A stereochemical and positional isotope exchange study of the mechanism of activation of isoleucine by isoleucyl-tRNA synthetase from Escherichia coli

Abstract
Isoleucyl-tRNA synthetase from E. coli catalyzes the activation of [18O2]isoleucine by adenosine 5''-[(R)-.alpha.-17O]triphosphate with inversion of configuration at P. Isoleucyl-tRNA synthetase does not catalyze positional isotope exchange in adenosine 5''-[.beta.-18O2]triphosphate in the absence of isoleucine or in the presence of the competitive inhibitor isoleucinol, which effectively eliminates the possibility of adenylyl-enzyme or adenosine metaphosphate intermediates being involved. Isoleucyl-tRNA synthetase catalyzes the activation of isoleucine by associative in line nucleotidyl transfer. The synthesis of adenosine 5''-[(R)-.alpha.-17O]diphosphate and its conversion to adenosine 5''-[(R)-.alpha.-17O]triphosphate is described and an explanation provided for the reported differences between the treatment of adenosine 5''-[(S)-.alpha.-thiodiphosphate] with cyanogen bromide and Br in [18O]water.

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