Methionyl‐tRNA Synthetase from Escherichia coli

Abstract

Enhancement of the nuclear relaxation rates by manganese has been used to derive manganesepurine-ring distances in the activation site of methionyl-tRNA synthetase. This is possible with the help of an abortive complex between the enzyme, methionine, adenosine, pyrophosphate and manganese which simulates an intermediate species of the activation reaction. It is found that the distances between the manganese ion and the purine ring are too high (>0.8 nm) to allow interaction between them. Thus, metal-purine interaction is involved neither in the catalytic mechanism nor in the stabilization of abortive synergistic complexes [S. Blanquet, G. Fayat and J. P. Waller (1975) Like all the aminoacyl-tRNA synthetases, methionyl-tRNA synthetase from Escherichiu coli requires a divalent ion (generally Mg2+) for adenylation of methionine. This metal ion promotes, in the same site, the synergistic formation of abortive complexes of substrates and/or analogs of substrates [1,2]. In a previous report [ 3 ] , we have shown that Mn2+ could substitute for Mg2+ in the adenylation site of trypsinmodified [4] methionyl-tRNA synthetase, both metals displaying similar properties for methionine activation and for formation of abortive complexes. Among these complexes, the enzyme . methionine . adenosine . pyrophosphate-metal (E . Met. Ado . PPi-Me2+) was shown to simulate the enzyme . methionyl-adenylate . pyrophosphate-metal (E . Met-AMP . PPi-Me2 ') intermediate of the activation reaction as regards its geometric, charge and thermodynamic properties [3,5]. In this article, we use the effects of the paramagnetic manganous ion on the relaxation times of the neighbouring protons to demonstrate that the metal ion does not interact with the purine ring of adenosine in the E . Met . Ado . PPi-Mn2+ complex. MATERIALS AND METHODSNative methionyl-tRNA synthetase was purified as described earlier [6] and converted into its trypsinAbbreviations. E , enzyme; Met, rnethionine; Met-AMP, methionyl-adenylate; PPi, pyrophosphate ; EPR, electron paramagnetic resonance; NMR, nuclear magnetic resonance.Enzyme. Methionyl-tRNA synthetase or L-methionine : tRNA Met ligase (AMP forming) (EC 6.1.1.10). modified fragment [4]. After exchange of its C-4 proton by heating at 90 "C in 2 H z 0 for 24 h, imidazole ( 5 mol/l) was neutralized with HCl until a pH-meter reading of 7.45 was reached after dilution 1 : 500 in 2 H~0 .The NMR spectrum of this 10 mM imidazole-HC1 buffer does not interfere with those of adenosine and tubercidine. The purified enzyme, stored at -15 "C in 20 mM potassium phosphate buffer pH 7.5, 10 niM 2-mercaptoethanol, 50 % glycerol, was extensively dialyzed against the imidazole-HC1 buffer and concentrated by ultrafiltration if necessary.The experiments were performed in 5-mm tubes at 20 "C in a Varian XL-100 NMR spectrometer operating in the Fourier transform mode with a disk. The 180-~-90 TI measurement method was used with fit of the experimental data to a three-parameter exponential curve with a non-linear iterative regression program. T2...