The role of tryptophan residues in Escherichia coli arginyl-tRNA synthetase.
The effect of N-bromosuccinimide (NBS) on the activity of Escherichia coli arginyl-tRNA synthetase (ArgRS) was studied. The results showed that only one tryptophan residue was easy of access to the reagent and was closely related to enzyme activity. When all the five tryptophan residues in ArgRS were changed via site-directed mutagenesis singly into Ala, the aminoacylation activity of the Trp162 mutated enzyme decreased seriously, while the other four mutant enzymes retained almost the same activity as the native one. The oxidation of the five mutant enzymes with NBS demonstrated that only the mutation of Trp162 resulted in the loss of sensitivity to the reagent. These results strongly suggest that Trp162 is more accessible to NBS and is related to enzyme activity. Furthermore, the far-UV CD spectroscopy of the mutant enzyme ArgRS162WA showed little change in its secondary structure. Finally, studies on the kinetics of the mutant enzyme ArgRS162WA in aminoacylation reaction showed that the reduction in activity could be attributed to the decrease in the values of kcat and kcat/Km for arginine. The thermodynamic calculation indicates that this mutation causes a decrease of the binding energy by 2.7 kJ/mol. Our data suggest that Trp162 is involved in the binding of arginine and in the transition state stabilization.
Zhang
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State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, 320 Yue-yang Road, Shanghai 200031, China.
Wang
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Wang
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D000215:Acylation; D001126:Arginine-tRNA Ligase / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D001974:Bromosuccinimide / Q000494:pharmacology; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*
D001426:Bacterial Proteins; D014364:Tryptophan; D001126:Arginine-tRNA Ligase; D001974:Bromosuccinimide