Méthionyl‐tRNA synthétase des embryons de blé: dissociation en sous‐unités

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Abstract
Methionyl‐tRNA Synthetase from Wheat Embryo: Dissociation into Subunits Wheat‐embryo methionyl‐tRNA synthetase is a dimeric protein of β2 structure. When highly diluted, it loses the capacity to catalyze ATP‐[32P]PPi exchange and to aminoacylate tRNAMet: at low enzymatic concentrations the rates of formation of [32P]ATP and [14C]methionyl‐tRNAMet are lower than those predicted by extrapolating the rates determined at higher enzyme concentrations. The difference between observed and expected rates becomes greater with decreasing enzyme concentration. Filtration of purified, dilute enzyme preparations on Sephadex G‐200 results in the separation of dimer and monomer fractions. The proportion of monomer present increases with increasing pre‐incubation times before the assay and demonstrates an equilibrium between active dimers and inactive monomers. The above‐mentioned loss of enzymatic activity is explained by the equilibrium being shifted towards the production of monomers. Data previously gathered for Escherichia coli prolyl‐tRNA synthetase and for bovine‐pancreatic tryptophanyl‐tRNA synthetase, coupled with the present results, suggests that the dissociation of dimeric synthetases may be a general phenomenon in eukaryotes as well as in prokaryotes. The number of sub‐units and the dissociation constant were obtained at equilibrium, according to relations adapted to the case of oligomeric enzymes (KD≃ 13 nM at 25 °C and pH 7.5). Rate constants were determined by kinetic studies of the attainment of equilibrium. The rate constant k1 for monomolecular dissociation was determined to be 1.85 · 10−3 s−1 and k2 for the bimolecular association to be 0.145 · 106 M−1 s−1. The KD calculated from k1 and k2 was coherent with the experimentally determined value, at equilibrium. The sub‐unit interactions, which involve only a small quantity of energy (ΔG°≃+ 11 kcal mol−1; +45 kJ mol−1) at 25 °C, depend on the ionic environment of the medium and the presence of substrates. Alkaline pH favors monomer production, while the presence of methionine, (Mg‐ATP)2− and tRNAMet protect the synthetase from dissociation. 2‐mercaptoethanol and dithioerythritol prevent only slightly the loss of activity. Bovine serum albumin, however, protects the enzyme from dissociation under dilute conditions.