AFFINITY LABELING OF ESCHERICHIA-COLI HISTIDYL-TRANSFER-RNA SYNTHETASE WITH REACTIVE ATP ANALOGS - IDENTIFICATION OF LABELED AMINO-ACID-RESIDUES BY MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY

Recent affinity labeling studies have revealed that dimeric histidyl-t RNA synthetase from Escherichia coli displayed half-of-the-sites react ivity toward labeling with pyridoxal 5'-phosphate [Kalogerakos, T, Hou ntondji, C., Berne, P.F, Dutka, S. & Blanquet, S. (1994) Biochimie (Pa ris) 76, 33-44]. In the present report, affinity labeling studies were conducted by using other ATP analogues such as pyridoxal 5'-diphospho -5'-adenosine (pyridoxal-ppAdo), pyridoxal 5'-triphospho-5'-adenosine (pyridoxal-pppAdo), pyridoxal 5'-diphosphate (pyridoxal-P-2) and 5'-p- fluorosulfonylbenzoyl adenosine (FSO(2)BzAdo). The histidine-dependent isotopic [P-32]PPi/ATP exchange activity of His-tRNA synthetase was r apidly and completely lost upon incubation with either pyridoxal-ppAdo , pyridoxal-pppAdo or pyridoxal-P-2, followed by reduction with sodium borohydride. Complete inactivation of His-tRNA synthetase corresponde d to the incorporation of 2.8 mol of either pyridoxal-ppAdo or pyridox al-P-2/mol dimeric synthetase. Incubation of His-tRNA synthetase with FSO(2)BzAdo also resulted in a complete inactivation of the synthetase . However, contrasting with the pyridoxal derivatives, the plot of the residual enzymatic activity against the amount of covalently bound FS O(2)BzAdo appeared biphasic. In the early stages of inactivation, the relationship between the amount of residual activity and FSO(2)BzAdo i ncorporation was linear and extrapolated to a stoichiometry of 1.1 mol reagent/mol His-tRNA synthetase, suggesting that the labeling of one subunit was sufficient to inactivate one dimeric His-tRNA synthetase m olecule. At longer incubation periods, additional reagent incorporatio n occurred and culminated at 2.5 mol label/mol His-tRNA synthetase. Ex cess of MgATP protected the enzyme against inactivation by either stud ied reagent. The labeled amino acid residues were identified by matrix -assisted-laser-desorption-ionization mass spectrometry, by measuring the peptide mass increase caused by the reagents. An identical set of four lysyl residues (Lys2, Lys118. Lys369 and Lps370 of His-tRNA synth etase) was found attached to pyridoxal-ppAdo or pyridoxal-P-2. In addi tion, pyridoxal-ppAdo labeled the alpha-amino group of the N-terminal alanine. In a His-tRNA synthetase sample having incorporated 2.5 mol F SO(2)BzAdo/mol), the labeled amino acid residues were Lys118, Lys196, Tyr262 (or Tyr263), Lys369 and Lys377. Whatever the used reagent, Lys1 18 appeared to be the predominantly labeled residue. Lys118 belongs to fragment 112-124 (RHERPQK-GRYRQF) corresponding to motif 2 of class 2 aminoacyl-tRNA synthetases. The other modified lysyl residues (lysine s 369, 370 and 377) are close to the catalytic motif 3, in the C-termi nal region of the synthetase. Tyr262 and Tyr263 belong to a fragment 2 56-263 (LVRGLDYY) highly conserved among all known His-tRNA synthetase primary structures. Examination of the recently solved structure of c rystalline E. coli His-tRNA synthetase [Arnez, J. G., Harris, D. C., M itschler, A., Rees, B., Francklyn, C. S. &, Moras, D. (1995) EMBO J. 1 4, 4143-4155] shows that, with the exception of lysines 369, 370 and 3 77, the location of which may account for peculiar accessibility and r eactivity, all the amino acid residues identified in this study map ne ar the enzyme nucleotide-binding site, at the N-terminal catalytic dom ain of the synthetase.