A synergistic coupling was observed for ligands binding to the specific sites for L-phenylalanine and ATP. Dissociation constants were measured by the use of fluorescence titration and equilibrium dialysis techniques. It is found that L-phenylalaninol, L-phenylalanine methylester, and phenylethylamine bind with an increased affinity when ATP, ADP, AMP or adenosine were present. With L-phenylalanine, synergistic binding was only observed for adenosine ; ATP has probably no effect. Results of competition experiments, the stoichiometry of complex formation and the magnitude of dissociation constants indicate that synergistic coupling involves the binding sites for ATP and amino acid a t the active site.The reaction mechanism of an aminoacyl-tRNA synthetase is understood as a two-step process. Formation of aminoacyl-adenylate from amino acid and ATP is subsequently followed by transfer of the aminoacyl moiety to cognate tRNA. ATP and amino acid have been found to react in an ordered sequence [l-31, or at random [4-61. We have investigated by equilibrium measurements with L-phenylalanyl-tRNA synthetase as an example for random mechanism, whether the binding of substrates and structurally related compounds to the sites for amino acid and ATP is in fact independent as suggested from kinetic experiments [41.

The molecular weight of L-phenylalanyl-tRNA synthetase was redetermined. A new value of 267 000 was obtained by high-speed analytical ultracentrifugation and Sephadex G-200 gel chromatography. The value is in contrast to the molecular weight of 180000 determined previFrom the molecular weight 267000 and the sedimentation coefficient S Z O ,~ = 8.6 S the ratio f / f o = 1.67 was calculated. The magnitude of f / f o is in accord with the molecular weight 183000 obtained from sucrose gradient centrifugation. The results indicate that the enzyme is highly hydrated and/or assumes the shape of a rod or a disc.The subunit composition was reinvestigated by sodium dodecylsulfate gel electrophoresis, guanidinium chloride gel filtration and by reactivation experiments of pH-2 inactivated enzyme. The results are consistent with a subunit structure of the type. Molecular weights of the subunits are 39000 and 94000, respectively. ously.With the intention to find an explanation for the asymmetry between the number of active sites and the subunit composition of L-phenylalanyl-tRNA synthetase [1-31 and prompted by the finding of inconsistencies by others [4] with earlier results [2, 5-71, we became engaged in the reinvestigation of the molecular weight and subunit structure of this enzyme.

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 atration revealed that phenylalanine and the nucleotide are bound in about equimolar amounts and in a stoichiometric ratio. Stable complexes between tRNAPhe and phenylalanyltRNA synthetase could be separated from free tRNAPhe via sucrose-gradient zone-centrifugation or gel filtration. Determination of the amount of tRNAPhe bound to phenylalanyl-tRNA synthetase (molecular weight 181 000) by absorption at 260 nm or by the amino-acid-acceptance activity yielded ratios close to one. tRNAPhe e 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 Ka of 30 pM 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 x lo7 M-I at pH 6.0 and 37 "C. The dependence of the n-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.

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