Lysyl‐tRNA synthetase from yeast

Discrimination factors ( D ) which are characteristic for discrimination between lysine and 19 naturally occuring non‐cognate amino acids have been determined from k cat and K m values for native and phosphorylated lysyl‐tRNA synthetases from yeast. Generally, both species of this class II aminoacyl‐tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90–1700, D values of the phosphorylated species in the range 40–770. The phosphorylated enzyme acts faster and less accurately. In aminoacylation of tRNA Lys ‐C‐C‐A(2′NH 2 ) discrimination factors D 1 vary over 30–980 for the native and over 8–300 for the phosphorylated enzyme. From AMP formation stoichiometry and D 1 values pretransfer proof‐reading factors ( II 1 ) of 1.1–56 were calculated for the native enzyme, factors of 1.0–44 for the phosphorylated species. Post‐transfer proof‐reading factors ( II 2 ) were calculated from D values and AMP formation stoichiometry in acylation of tRNA Lys ‐C‐C‐A. Pretransfer proof‐reading is the main correction step, posttransfer proof‐reading is less effective or negligible ( II 2 ∼ 1–8). Initial discrimination factors ( I ), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (ΔΔ G I ), were calculated from discrimination and proof‐reading factors. In contrast to class I synthetases, for lysyl‐tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three‐step process instead of the four‐step recognition observed for the class I synthetases. Plots of ΔΔ G I values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical ‘stopper model’ of these sites.