A stereochemical and positional isotope-exchange study of the mechanism of activation of methionine by methionyl-tRNA synthetase from Escherichia coli.
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 Mg[18O2]PPi), demonstratings that the positional isotope exchange occurs at least in part in the E X Met-AMP X Mg[18O2]PPi complex.
D000604:Amino Acyl-tRNA Synthetases / Q000502:physiology*; D001711:Biotransformation; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D007553:Isotope Labeling; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D008718:Methionine-tRNA Ligase / Q000502:physiology*; D010103:Oxygen Isotopes; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D010103:Oxygen Isotopes; D008715:Methionine; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase