Isoleucyl‐tRNA Synthetase from Baker's Yeast

Abstract

The aminoacylation of 3 modified tRNAIle species with isoleucine and with valine by isoleucyl-tRNA synthetase was investigated by initial rate kinetics. For aminoacylation of tRNAIle-C-C-3''dA with isoleucine, a bi-bi uni-uni ping-pong mechanism was found by bisubstrate kinetics and inhibition by products and by 3''dATP; for aminoacylation with valine, a bi-uni uni-bi ping-pong mechanism. For isoleucylation of tRNAIle-C-C-A(3''NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni-bi uni-uni-uni ping-pong mechanism; for valylation of this tRNA, a bi-bi uni-uni ping-pong mechanism is observed by bisubstrate kinetics and product inhibition. tRNAIle-C-C-2dA was aminoacylated under modified conditions with isoleucine in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P[NH]P as product analog and by isoleucinol. Aminoacylation with valine is achieved in a rapid-equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3''dATP and valinol. All 6 reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle-C-C-A. The Km values of the 3 substrates and the kcat values of the 6 reactions are given. For aminoacylation at the terminal 2''OH group of the tRNA, differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine were calculated which result in discrimination factors of 181 and 167. For aminoacylation at the terminal 3''-OH group, a difference of only 4.43 kJ and a low discrimination factor of only 6 is observed. Maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2''-OH group and conclusions drawn from experiments with modified tRNA concerning 2'',3''-specificity led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNA. The stability of Ile-tRNAIle-C-C-2''dA and Val-tRNAIle-C-C2''dA, the lesser stability of Val-tRNAVal-C-C-2''dA and the instability of Thr-tRNAVal-C-C-2''dA are consistent with postulations for a pre-transfer proofreading step for isoleucyl-tRNA synthetase and a post-transfer hydrolytic editing step for valyl-tRNA synthetase at the terminal 3''OH group of the tRNA.