Activation of methionine by Escherichia coli methionyl-tRNA synthetase.
In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)
Ghosh
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G
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
Pelka
H
H
Schulman
L H
LH
Brunie
S
S
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000096:biosynthesis; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000235:genetics; D008715:Methionine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism*; D008718:Methionine-tRNA Ligase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013379:Substrate Specificity; D046249:Transfer RNA Aminoacylation; D014364:Tryptophan / Q000235:genetics
D001426:Bacterial Proteins; D005973:Glutamine; C011501:methioninyl adenylate; D001224:Aspartic Acid; D000249:Adenosine Monophosphate; D014364:Tryptophan; D001120:Arginine; D008715:Methionine; D008718:Methionine-tRNA Ligase; D000409:Alanine