Demonstration of two reaction pathways for the aminoacylation of tRNA. Application of the pulsed quenched flow technique.

TLDR: A rapid mixing and quenching device is described which operates efficiently in the range of 150 msec to several minutes as well as the usual time scale of 5-150 msec of the conventional apparatus to measure the initial rate of acylation by the tyrosyl-tRNA synthetase of Escherichia coli during the first turnover of the enzyme.

Abstract: A rapid mixing and quenching device is described which operates efficiently in the range of 150 msec to several minutes as well as the usual time scale of 5-150 msec of the conventional apparatus. This has been used to measure the initial rate of acylation of tRNATyr by the tyrosyl-tRNA synthetase of Escherichia coli during the first turnover of the enzyme, and also the rate constants of the partial reactions of amino acid activation and transfer to the tRNA. It is shown that at saturating conenctration of tRNA the reaction proceeds by a ternary complex mechanism. The rate-determining step is either the aminoacyltion process or a step preceding it. At low concentrations of tRNA the reaction proceeds by the stepwise process of formation of tyrosyl adenylate followed by acylation of the tRNA. The rate constants for these partial reactions are faster than that for the ternary complex reaction. But the prior binding of tRNA greatly decreases the rate of tyrosyl adenylate formation. Both pathways are probably important at physiological concentrations. 88% of the tyrosine from the tyrosyl adenylate complex is transferred to tRNA. The presence of added tyrosine and ATP reduces this to 78%. However, the addition of aliquots of ATP to a mixture of enzyme, tyrosine, and a saturating concentration of tRNA (i.e., ternary complex conditions) leads to at least 0.97 mol of tRNA being acylated/mol of ATP hydrolyzed. Trapping experiments show that the 12% of adenylated that is not transferred to tRNA is hydrolyzed on the enzyme rather than expelled into solution.