ESTABLISHING THE MISACYLATION-DEACYLATION OF THE TRANSFER-RNA PATHWAY FOR THE EDITING MECHANISM OF PROKARYOTIC AND EUKARYOTIC VALYL-TRANSFER RNA-SYNTHETASES

The valyl-tRNA synthetases from Bacillus stearothermophilus and Escherichia coli edited the mistaken activation of L-.alpha.-aminobutyrate (.alpha.But) by 1st misacylating tRNAVal and then specifically deacylating the .alpha.But-tRNAVal. This pathway was established initially by rapid quenching and sampling experiments which detected the transiently formed .alpha.But-tRNAVal on the addition of tRNAVal to the preformed and isolated E[enzyme].cntdot..alpha.But-AMP complex. The misacylation/deacylation pathway of editing occurred in the normal reaction in the steady state by isolation of the mischarged tRNA. For example, [14C]-.alpha.But-tRNAVal (E. coli) can be isolated from a steady-state reaction mixture containing [14C]-.alpha.But (0.9 mM), enzyme (71 .mu.M), tRNAVal (72 .mu.M), and ATP (8 mM), at the steady-state concentration predicted from the measured deacylation rate constant (50 s-1) and the steady-state kinetic constants for the ATP/pyrophosphatase activity under these conditions. The same pathway appears to operate for the rejection of threonine by the eukaryotic valyl-tRNA synthetase since, on mixing the enzyme-bound threonyl adenylate and tRNA from yeast, transiently formed Thr-tRNAVal is observed and its deacylation rate constant estimated to be greater than 80 s-1. The rate-determining step of the valyl-tRNA synthetase catalyzed deacylation of .alpha.But-tRNAVal is different in nature and in magnitude from that of Val-tRNAVal. The rate of the editing reaction is independent of pH between 6-7.8 and is unaffected by the addition of aminoacyl adenylate, while the rate of deacylation of Val-tRNAVal follows an ionization of pKa = 8 and is decreased by the addition of aminoacyl adenylate. A unified scheme is presented that could account for the specificity in the deacylation reaction.