Open AccessTyrosyl‐tRNA synthetase from baker's yeast
Author(s) -
FREIST Wolfgang,
STERNBACH Hans
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb14391.x
Subject(s) - aminoacylation , transfer rna , stereochemistry , ribose , chemistry , amino acid , substrate (aquarium) , moiety , enzyme , biochemistry , biology , rna , ecology , gene
The order of substrate addition to tyrosyl‐tRNA synthetase from baker's yeast was investigated by bisubstrate kinetics, product inhibition and inhibition by dead‐end inhibitors. The kinetic patterns are consistent with a random bi‐uni uni‐bi ping‐pong mechanism. Substrate specificity with regard to ATP analogs shows that the hydroxyl groups of the ribose moiety and the amino group in position 6 of the base are essential for recognition of ATP as substrate. Specificity with regard to amino acids is characterized by discrimination factors D which are calculated from k cat and K m values obtained in aminoacylation of tRNA Tyr ‐C‐C‐A. The lowest values are observed for Cys. Phe. Trp ( D = 28000 – 40000), showing that, at the same amino acid concentrations, tyrosine is 28000 – 40000 times more often attached to tRNA Tyr ‐C‐C‐A than the noncognate amino acids. With Gly, Ala and Ser no misacylation could be detected ( D > 5); D values of the other amino acids are in the range of 1—5. Lower specificity is observed in aminoacylation of the modified substrate tRNA Tyr ‐C‐C‐A(3'NH 2 ) ( D 1 = 500—55000). From kinetic constants and AMP‐formation stoichiometry observed in aminoacylation of this tRNA species, as well as in acylating tRNA Tyr ‐C‐C‐A hydrolytic proof‐reading factors could be calculated for a pretransfer ( II 1 ) and a post‐transfer ( II 2 ) proof‐reading step. The observed values of II 1 = 12 – 280 show that pretransfer proof‐reading is the main correction step whereas post‐transfer proof‐reading is marginal for most amino acids ( II 2 = 1–2). Initial discrimination factors caused by differences in Gibbs free energies of binding between tyrosine and noncognate amino acids are calculated from discrimination and proof‐reading factors. Assuming a two‐step binding process, two factors ( I 1 and I 2 ) are determined which can be related to hydrophobic interaction forces. The tyrosine side chain is bound by hydrophobic forces and hydrogen bonds formed by its hydroxyl group. A hypothetical model of the amino acid binding site is discussed and compared with results of X‐ray analysis of the enzyme from Bacillus stearothermophilus .