Amino acid analogues as substrates of a rabbit reticulocyte aminoacyl-tRNA synthetase preparation

Abstract

l-Methionine-dependent ATP-PP 1 exchange catalyzed by a partially purified aminoacyl-tRNA synthetase preparation from rabbit reticulocytes had a K m (methionine) of 0.2 m M. The amino acid analogues seleno- dl-methionine, α-methyl- dl-methionine, l-ethionine and seleno- dl-ethionine also promoted ATP-PP 1 exchange, with K m values about 2- to 100-fold greater than that of methionine. A number of N-substituted derivatives of methionine were inactive as either substrates or inhibitors. Evidence is presented that methionyl-tRNA synthetase of rabbit reticulocytes can exist in different polymeric states. The best preparations obtained contained leucylvalyl and isoleucyl-tRNA synthetase activities. Using such preparations, phosphonic acid analogues of isoleucine, valine and leucine were shown, for the first time, to be specific inhibitors of the corresponding aminoacyl-tRNA synthetases. The inhibitory effects of l-methioninyl adenylate and l-leucinyl adenylate on norleucine-dependent ATP-PP 1 exchange suggested that l-norleucine was a substrate of both methionyl- and leucyl-tRNA synthetases, a conclusion substantiated by heat denaturation studies and the fact that l-norleucine-dependent ATP-PP 1 exchange was strongly inhibited by the phosphonic acid analogue of leucine.