Modification of aminoacyl-tRNA synthetases with pyridoxal-5'-phosphate. Identification of the labeled amino acid residues

Abstract

The isotopic [32P]PPi-ATP exchange activity of isoleucyl-, valyl-, histidyl-, tyrosyl- and methionyl-tRNA synthetases from Escherichia coli are lost upon incubation in the presence of pyridoxal-5'-phosphate (PLP). When the residual activity of either isoleucyl-, valyl- or methionyl-tRNA synthetase (monomeric truncated form) was plotted as a function of the number of PLP molecules incorporated per enzyme molecule, the plots obtained appeared biphasic. Below 50% inactivation of these enzymes, PLP incorporation varied linearly with the isotopic exchange measurements, and extrapolation of the first half of the plot indicated a stoichiometry of 1.10 +/- 0.05 mol of PLP incorporated per mol of 100% inactivated synthetase. Beyond 50% inactivation, the graph deviated from its initial slope, and up to 4-5 mol of PLP were incorporated per mol of synthetase at the highest used PLP concentrations. In the cases of homodimeric histidyl- and tyrosyl-tRNA synthetases, extrapolation of the graph at 100% inactivation indicated 2.8 +/- 0.1 and 2.4 +/- 0.1 mol of PLP incorporated per mol of enzyme, respectively. PLP-labeled peptides were obtained through trypsin digestion and RPLC purification, prior to Edman degradation analysis. PLP-labeled residues were identified as lysines 132, 332, 335 and 402 of monomeric methionyl-tRNA synthetase, lysines 332, 335, 402, 465, 596 and 640 of native dimeric methionyl-tRNA synthetase, lysines 22, 117, 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557, 559, 593 and 909 of valyl-tRNA synthetase, lysines 2, 118, 369 and 370 of histidyl-tRNA synthetase, and lysine 237 of tyrosyl-tRNA synthetase. In addition, the amino terminal residue of the polypeptide chain(s) of either isoleucyl-, valyl-, histidyl- or methionyl-tRNA synthetases was found labeled. Among these residues, lysines 332, 335 and 402 of monomeric methionyl-tRNA synthetase as well as lysines 332, 335, 402 and 596 of dimeric methionyl-tRNA synthetase, lysines 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557 and 559 of valyl-tRNA synthetase, lysines 2, 369 and 370 of histidyl-tRNA synthetase, and lysine 237 of tyrosyl-tRNA synthetase were labeled in the presence of PLP concentrations smaller than or equal to 1 mM, and are shown to be critical for the activity of the enzymes. It is concluded that these residues participate to the binding sites of the phosphates of ATP on the studied synthetases.