Thermodynamic studies on the specificity of L-isoleucine-tRNA ligase of Escherichia coli MRE 600. Calorimetric investigations on binding of amino acids and isoleucinol to the enzyme.
The association enthalpies, delta Ha, involved in the reactions between L-isoleucine:tRNA ligase (AMP-forming) from Escherichia coli MRE 600 (EC 6.1.1.5) and various amino acids have been determined calorimetrically in 50 mM potassium phosphate buffer, at pH 7.5, in the presence of 1 mM dithioerythritol. The delta Ha values for binding of L-isoleucine, L-leucine, L-valine, L-norvaline and L-2-amino-3S, 4-dimethyl pentanoic acid agree within the limits of experimental error in magnitude (3.7 +/- 0.9 kcal mol-1 or 15.5 +/- 3.8 kJ mol-1 at 25 degrees C) and variation with temperature (delta cp = -430 +/- 20 cal mol-1 K-1 or 1799 +/- 84 J mol-1 K-1). In view of the large differences in the equilibrium constants for the corresponding binding equilibria, the identical association enthalpies suggest that the enthalpic contribution to the Gibbs free energy of binding, delta Ga, cannot be responsible for the specificity of the interaction of the enzyme with the amino acids. It has rather to be inferred that the entropic term, delta Sa, is decisive in discriminating the correct amino acid. Analogous calorimetric binding studies on the reaction between L-isoleucinol and the enzyme suggest that the absence of the carboxyl group renders the association enthalpy more positive (by 4-5 kcal mol-1 or 16.7-20.9 kJ mol-1) with respect to that of the amino acids. The variation with temperature of the delta Ha values, however, practically parallels that of the amino acids.
D000596:Amino Acids / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007532:Isoleucine / Q000031:analogs & derivatives* / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D010421:Pentanoic Acids / Q000378:metabolism; D014176:Protein Biosynthesis; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D014633:Valine / Q000031:analogs & derivatives / Q000378:metabolism
D000596:Amino Acids; D010421:Pentanoic Acids; D007532:Isoleucine; C014060:isoleucinol; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase; D014633:Valine