Valyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Val)-C-C-A and tRNA(Val)-C-C-A(3'NH2).
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 tRNA(Val)-C-C-A. The lowest values were found for Trp, Ser, Cys, Lys, Met and Thr (D = 90-870), showing that valine is 90-870 times more frequently attached to tRNA(Val)-C-C-A than the noncognate amino acids at the same amino acid concentrations. The other amino acids exhibit D values between 1,100 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 tRNA(Val)-C-C-A(3'NH2) discrimination factors D1 are in the range of 40-1260. From D1 values and AMP formation stoichiometry, pretransfer proof-reading factors pi 1 were determined: post-transfer proof-reading factors II 2 were determined from D values and AMP formation stoichiometry in acylation of tRNA(Val)-C-C-A. II 1 values (7-168) show that pretransfer proof-reading is the main correction step, post-transfer proof-reading (II 2 approximately 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.
Freist
W
W
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
Cramer
F
F
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000032:analysis*; D001665:Binding Sites / Q000235:genetics; D004735:Energy Transfer; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D012345:RNA, Transfer, Amino Acid-Specific / Q000032:analysis*; D012346:RNA, Transfer, Amino Acyl / Q000032:analysis*; D012366:RNA, Transfer, Val / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D014637:Valine-tRNA Ligase / Q000032:analysis* / Q000235:genetics
D000596:Amino Acids; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; D012366:RNA, Transfer, Val; D000604:Amino Acyl-tRNA Synthetases; D014637:Valine-tRNA Ligase