This reaction is reversible and may be conveniently traced by pyrophosphate exchange: if [32P]-pyrophosphate is added to the reaction mixture, the label is soon detected in [32P]-ATP. Aminoacyl adenylate formed in the reaction remains bound to the enzyme and is not released into solution.

The reaction, and consequently the overall reaction, is markedly shifted in the direction of aminoacyl adenylate and aminoacyl-tRNA formation due to the hydrolysis of the inorganic pyrophosphate which is catalyzed by pyrophosphatase. Therefore, the production of pyrophosphate in the amino acid activation step and the subsequent hydrolysis of the pyrophosphate to the inorganic orthophosphate play an important part in providing the energy that ensures the direction of the entire process.

A reaction catalyzed at the second stage by the same ARSase involves the so-called accepting of the amino acid where the 2'- or 3'-hydroxyl of the ribose residue of the tRNA 3'-terminal adenosine attacks the anhydride group of the aminoacyl adenylate, resulting in the formation of an ester bond between the aminoacyl residue and the tRNA, with the accompanying release of AMP (Fig. 3.12):

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