ARGININE-395 IS REQUIRED FOR EFFICIENT INVIVO AND INVITRO AMINOACYLATION OF TRANSFER-RNAS BY ESCHERICHIA-COLI METHIONYL-TRANSFER RNA-SYNTHETASE

We have previously shown that the anticodon of methionine tRNAs contains the major recognition site required for aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase (MetRS) and have located part of the anticodon binding domain on the enzyme at a site close to Trp461 [Schulman, L. H., & Pelka, H. (I 988) Science 242, 765-768; Ghosh, G., Pelka, H., & Schulman, L. H. (I 990) Biochemistry 29, 2220-2225]. In order to gain information about other possible sites of contact between MetRS and its tRNA substrates, we have examined the effects of mutations at a series of positively charged residues on the surface of the C-terminal domain of the enzyme. Conversion of Arg356, Arg366, Arg380, or Arg453 to Gln had little or no effect on enzyme activity. Similarly, conversion of Lys402 or Lys439 to Asn failed to significantly alter aminoacylation activity. Conversion of Arg380 to Ala or Arg442 to Gln produced a 5-fold reduction in k(cat)/K(m) for aminoacylation of tRNA(fMet), with no effect on methionine activation, indicating a possible minor role for these residues in interaction of the enzyme with the tRNA substrate. In contrast, mutation of a phylogenetically conserved residue, Arg395, to Gln increased the K(m) for aminoacylation of tRNA(fMet) about 30-fold and reduced k(cat)/K(m) by 25 000-fold. The mutant enzyme was also shown to be highly defective by its inability to complement a strain of E. coli having an altered chromosomal MetRS gene. Examination of the kinetic parameters for ATP-PP(i) exchange catalyzed by the Gln395 enzyme showed little or no effect of the mutation on interaction of MetRS with methionine or ATP, indicating that the major role of Arg395 is in tRNA recognition. Mutation of a second conserved residue, Asn391, to Ala specifically increased the K(m) for aminoacylation of tRNA(fMet) 20-fold with little or no effect on the other kinetic parameters. Examination of the crystal structure of MetRS [Brunie, S., Zelwer, C., & Risler, J.-L. (1990) J. Mol. Biol. 216, 411-424] shows that Trp461 is located on a separate peptide, at a distance of 10-20 angstrom from Asn391 and Arg395. Extensive molecular dynamics simulation studies have revealed that the peptide loop containing Trp461 is the most flexible part of MetRS and that residues in this loop can move more than 5 angstrom with little energy cost. This suggests that tRNA binding may induce a conformational change in MetRS that allows simultaneous interaction of the anticodon loop of the tRNA with the two peptides containing Trp461 and Asn391-Arg395. Such a substrate-induced conformational change may be an important feature of the tRNA recognition mechanism.