Biochemical and Immunological Characterization of Threonyl‐tRNA Synthetase of Two Borrelidin‐Resistant Mutants of Escherichia coli K12

Abstract

The threonyl‐tRNA synthetase of two wild‐type Escherichia coli K12 strains and of two borrelidin‐resistant mutants thereof has been purified about 100‐fold. A neutralizing antiserum has been prepared against one of the wild‐type threonyl‐tRNA synthetases. The comparative characterization of the threonyl‐tRNA synthetases of the two wild strains and the two borrelidin‐resistant mutants revealed that the threonyl‐tRNA synthetase of one borrelidin‐resistant mutant (K12B borr 2) exhibits a lowered Km value for threonine and ATP and an increased K1 value for borrelidin with respect to threonine. Furthermore the activity of the threonyl‐tRNA synthetase of this mutant k more sensitive to higher pH values, is more rapidly heat‐inactivated and shows a different shape of its antiserum‐neutralization curve than the threonyl‐tRNA synthetase of the parental strain (K12B). The threonyl‐tRNA synthetase of the other borrelidin‐resistant mutant (K12B borr 3) was investigated in the same way and it did not show any difference to the threonyl‐tRNA synthetase of the parental strain, except that the specific activity of the enzyme in crude extracts of this mutant is 5 times as high as the corresponding value in crude extracts of K12B. From these data it is concluded that borrelidin resistance in K12B borr‐2 is due to a structurally altered threonyl‐tRNA synthetase (structural mutant) whereas in K12B borr‐3 it is due to the constitutive increase of the threonyl‐tRNA synthetase level by a factor of five (regulatory mutant). The inhibition of the threonyl‐tRNA synthetase by borrelidin with respect to threonine was noncompetitive in all four cases. In order to observe a considerable heat inactivation of the threonyl‐tRNA synthetase within 10 min a temperature of 60°C was necessary. The heat‐inactivation process was not affected by the presence of threonine, but it was hastened by the presence of ATP or tRNA. Copyright © 1973, Wiley Blackwell. All rights reserved