Substrate Complexes of Phenylalanyl‐tRNA Synthetase from Escherichia coli

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Abstract
The stoichiometry of substrate binding to phenylalanyl‐tRNA synthetase from Escherichia coli K10 was investigated. Isolation of the phenylalanyl · adenylate · enzyme complex by means of gel filtration revealed that phenylalanine and the nucleotide are bound in about equimolar amounts and in a stoichiometric ratio. Stable complexes between tRNAPhe and phenylalanyl‐tRNA synthetase could be separated from free tRNAPhevia sucrose‐gradient zone‐centrifugation or gel filtration. Determination of the amount of tRNAPhe bound to phenylalanyl‐tRNA synthetase (molecular weight 181000) by absorption at 260 nm or by the amino‐acid‐acceptance activity yielded ratios close to one. tRNAPhe· enzyme complexes were most stable at pH values below 6.5; complex formation between phenylalanyl‐tRNA synthetase and non‐cognate tRNA species was not observed. For determination of the affinity and ratio of binding of phenylalanine the non‐equilibrium dialysis method was employed: a Kd of 30 μM was obtained; by the criteria of this method one binding site for the amino acid could be demonstrated.Phenylalanyl‐tRNA synthetase of E. coli is protected against heat inactivation by Mg, ATP, ATP and phenylalanine or tRNAPhe. By means of the protection constant, the apparent affinity constant for tRNAPhe was determined to be 3 × 107 M−1 at pH 6.0 and 37 °C. The dependence of the π‐values obtained on pH indicates that the affinity of phenylalanyl‐tRNA synthetase for tRNA drastically increases at more acid pH. The same tendency was obtained upon determination of the pH‐dependence of the Km values for tRNAPhe.Out of the 24 SH‐groups of phenylalanyl‐tRNA synthetase which are reacting with 5,5′‐dithio‐bis(2‐nitrobenzoic acid) in the presence of 8 M urea only less than one half of them are oxidized on the native enzyme. The kinetics of titration of the sulfhydryl is grossly changed in the presence of ATP, ATP and phenylalanine or tRNAPhe. In addition, in the presence of ATP and phenylalanine or of tRNAPhe 2‐3 SH‐groups are completely protected from oxidation. Under this condition loss of activity which accompanies the SH‐group oxidation is greatly diminished.