title: L-phenylalanine:tRNA ligase of Escherichia coli K10. A rapid kinetic investigation of the catalytic reaction.
abstract: 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.
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000494:pharmacology; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D000596:Amino Acids; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase; D008274:Magnesium