THE ROLE OF TRYPTOPHAN RESIDUES IN ESCHERICHIA-COLI ARGINYL-TRANSFER-RNA SYNTHETASE

The effect of N-bromosuccinimide (NBS) on the activity of Escherichia coli arginyl-tRNA synthetase (ArgRS) was studied. The results showed t hat only one tryptophan residue was easy of access to the reagent and was closely related to enzyme activity. When all the five tryptophan r esidues in ArgRS were changed via site-directed mutagenesis singly int o Ala, the aminoacylation activity of the Trp(162) mutated enzyme decr eased seriously, while the other four mutant enzymes retained almost t he same activity as the native one. The oxidation of the five mutant e nzymes with NBS demonstrated that only the mutation of Trp(162) result ed in the loss of sensitivity to the reagent. These results strongly s uggest that Trp(162) is more accessible to NBS and is related to enzym e activity. Furthermore, the far-UV CD spectroscopy of the mutant enzy me ArgRS162WA showed little change in its secondary structure. Finally , studies on the kinetics of the mutant enzyme ArgRS162WA in aminoacyl ation reaction showed that the reduction in activity could be attribut ed to the decrease in the values of k(cat) and k(cat)/K-m 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 Tr p(162) is involved in the binding of arginine and in the transition st ate stabilization. (C) 1998 Elsevier Science B.V. All rights reserved.