Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Abstract

Discrimination between cognate and non-cognate tRNAs by aminoacyl-tRNA synthetases occurs at several steps of the aminoacylation pathway. We have measured changes of solvation and counterion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-tRNA synthetases. The decrease in the association constant with increasing KCI concentration is relatively small for cognate tRNA binding when compared to known DNA-protein interactions. The electroneutral nature of the tRNA binding domain may be largely responsible for this low ion release stoichiometry, suggesting that a relatively large electrostatic component of the DNA-protein interaction free energy may have evolved for other purposes, such as, target search. Little change in solvation upon tRNA binding is seen. Non-cognate tRNA binding actually increases with increasing KCI concentration indicating that charge repulsion may be a significant component of binding free energy. Thus, electrostatic interactions may have been used to discriminate between cognate and non-cognate tRNAs in the binding step. The catalytic constant of glutaminyl-tRNA synthetase increases with increasing osmotic pressure indicating a water release of 8.4 ± 1.4 mol/mol in the transition state, whereas little change is seen in the case of glutamyl-tRNA synthetase. We propose that the significant amount of water release in the transition state, in the case of glutaminyl-tRNA synthetase, is due to additional contact of the protein with the tRNA in the transition state.