Phenylalanyl-tRNA synthetase from yeast. Steady-state kinetic investigation of the reaction mechanism.

Abstract

It has been shown that the initial rates, v, of the formation of aminoacyl‐tRNA, catalyzed by phenylalanine‐tRNA synthetase, do not depend on the concentration of the substrates ATP and phenylalanine in a simple way. In contrast a normal Michaelis‐Menten behaviour has been observed for the substrate tRNA. The reciprocal plots of v versus v/[S] for ATP and phenylalanine are curved and only approach linearity at low and high concentrations of the varied substrate. From these limiting slopes two Km and two V values corresponding to low and high concentrations of ATP and phenylalanine have been determined. Since the reciprocal plots for tRNA are linear, only one Km and V value has been obtained for this substrate. In order to show that this kinetic behaviour is an intrinsic property of the enzyme and not an artifact, a new procedure for the purification of the enzyme has been devised and the purity and stability of the preparation carefully checked. In addition the influence of ammonium ions and of spermine on the initial rates has been measured to verify that the non‐linearity of the reciprocal plots is not the result of the substrate acting as a base. Inhibition experiments with tRNAox (a modified tRNA obtained from native tRNA by periodate oxidation of the 2′:3′‐diol in the 3′‐terminal ribose to the corresponding dialdehyde) at low and high phenylalanine concentrations have shown that it is also not a consequence of two fundamentally different reaction mechanisms being followed at the two concentration ranges. On the basis of this and other evidence it is proposed that two mutually interacting sites are engaged, of which one or both are operative, depending on the concentration of the substrates. Studies of pyrophosphate inhibition at low tRNA concentration have provided evidence that the aminoacylation of tRNA proceeds via two catalytic steps in which aminoacyl‐AMP occurs as an intermediate. At high tRNA concentrations it has been found that this substrate is adding prior to the dissociation of pyrophosphate.