AFFINITY LABELING OF AMINOACYL-TRANSFER RNA-SYNTHETASES WITH ADENOSINE TRIPHOSPHOPYRIDOXAL - PROBING THE LYS-MET-SER-LYS-SER SIGNATURE SEQUENCE AS THE ATP-BINDING SITE IN ESCHERICHIA-COLI METHIONYL-TRANSFER RNA AND VALYL-TRANSFER RNA-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-PP(i) 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-PP(i) 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]. This indicates that, in the case of the two studied enzymes, the ATP-dependent activation of the amino acid and its subsequent transfer to the 3'-adenosine of tRNA take place in the same region. Furthermore, comparison of the primary structure of MetRS around Lys-402 to that of ValRS around Lys-593 reveals little significant similarity, with four identities and two conservative replacements out of 11 amino acid residues.