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J. Biol. Chem., Vol. 266, Issue 26, 17136-17141, 09, 1991
G Ghosh, S Brunie and LH Schulman
The crystal structure of a fully biologically active monomeric form of
Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has
recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990)
J. Mol. Biol. 216, 411-424), revealing details of the active site of the
enzyme, including the location of amino acid residues potentially involved
in substrate binding. In the present paper, the role of 3 active site
residues in interaction with methionine, ATP, and tRNA(fMet) and in
catalysis of methionyl-adenylate has been explored using site-directed
mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP
cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in
kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine
side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala
produced a 14-fold increase in the Km for ATP with only a small (2-3-fold)
change in the other kinetic parameters, indicating that the major role of
this residue is in formation of the initial complex with ATP and/or in
stabilization of the methionyl- adenylate reaction intermediate. Mutation
of the adjacent residue Tyr358 to Ala had no effect on the Km values for
methionine or ATP but produced nearly a 2000-fold decrease in the rate of
ATP-PPi exchange. This mutation also dramatically reduced the rate of
pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of
pyrophosphate without increasing the Km for PPi. None of the mutations
affected the Km for tRNAfMet in the aminoacylation reaction. The results
suggest that Tyr358 may enhance the rate of methionyl-adenylate formation
by binding to the alpha-phosphate of ATP in the transition state.
Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction
requires a significant change in the conformation of this region of the
active site compared to the structure found in the MetRS.ATP complex. Such
a shift is consistent with an induced-fit mechanism for methionine
activation. Primary sequence comparisons of methionine-specific enzymes
from yeast and bacterial sources reveals that Tyr358 is conserved in all of
the known MetRS sequences.
Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
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