A Stereochemical and Positional Isotope‐Exchange Study of the Mechanism of Activation of Methionine by Methionyl‐tRNA Synthetase from Escherichia coli

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Abstract
Methionyl‐tRNA synthetase from Escherichia coli catalyses the activation of [18O2]methionine by adenosine 5′‐[(R)‐α17O]triphosphate with inversion of configuration at Pα. Furthermore methionyl‐tRNA synthetase does not catalyse positional isotope exchange in adenosine 5′‐[β‐18O2]triphosphate in the absence of methionine or in the presence of the competitive inhibitor, methioninol, which eliminates the possibility of either adenylyl‐enzyme or adenosine metaphosphate intermediates being involved. These observations require that methionyl‐tRNA synthetase catalyses the activation of methionine by an associative ‘in‐line’ nucleotidyl transfer mechanism. A kinetic study of positional isotope exchange in adenosine 5′‐[β‐18O2]triphosphate in the presence of methionine, Mg2+ and methionyl‐tRNA synthetase showed that torsional equilibration (18O exchange into the Pα—O—Pβbridge) occurs faster than tumbling (18O exchange into P7 by rotation about the C2 axis of Mg[18O2]PPi), demonstratings that the positional isotope exchange occurs at least in part in the E · Met‐AMP · Mg[18O2]PPi complex.

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