Chemical modification and mutagenesis studies on zinc binding of aminoacyl-tRNA synthetases.
Thermus thermophilus methionyl-tRNA synthetase consists of two identical subunits with a potential Zn(2+)-binding sequence of Cys-X2-Cys-X13-Cys-X2-His (Nureki, O., Muramatsu, T., Suzuki, K., Kohda, D., Matsuzawa, H., Ohta, T. Miyazawa, T., and Yokoyama, S. (1991) J. Biol. Chem. 266, 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 with 4-(2-pyridylazo)resorcinol. Site-directed mutagenesis of Cys and His residues in the Zn(2+)-binding sequence reduced the aminoacylation activity; the kcat value was markedly decreased, and the Km values for L-methionine and tRNAf(Met) were increased. Similarly, Cys modification released two Zn2+ ions from T. thermophilus and Escherichia coli isoleucyl-tRNA synthetases and E. coli threonyl-tRNA synthetase, which have Zn(2+)-binding motifs, and impaired their activities. By contrast, three other aminoacyl-tRNA synthetases that lack Zn(2+)-binding motif neither released Zn2+ ion nor lost their activities upon Cys modification. These results indicate that the Zn(2+)-binding sequences are important for catalysis and recognition in the aminoacylation reactions of a subgroup of aminoacyl-tRNA synthetases.
Nureki
O
O
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Kohno
T
T
Sakamoto
K
K
Miyazawa
T
T
Yokoyama
S
S
D000215:Acylation; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010663:Phenylmercury Compounds / Q000737:chemistry; D016963:Thermus thermophilus / Q000201:enzymology; D015032:Zinc / Q000378:metabolism*
D010663:Phenylmercury Compounds; C002910:4-hydroxymercuribenzenesulfonate; D000604:Amino Acyl-tRNA Synthetases; D015032:Zinc