Aminoacyl-tRNA synthetases facilitate a two-step reaction. The process starts with amino acid activation, in which the amino acid and ATP bind to the enzyme's catalytic site. This leads to a condensation reaction between the α-carboxylate oxygen of the amino acid and the α-phosphate group of ATP, forming aminoacyl-adenylate (aa-AMP) and inorganic pyrophosphate (PPi), which is released from the active site. Although tRNA is typically not involved in this step, certain synthetases require the presence of specific tRNA species for efficient amino acid activation. In the second step, the hydroxyl group of adenine 76 nucleotide in the tRNA molecule attacks the carbonyl carbon of the adenylate, resulting in the formation of aminoacyl-tRNA and the release of AMP. After the second step of the reaction, the resulting tRNA molecule becomes "charged" with the amino acid and is ready to participate in protein synthesis. the overall reaction is: aa + ATP + tRNA ⟶ aa-tRNA + AMP + PPi.