Modification of phenylalanyl-tRNA synthetase from Escherichia coli by histidine-specific reagents. Effects on structure and function.

Abstract

Phenylalanyl‐tRNA synthetase from Escherichia coli was subjected to chemical modification with diethylpyrocarbonate at pH 6.0 and to photochemical oxidation in the presence of rose Bengal, both methods giving rise to loss of enzymic activity. The highly sensitive reaction of the enzyme with either reagent is pseudo‐first order. The specificity of the modification methods employed has been investigated.All substrates were studied for their ability to protect the enzyme against inactivation. Presence of phenylalanine, ATP and Mg2+ provides the most pronounced effect. Under this condition the tRNA‐aminoacylation activity may be completely abolished, whereas the pyrophosphate–ATP exchange activity is only partly affected. However, such enzyme is still able to form a specific complex with tRNAPhe. The results presented therefore suggest that histidine residues of phenylalanyl‐tRNA synthetase accessible to modification by diethylpyrocarbonate or to photooxidation do not seem to be involved in the binding of tRNA but might possibly take part in the esterification reaction.Quantitative analysis showed that a total of 50 histidine residues are accessible to diethylpyrocarbonate in the unprotected enzyme under non‐denaturing conditions. In the presence of phenylalanine, ATP and Mg2+ the loss of aminoacylation activity is correlated with the modification of 2–4 residues.During extensive modification of the enzyme a dissociation process takes place. It was found that the observed loss of quaternary structure is not correlated with the decrease of the aminoacylation activity of the enzyme upon modification.