ACTIVATION OF METHIONINE BY ESCHERICHIA-COLI METHIONYL-TRANSFER RNA-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-PP(i) exchange catalyzed by MetRS with little or no effect on the K(m)'s for methionine or ATP or on the K(m) 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 K(m) for methionine and a 2000-fold decrease in K(cat) for ATP-PP(i) exchange, with no change in the K(m) 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. Consistent with this, substitution of Ala for Trp305 resulted in a specific loss of affinity for methionine and an overall 100-fold decrease in K(cat)/K(m) for ATP-PP(i) exchange. Comparison of the sequences in this domain of the Met, Ile, Leu, and Val synthetases shows that there is little homology between all members of this group but strong homology between enzymes specific for the same amino acids. This supports the idea that the region close to and including Trp305 forms part of the domain involved in discrimination of the methionine side chain by MetRS and suggests that corresponding sequences in the other enzymes play a similar role in distinguishing between the closely related side chains of this group of amino acids.