporation of 0.98 mol of AP3-PL/mol of monomeric trypsin-modified MetRS. ATP or MgATP protected the enzyme from inactivation. The labeling with AP3-PL was also applied to E. coli valyl-tRNA synthetase (ValRS). Both the tRNA(Val) aminoacylation and the valine-dependent ATP-PPi exchange activities were abolished by the incorporation of 0.91 mol of AP3-PL/mol of monomeric ValRS. AP3-PL was found attached to lysine residues 335, 402, and 528 in the primary structure of MetRS. In the case of ValRS, the AP3-PL-labeled residues corresponded to lysines 557, 593, and 909. We therefore conclude that these lysines of MetRS and ValRS are directed toward the ATP-binding site of these synthetases, more specifically at or close to the subsite for the gamma-phosphate of ATP. AP3-PL-labeled Lys-335 of MetRS and Lys-557 of ValRS belong to the consensus tRNA CCA-binding Lys-Met-Ser-Lys-Ser sequence [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].(ABSTRACT TRUNCATED AT 250 WORDS)
Hountondji
C
C
Laboratoire de Biochimie, URA CNRS 240, Ecole Polytechnique, Palaiseau, France.
Schmitter
J M
JM
Fukui
T
T
Tagaya
M
M
Blanquet
S
S
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin; D014637:Valine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000345:Affinity Labels; D010446:Peptide Fragments; C049517:adenosine triphosphopyridoxal; D000255:Adenosine Triphosphate; D014357:Trypsin; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase; D014637:Valine-tRNA Ligase