Kinetic demonstration of the intermediate role of aminoacyl-adenylate-enzyme in the formation of valyl transfer ribonucleic acid

Abstract

The question whether aminoacyl-tRNA synthetases act in a stepwise or in a concerted mechanism has been investigated kinetically with the valine enzyme of Escherichia coli, which had been used in previous studies by others who concluded that the physiological mechanism is concerted. An exchange between aminoacyl-tRNA and tRNA, dependent upon AMP, was studied. PP-i inhibits this exchange completely in the presence of Mg2+ and AMP but in the absence of added Mg2+ or with dAMP as the nucleotide the inhibition by PP-i is only partial; this is compatible with a stepwise, not a concerted, reaction. Exchange of isotopically labeled substrates in a system at chemical equilibrium also shows effects of substrate concentrations on rates in agreement with the predictions of a stepwise mechanism.