Methionyl-Transfer-RNA Transformylase from Escherichia coli

A large-scale purification procedure of methionyl-tRNA transformylase (10-formyltetrahydro-folate:l-methionyl-tRNA N-formyltransferase) from Escherichia coli is described. The purification (10000-fold) produces 22 mg homogeneous enzyme from 5 kg wet cells with a yield of 45%. The specific activity of the enzyme in the reaction of formylation of methionyl-tRNAMetf is 30-fold higher than that reported by others [Dickerman, H. W., Steers, E., Jr, Redfield, B. G. and Weissbach, H. (1967) Biol. Chem. 242, 1522-1525]. The enzyme is a monomer of molecular weight 32000, based on high-speed equilibrium sedimentation, small-angle neutron scattering, molecular sieving on Sephadex and gel electrophoresis in the presence of sodium dodecyl sulfate. Its absorption coefficient has been determined as A280nm=1.4 ± 0. 1 cm2. mg−1. The Michaelian parameters of the transformylation reaction have been measured for each substrate at 25 °C, pH 7. 6, in the presence of 150 mM KC1 and 7 mM MgCl2. Km values are 13.5 ± 1.5 μ and 0.35 ± 0.05 μ for 10-formyltetrahydrofolate and for methionyl-tRNAMetf respectively. A V value of 20 ± 2 s−1 is found at saturation of both substrates. The tryptophan fluorescence of transformylase is remarkably sensitive to the bindings of 10-formyltetrahydrofolate and tRNAMetf This property enables one to determine 1:1 stoichiometries of the enzyme with either 10-formyltetrahydrofolate, tRNAMetf at high ionic strength. Stability of the tRNAMetf transformylase complex is improved upon aminoacylation of tRNA. The observed association constant for the interaction between enzyme and tRNAMetf is sensitive to the concentration of cations (K+ or/and Mg2+). This behaviour has allowed us to estimate to 4.6 ± 0.7 as the number of ion pairs formed between tRNAMetf and transformylase.