Methionyl-tRNA synthetase from Escherichia coli: substituting magnesium by manganese in the L-methionine activating reaction.
While Mg2+ can be efficiently replaced by Ni2+, Co2+ and Mn2+ in the ATP-PPi isotopic exchange reaction catalysed by methionyl-tRNA synthetase from Escherichia coli, the latter ion was selected for detailed analysis of the L-methionine activation reaction. In order to avoid artefactual results due to the slow aggregation of Mn2+ with pyrophosphate, this process was investigated by electron paramagnetic resonance and conditions were determined where it does not interfere with enzymic experiments. The thermodynamic parameters derived from steady-state (ATP-PPi isotopic exchange, fluorescence at equilibrium) or prestationary (fluorescence stopped-flow) experiments are compared to those obtained in the presence of Mg2+ [Hyafil et al. (1976) Biochemistry, 15, 3678-3685]. While the standard deltaG for the reaction (E-Met-ATP-Me2+equilibriumE-Met approximately AMP-PPi-Me2+) is close to zero in the case of Mg2+, Mn2+ slows down the rate of adenylate reversion and thus shifts the reaction towards the latter species. The deltaG for the formation of the E-Met approximately AMP complex does not depend on the metal used, suggesting that the divalent ion does not participate in the structuration of this complex. Substituting Mn2+ for Mg2+ decreases notably the dissociation constant of PPi-Me2+ from the E-Met approximately AMP-PPi-Me2+ species and from its abortive analog E-Met-Ado-PPi-Me2+. Similarly the dissociation constant of ATP-Me2+ from another dead-end analog E-methioninol-ATP-Me2+ is decreased by Mn2+. Involvement of the purine N7 atom in the binding of the metal ion to the active site of methionyl-tRNA synthetase is ruled out by the use of 7-deaza-adenosine. The role of the metal in the catalytic process of methionine activation and its relevance to the specificity of the reaction is then discussed in the light of the results obtained without metal and with Mg2+ and Mn2+.
D000241:Adenosine; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D002151:Calorimetry; D002413:Cations, Divalent; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D008433:Mathematics; D008715:Methionine; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D046249:Transfer RNA Aminoacylation; D014372:Tubercidin
D002413:Cations, Divalent; D008345:Manganese; D008715:Methionine; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase; D008274:Magnesium; D000241:Adenosine; D014372:Tubercidin