CHEMICAL MODIFICATION AND MUTAGENESIS STUDIES ON ZINC-BINDING OF AMINOACYL-TRANSFER RNA-SYNTHETASES

Thermus thermophilus methionyl-tRNA synthetase consists of two identic al subunits with a potential Zn2+-binding sequence of Cys-X2-Cys-X13-C ys-X2-His (Nureki, O., Muramatsu, T., Suzuki, K., Kohda, D., Matsuzawa , H., Ohta, T. Miyazawa, T., and Yokoyama, S. (1991) J. Biol. Chem. 26 6, 3268-3277). Upon chemical modification of the 3 Cys residues of T. thermophilus MetRS with sodium p-(hydroxymercuri)phenylsulfonate, one Zn2+ ion was released from one subunit of the molecule, as monitored w ith 4-(2-pyridylazo)resorcinol. Site-directed mutagenesis of Cys and H is residues in the Zn2+-binding sequence reduced the aminoacylation ac tivity; the k(cat) value was markedly decreased, and the K(m) values f or L-methionine and tRNA(f)Met were increased. Similarly, Cys modifica tion released two Zn2+ ions from T. thermophilus and Escherichia coli isoleucyl-tRNA synthetases and E. coli threonyl-tRNA synthetase, which have Zn2+-binding motifs, and impaired their activities. By contrast, three other aminoacyl-tRNA synthetases that lack Zn2+-binding motif n either released Zn2+ ion nor lost their activities upon Cys modificati on. These results indicate that the Zn2+-binding sequences are importa nt for catalysis and recognition in the aminoacylation reactions of a subgroup of aminoacyl-tRNA synthetases.