Arginine-395 is required for efficient in vivo and in vitro aminoacylation of tRNAs by Escherichia coli methionyl-tRNA 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. (1988) Science 242, 765-768; Ghosh, G., Pelka, H., & Schulman, L.H. (1990) 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 kcat/Km for aminoacylation of tRNAfMet, 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 Km for aminoacylation of tRNAfMet about 30-fold and reduced kcat/Km 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Ghosh
G
G
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
Kim
H Y
HY
Demaret
J P
JP
Brunie
S
S
Schulman
L H
LH
D000595:Amino Acid Sequence; D000926:Anticodon / Q000378:metabolism; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008718:Methionine-tRNA Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D000926:Anticodon; D009838:Oligodeoxyribonucleotides; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; D001120:Arginine; D008715:Methionine; D008718:Methionine-tRNA Ligase