The kinetics of the amino acid activation and the transfer of the amino acid to tRNA have been investigated for L-phenylalanine:tRNA ligase of Escherichia coli K10 by stopped-flow and radioactive techniques. The rapid kinetics were followed by the observation of the displacement of the fluorescent dye, 6-p-toluidinylnaphthalene-2-sulfonate from the binding site of L-phenylalanine under conditions where a single active site of the enzyme was involved. The following results are of particular interest. (1) Equilibrium binding of L-phenylalanine and tRNAPhe indicates in each case two sites of interaction with an approximately tenfold difference of the binding affinity. (2) Experimental conditions of the kinetic investigation were chosen to favor reactions at the high affinity binding sites. Under those conditions, the rate constants have been evaluated at 1 mM magnesium to be in the range 12-25 sec-1 for the activation reaction and 42-77 sec-1 for the reverse, the variation of the values depending on those of the dissociation constants used for computation. The rate constant for the transfer reaction is 0.05 sec-1 and for the reverse 0.19 sec-1. The forward reaction is rate limiting for the overall reaction at single turnover and steady-state conditions. (3) All rate constants depend on the concentration of magnesium. Evidence is provided that the transfer occurs via a productive enzyme-tRNAPhe complex which is in a magnesium-dependent equilibrium with an unproductive complex, high magnesium favoring the former. The position of the tRNA-CCA end in the productive complex is such, that the fluorescent dye can be displaced by Phe-tRNAPhe. The thermodynamics of the overall reaction have been treated on the basis of the partial reactions. The free enthalpy of the completed reaction was calculated to be very close to zero. The significance of the adenylate intermediate is discussed with respect to the product inhibition expected on the basis of the tendency of tRNAPhe and L-phenylalanine to form tight complexes with the enzyme.

Formation of binary and ternary enzyme-ligand complexes was investigated for amino acid:tRNA ligases specific for L-isoleucine, L-leucine, and L-phenylalanine. Each of the enzymes exhibited synergistic binding when a substrate was substituted by a structurally related compound. The strength of coupling between the sites binding the amino acid and ATP was strongly dependent on the structure of ligands. The phenomenon was observed with the L-leucine and L-phenylalanine-specific enzymes only in the presence of magnesium. Spermine was inhibitory for L-phenylalanine:tRNA ligase. From the variation which structure of the strength of the observed synergism a correlation scheme was derived considering the ammonium group, the carboxylate group and the side chain of the amino acid, and the adenosine and triphosphate moieties of ATP. The strength of coupling between the subsites binding various combinations of these moieties was evaluated. We found that binding of the subgroups of the amino acid exerts an intramolecular synergism. The strength intramolecular synergism was similar to the strength of the intermolecular synergism observed for the simultaneous binding of an amino alcohol and ATP (or MgATP-2-). We have derived a molecular mechanism for the formation of the ternary enzyme-amino acid-ATP (or MgATP-2-) complex taking into account the synergistic phenomena. The complex is considered to involve electrostatic repulsion between the amino acid carboxylate and the ATP triphosphate moieties. When one of the negatively charged groups have been eliminated, the enzymatic rearrangement which facilitates the formation of this complex may be seen as a synergistic coupling.

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.

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