The Interaction of Tryptophanyl-tRNA Synthetase with the Triazine Dye Brown MX-5BR

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Abstract
Brown MX-5BR specifically and irreversibly inactivates tryptophanyl tRNA synthetase from Bacillus stearothermophilus at pH 8.5. The enzyme is protected from inactivation by the substrates tryptophan and ATP and to lesser extents by ADP, AMP, the product PPi and other nucleotides such as GTP. The Kd of the pure reactive dye for the enzyme was 6.7 .times. 10-6 M. The Km values of the 2 substrates tryptophan and MgATP were 1 .times. 10-5 M and 5 .times. 10-5 M, respectively. The aminated dye is a competitive inhibitor of tryptophanyl tRNA synthetase with respect to both tryptophan and MgATP with Ki values of 2 .times. 10-4 M against both substrates. The use of this dye as an active-site-directed affinity label is discussed.