Kinetic analysis of the isoleucyl-tRNA synthetase mechanism: the next reaction cycle can start before the previous one ends.

Aminoacyl-tRNA synthetases join correct amino acids to their cognate tRNA at the start of the protein synthesis. Through the kinetic analysis, it is possible to estimate how their functional details correspond to the known structural features. Kinetic analysis of the isoleucyl-tRNA synthetase (IleRS) from Escherichia coli was accomplished. Sixteen different steady-state two-ligand experiments were statistically analysed simultaneously so that the same rate equations and same rate and dissociation constants applied to all experiments. The so-called rapid equilibrium segments procedure was used to derive the rate equations. The final best-fit mechanism included the normal activation and transfer steps, and reorganization of the steps between them and after the transfer step. In addition, the analysis strongly suggested an additional activation step, formation of a new isoleucyl-AMP before the isoleucyl-tRNA was freed from the enzyme. The removal of Ile-tRNA was possible without the formation of Ile-AMP if both isoleucine and ATP were bound to the E-Ile-tRNA complex, but this route covered only 11% of the total formation of Ile-tRNA. In addition to the Mg2+ in MgATP or MgPPi, only two tRNA-bound Mg2+ were required to explain the magnesium dependence in the best-fit mechanism. The first Mg2+ could be present in all steps before the second activation and was obligatory in the first reorganizing step and transfer step. The second Mg2+ was present only at the transfer step, whereas elsewhere it prevented the reaction, including the activation reactions. Chloride inhibited the IleRS reaction, while 100 mm KCl caused 50% inhibition if the ionic strength was kept constant with K-acetate. The Kmapp (tRNA) value was increased from 0.057 to 1.37 µm when the KCl concentration was increased from 0 to 200 mm. The total rate equation helps to understand the reaction route and how the simultaneous presence of Ile-tRNA and Ile-AMP can cause new possibilities to proofreading mechanisms of this enzyme. Enzyme: Isoleucyl-tRNA synthetase (EC 6.1.1.5).

Magnesium dependence of the measured equilibrium constants of aminoacyl-tRNA synthetases.

The apparent equilibrium constants (K') for six reactions catalyzed by aminoacyl-tRNA synthetases from Escherichia coli were measured, the equations for the magnesium dependence of the equilibrium constants were derived, and best-fit analyses between the measured and calculated values were used. The K' values at 1 mM Mg(2+) ranged from 0.49 to 1.13. The apparent equilibrium constants increased with increasing Mg(2+) concentrations. The values were 2-3 times higher at 20 mM Mg(2+) than at 1 mM Mg(2+), and the dependence was similar in the class I and class II synthetases. The main reason for the Mg(2+) dependence is the existence of PP(i) as two magnesium complexes, but only one of them is the real product. AMP exists either as free AMP or as MgAMP, and therefore also has some effect on the measured equilibrium constant. However, these dependences alone cannot explain the measured results. The measured dependence of the K' on the Mg(2+) concentration is weaker than that caused by PP(i) and AMP. Different bindings of the Mg(2+) ions to the substrate tRNA and product aminoacyl-tRNA can explain this observation. The best-fit analysis suggests that tRNA reacts as a magnesium complex in the forward aminoacylation direction but this given Mg(2+) ion is not bound to aminoacyl-tRNA at the start of the reverse reaction. Thus Mg(2+) ions seem to have an active catalytic role, not only in the activation of the amino acid, but in the posttransfer steps of the aminoacyl-tRNA synthetase reaction, too.

Analysis of the kinetic mechanism of arginyl-tRNA synthetase.

A kinetic analysis of the arginyl-tRNA synthetase (ArgRS) from Escherichia coli was accomplished with the goal of improving the rate equations so that they correspond more closely to the experimental results. 22 different steady-state kinetic two-ligand experiments were statistically analysed simultaneously. A mechanism and values for the ArgRS constants were found where the average error was only 6.2% and ranged from 2.5 to 11.2% in the different experiments. The mechanism included not only the normal activation and transfer reactions but also an additional step which may be a conformational change after the transfer reaction but before the dissociation of the product Arg-tRNA from the enzyme. The forward rate constants in these four steps were low, 8.3-27 s(-1), but the reverse rate constants of the activation and transfer reactions were considerably higher (230 and 161 s(-1)). Therefore, in the presence of even low concentrations of PP(i) and AMP, the rate limitation occurs at the late steps of the total reaction. AMP increases the rate of the ATP-PP(i) exchange reaction due to the high reverse rate in the transfer reaction. The rate equation obtained was used to calculate the steady-state enzyme intermediate concentrations and rates between the intermediates. Three different Mg2+ binding sites were required to describe the Mg2+ dependence. One of them was the normal binding to ATP and the others to tRNA or enzyme. The measured Mg2+ dependence of the apparent equilibrium constant of the ArgRS reaction was consistent with the Mg2+ dependences of the reaction rates on the rate equation. Chloride inhibits the ArgRS reaction, 160 mM KCl caused a 50% inhibition if the ionic strength was kept constant with K-acetate. KCl strongly affected the K(m)(app) (tRNA) value. A difference was detected in the progress curves between the aminoacylation and ATP-PP(i) exchange rates. When all free tRNA(Arg) had been used from the reaction mixture, the aminoacylation reaction stopped, but the ATP-PP(i) exchange continued at a lowered rate.