Enzymatic Carboxyl Activation of Amino Acids* by Mahlon

Previous work in this laboratory (l-3) had revealed that the incorporation of C14-labeled amino acids into the protein of rat liver microsome fraction was dependent upon ATP’ and enzymatic components of the soluble protein fraction. Part of this enzymatic requirement was accounted for by enzymes which would generate ATP from a precursor such as phosphocreatine, phosphopyruvate, or phosphoglycerate. It was apparent, however, that, after fortification of the incorporation system with the precursors and the appropriate ATP generating enzymes, heatlabile, non-dialyzable components of the soluble fraction were still required. It therefore seemed reasonable to subject to experimental test the possibility that a mechanism for amino acid activation by ATP resides in this soluble protein fraction. Preliminary results of such a study (4) revealed that the dialyzed soluble protein fraction of rat liver catalyzes an exchange of PP32 with ATP which is enhanced several fold by the addition of a group of pure L-amino acids. The microsome fraction also catalyzes a PP32 exchange which is not, however, influenced by amino acids. It was found, furthermore, that AMP fails both to inhibit the amino acid-dependent exchange and to exchange with ATP (by using Cl*-labeled AMP). These results suggested that the amino acids were being activated as an amino acyl AMP compound. This possibility was given further support by the finding that a-amino hydroxamic acids are formed in the presence of high hydroxylamine concentrations, with concomitant loss of ATP. Since the amino acids did not cause a net splitting of ATP unless hydroxylamine was present, it was proposed that the amino acyl AMP is bound on the enzyme surface and dissociates from t,he enzyme t’o only a small extent if at all. The L-amino acid effect on exchange and OJI hydroxamic acid formation was additive with different amino acids, and D-amino acids were inert in the system.