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  • Title: 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.
    Author: Hountondji C, Schmitter JM, Fukui T, Tagaya M, Blanquet S.
    Journal: Biochemistry; 1990 Dec 25; 29(51):11266-73. PubMed ID: 2271710.
    Abstract:
    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)
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