Hydrolytic Action of Aminoacyl-tRNA Synthetases from Baker's Yeast. “Chemical Proofreading” of Thr-tRNAVal by Valyl-tRNA Synthetase Studied with Modified tRNAVal and Amino Acid Analogues

Abstract

The properties of native and of two modified tRNAVal species in the correction of misactivated threonine by valyl-tRNA synthetase have been studied. Whereas Thr-tRNAVal-C-C-A could not be isolated in the valyl-tRNA synthetase catalyzed reaction, Thr-tRNAVal-C-C-3′dA is isolable in up to 50% yield in this system and tRNAVal-C-C-3′NH2A is fully aminoacylated with threonine by the same enzyme. The hydrolysis of preformed Thr-tRNAVal-C-C-A by free valyl-tRNA synthetase is 30 times faster than the corresponding breakdown of Val-tRNAVal-C-C-A. This hydrolytic activity is also observed with Thr-tRNAVal-C-C-3′dA although the rate is reduced to that of the reaction of Val-tRNAVal-C-C-A. Modification of the threonine to O-methylthreonine, which is also a substrate for valyl-tRNA synthetase, leads to stabilization of the O-methylthreonyl-tRNA esters. The AMP/PP independent hydrolysis under aminoacylating conditions, which is a measure of the correction process, indicates that O-MeThr-tRNAVal-C-C-A is only very slowly corrected while the tRNAVal-C-C-3′dA and tRNAVal-C-C-3′NH2A esters are completely stable. Removal of the methoxy group of O-methylthreonine as in α-aminobutyric acid increases the rate of the hydrolytic reaction and once again α-Abu-tRNAVal-C-C-A and α-Abu-tRNAVal- C-C-3′dA are unstable under aminoacylating conditions and not isolable. © 1977, American Chemical Society. All rights reserved.