Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD.
A reactive bis-dichloro derivative of the Procion dye Green HE-4BD was shown to inactivate irreversibly methionyl-tRNA synthetase (MTS) from Escherichia coli and also tryptophyl-tRNA synthetase (WTS) and tyrosyl-tRNA synthetase (YTS) from Bacillus stearothermophilus at pH 8.5 and 37 degrees C. At a 5-fold excess of reactive dye over enzyme subunit concentration MTS was quantitatively inactivated within 20 min in the ATP/pyrophosphate exchange assay, whereas WTS and YTS show an 80% loss of activity over the same time period. The inactivation is affected by the addition of substrates, which either protect (WTS and YTS) or promote (YTS with tyrosine) the dye-mediated enzyme inactivation. Green HE-4BD-OH was shown to be a competitive inhibitor of MTS with respect to MgATP, methionine and tRNA substrates.
McArdell
J E
JE
Division of Biotechnology, Centre for Applied Microbiology and Research, Salisbury, Wilts, U.K.
Duffield
M
M
Atkinson
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004396:Coloring Agents; D004926:Escherichia coli; D001411:Geobacillus stearothermophilus; D008718:Methionine-tRNA Ligase / Q000378:metabolism; D014227:Triazines; D014369:Tryptophan-tRNA Ligase / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism
D004396:Coloring Agents; D014227:Triazines; C026268:procion green H-E4BD; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase; D008718:Methionine-tRNA Ligase; D014369:Tryptophan-tRNA Ligase