| Abstract: | Methionyl-tRNA synthetase from Escherichia coli catalyses the activation of 18O2]methionine by adenosine 5'-(R)-alpha 17O]triphosphate with inversion of configuration at P alpha. Furthermore methionyl-tRNA synthetase does not catalyse positional isotope exchange in adenosine 5'-beta-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'-beta-18O2]triphosphate in the presence of methionine, Mg2+ and methionyl-tRNA synthetase showed that torsional equilibration (18O exchange into the P alpha--O--P beta bridge) occurs faster than tumbling (18O exchange into P gamma by rotation about the C2 axis of Mg18O2]PPi), demonstratings that the positional isotope exchange occurs at least in part in the E X Met-AMP X Mg18O2]PPi complex. |
