Mechanism of Action of Amino Acid Transfer Ribonucleic Acid Ligases

Abstract The first reaction in protein biosynthesis has been presumed to be the interaction of ATP, amino acid, and enzyme (amino acid-activating enzyme, aminoacyl transfer RNA ligase, tRNA synthetase) to form an enzyme-bound aminoacyl adenylic anhydride. Such aminoacyl anhydrides could react forward with hydroxylamine or with tRNA or backward with [32P]-PPi. Experiments, primarily involving inhibitors, cast serious doubt on the existence of such a common intermediate in the enzyme-catalyzed formation of amino acid hydroxamate or [32P]-ATP from amino acid, ATP, and hydroxylamine, or [32P]-PPi, respectively. Studies with several enzymes indicate the probability of a general base-stimulated concerted reaction in which amino acid hydroxamate, AMP, and PPi are formed with no discrete intermediates. The esterification of tRNA resembles hydroxamate formation more than it resembles ATP-PPi exchange, strengthening the argument that the physiologically significant reaction does not involve an enzyme-bound aminoacyl adenylate. When tRNA is esterified in the absence of other bases, it is probable that one of the terminal ribose hydroxyls serves as a general base while the other is being acylated.