Valyl‐tRNA synthetase from yeast

Abstract

For discrimination between valine and the 19 naturally occurring noncognate amino acids, as well as between valine and 2‐amino‐isobutyric acid by valyl‐tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values in aminoacylation of tRNAVal‐C‐C‐A. The lowest values were found for Trp, Ser, Cys, Lys, Mct and Thr (D = 90–870), showing that valine is 90–870 times more frequently attached to tRNAVal‐C‐C‐A than the noncognate amino acids at the same amino acid concentrations. The other amino acids exhibit D values between 1100 and 6200. Generally, valyl‐tRNA synthetase is considerably less specific than isoleucyl‐tRNA synthetase, but this may be partly compensated in the cell by valine concentrations higher than those of noncognate acids.In aminoacylation of tRNAVal‐C‐C‐A(3′NH2 discrimination factors D1 are in the range of 40–1260. From D values and AMP formation stoichiometry, pretransfer proof‐reading factors II1 were determined; post‐transfer proof‐reading factors II2 were determined from D values and AMP formation stoichiometry in acylation of tRNAVal‐C‐C‐A.II1 values (7–168) show that pretransfer proof‐reading is the main correction step, posttransfer proof‐reading (II2=1–7) is less effective and in some cases negligible.Initial discrimination factors were calculated from discrimination and proof‐reading factors according to a two‐step binding process. These factors, due to different Gibbs free energies of binding can be related to hydrophobic interaction forces, and a hypothetical ‘stopper’ model of the amino‐acid‐binding site is discussed.