For discrimination between arginine and 19 other amino acids in aminoacylation of tRNAArg-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors ( D ) have been determined from k,,, and K, values. The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 timcs more often incorporated into tRNAArg-C-C-A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10000 and 60000.In aminoacylation oftRNAArg-C-C-A(3'NH2) discrimination factors D 1 are in the range 10-600. From these values and AMP formation stoichiometry, pretransfer proof-reading factors f I l were determined ; from D values and AMP stoichiometry in aminoacylation of tRNAArg-C-C-A, posttransfer proof-reading factors f I 2 could be calculated. Ill values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (n, Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors. According to a two-step binding process, two factors (II and 1,) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical 'stopper' model of the amino acid recognition site is discussed 1 -10) plays a marginal role.Arginyl-tRNA synthetases occupy a special position among aminoacyl-tRNA synthetases. As onc striking property they share with glutamyl-tKNA and glutaminyl-tRNA synthetases the requirement of the cognate tRNA for the A'I'P,/PPi exchange reaction; the reverse reaction of the first step in the generally accepted two-step mechanism of aminoacyl-tRNA synthetases [I -241.Correspondence to W. Freist, Max-Planck-lnstitut fur Experimentelle Medizin, Abteilung Chemie, Hermann-Rein-Strasse 3, D-3400 Giittingen. Federal Republic of Gcnnany Abbreviations. D, discrimination factor in aminoacylation of tRNA-C-C-A; D,, discrimination factor in aminoacylation of tRNA-C-C-A(3'NH2); I'. overall initial discrimination factor in aminoacylation of tRN A-C-C-A; n', overall proof-reading faclor in aminoacylation of tRNA-C-C-A; I,, initial discrimination factor of first initial discrimination step; 12, initial discrimination factor of second initial discrimination stcp; II ,, pretransfer proof-reading factor: Il,; posttransfcr proof-reading factor; X'. number of AMP molecules generatedimolecule aminoacyl-tRNA in aminoacylation of cognate tRNA-C-C-A with cognate amino acid; Y', number of AMP moleculcs gencrated/molecule aminoacyl-tRNA in aminoacylation or cognate tRNA-C-C-A with noncognate amino acid; XI, number of AMP molecules generatcdjmolecule aminoacyl-tRN A in aminoacylation of cognate tKNA-C-C-A(3'NH2) with cognate amino acid; Y 1 , numbcr of AMP molecules generated/molecule aminoacyl-tRNA in aminoacylation of cognate tRNA-C-C-A(3'NH2) with noncognatc amino acid.Enzymes. Arginyl-tRNA synthet...
