Methionyl-tRNA synthetase from Escherichia coli: active stoichiometry and stopped-flow analysis of methionyl adenylate formaiton.
Native dimeric methionyl-tRNA synthetase and its monomeric proteolytic fragment are shown to form and to bind 1 mol of methyionyl adenylate per polypeptide chain. Moreover, at 25 degrees C, each monomer of the dimeric native enzyme behaves independently, exhibiting the same parameters for the methionine activation reaction as does the monomeric modified enzyme. These results were obtained using several independent methods including equilibrium and nonequilibrium dialysis, active site and tryptophan fluorescence titrations, and stopped-flow by fluorescence. Stopped-flow resolution of the reversible methionine activation reaction also demonstrates that methionine and ATP-Mg2+ react without coupling to form a ternary enzyme-methionine-ATP-Mg2+ complex. This complex readily converts to enzyme-methionyl approximately adenylate-PP-Mg2+ with a standard free energy close to zero. It is concluded that the uncoupled enzyme-methionine-ATP-Mg2+ complex may resemble the transition state of the reaction at the expense of the additional state of the reaction at the expense of the additional synergistic binding energy provided by reciprocal coupling, within the site, of the methionine molecule with the adenosine and PP-Mg2+ parts of the ATP-Mg2+ molecule (Blanguet, S., Fayat, G., and Waller, J. P. (1975), J. Mol. Biol. 94, 1.).
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D003956:Dialysis; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism*; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014357:Trypsin
D011756:Diphosphates; D046911:Macromolecular Substances; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D008715:Methionine; D014357:Trypsin; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase; D008274:Magnesium