A stereochemical and positional isotope exchange study of the mechanism of activation of isoleucine by isoleucyl-tRNA synthetase from Escherichia coli.
Isoleucyl-tRNA synthetase from Escherichia coli catalyzes the activation of [18O2]isoleucine by adenosine 5'-[(R)-alpha-17O]triphosphate with inversion of configuration at phosphorus. Moreover, 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 either adenylyl-enzyme or adenosine metaphosphate intermediates being involved. Together, these observations require that 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 bromine in [18O]water.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007532:Isoleucine / Q000096:biosynthesis*; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013237:Stereoisomerism
D007532:Isoleucine; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase