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.

[Spectral characteristics of muscle aspartyl- and valyl-tRNA- synthetases and their complexes with substrates in normal conditions and after prolonged starvation]. / Spektral'nye kharakteristiki myshechnykh aspartil- i valil-tRNK- sintetaz i ikh kompleksov s substratami v norme i posle dlitel'nogo golodaniia.

Spectral characteristics of aminoacyl-tRNA-synthetases (ARSases) isolated from muscles of normal rabbits and of those fasted for a long time were studied by the methods of fluorescence and differential spectroscopy. Fluorescence spectra and differential absorption spectra of the compared proteins evidenced for more hydrophobic surrounding of tryptophanyls and their less accessibility for Cs+ ions in proteins of fasted animals. Interaction of aspartyl- and valyl-tRNA-synthetases from muscles of normal and long-fasted rabbits with substrates is accompanied by the essential quenching of tryptophan fluorescence of ARSases. Equilibrium constants of substrate binding calculated from the fluorescence quenching curves are higher for specific amino acids than for non-specific ones. The effect of a long-wave shift of fluorescence spectra under marginal excitation of tryptophan residues was used to determine structural differences of enzymes in norm and under fasting and to find their structural peculiarities during formation of aminoacyl adenylate. Aminoacyl-tRNA-synthetases (ARSases) are key enzymes of the protein biosynthesis. High specificity of their interaction with substrates is the basis for the accuracy of genetic information implementation, namely translation of the genetic code. Molecular mechanisms of substrates "recognition" by ARSases are the objects of great attention of researchers.

Structural studies on aminoacyl-tRNA synthetases. A tentative correlation between the subunit size and the occurrence of repeated sequences.

Recent studies have shown that those synthetases with subunits greater than 85,000 daltons contain extensive repeated sequences, whilst those with small subunits (40,000 daltons) do not. We have undertaken a comparative study of four aminoacyl-tRNA synthetases (glutamyl-, arginyl-, valyl-, and phenylalanyl-tRNA synthetases) with subunit sizes ranging from 56,000 to 130,000 daltons in an attempt to correlate the occurrence and extent of the repeats with the length of the polypeptide chain. Our results show that monomeric glutamyl-tRNA synthetase from Escherichia coli (56,000 daltons) contains few repeated sequences, whereas both subunits of yeast phenylalanyl-tRNA synthetase (alpha, 73,000 daltons; beta, 62,000 daltons) and yeast arginyl-tRNA synthetase (74,000 daltons) do have a significant amount of repeats. Thus 56,000 dalton appears to be the minimum size compatible with the existence of such repeats.