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.
Airas
R Kalervo
RK
Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland. kalervo.airas@utu.fi
D000255:Adenosine Triphosphate / Q000737:chemistry; D001126:Arginine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D011189:Potassium Chloride / Q000494:pharmacology; D046249:Transfer RNA Aminoacylation
D029968:Escherichia coli Proteins; D008024:Ligands; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D001126:Arginine-tRNA Ligase; D008274:Magnesium