par Parfaitand, Raymond;Grosjean, Henri
Référence European journal of biochemistry / FEBS, 30, 2, page (242-249)
Publication Publié, 1972
Article révisé par les pairs
Résumé : Arginyl‐tRNA synthetase from Bacillus stearothermophilus (NCA 1518) has been purified approximately 450‐fold to 90%, homogeneity. The enzyme has a molecular weight of 78000 as estimated by disc electrophoresis in denaturing conditions (dodecylsulfate). No subunits were revealed in these conditions. The Km values for arginine, ATP and tRNA have been determined. Like the arginyl‐tRNA synthetase from other organisms, this one from B. stearothermophilus requires tRNA to catalyse an arginine‐dependent ATP/[32P]PPi exchange reaction. Periodatetreated tRNA does not induce this exchange reaction. Aminoacylation is not completely inhibited by 2 mM pyrophosphate, indicating that the exchange reaction may be explained by the reversal of the overall aminioacylation reaction. Different methods tested failed to reveal any arginyl‐adenylate intermediary. By gel filtration, however, enzyme‐bound arginine could be isolated from a system containing all the substrates of the enzyme. The binding of arginine and ATP to arginyl‐tRNA synthetase has been studied by equilibrium partition and shows that arginine is bound only after the binding of tRNA and ATP. The binding of ATP does not depend on the presence of other substrates in the reaction mixture. One binding site for arginine and one for ATP are found per molecule of active enzyme. Periodate‐oxidized tRNA and phenoxyacetylarginyl‐tRNA did not induce the binding of arginine to the enzyme. Intact tRNA from other species which could be aminoacylated by arginyl‐tRNA synthetase from B. stearothermophilus, also induced the binding of arginine to the enzyme. Nucleotides other than ATP were not effective in the binding of arginine. It is concluded that the mechanism of action of this enzyme is a concerted one, as proposed by Loftfield and Eigner, in which arginine is bound after tRNA and ATP. Copyright © 1972, Wiley Blackwell. All rights reserved

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