On the use of affinity chromatography in demonstrating the transfer of thioester-bound D-phenylalanine from the light enzyme of gramicidin S synthetase to an acceptor site for this amino acid on the heavy enzyme.

Abstract

When a mixture of the light enzyme of gramicidin S synthetase containing thioester‐bound d‐[14C]phenylalanine and the heavy enzyme is passed through a Sepharose column containing carboxyl‐bound proline (substrate amino acid) as ligand, the two enzymes are retained as the result of the affinity between the heavy enzyme and the ligand and the affinity between the two enzymes. Upon elution and separation of the two enzymes it was demonstrated that the d‐[14C]phenylalanyl groups had been transferred to the heavy enzyme. This transfer did not take place when similar experiments were carried out using as ligands valine, ornithine or leucine which are also substrates for the heavy enzyme. Hence binding of proline to the heavy enzyme is required for transfer to take place. Upon incubation of the heavy enzyme containing the transferred d‐[14C]phenylalanyl group with the light enzyme, ATP and the five unlabelled substrate amino acids, radioactivity is incorporated into gramicidin S. This suggests that in the biosynthesis of gramicidin S, the d‐phenylalanyl group is transferred to an acceptor site on the heavy enzyme and subsequently to the imino group of thioester‐bound proline. In view of what is known about gramicidin S synthesis it seems likely that the acceptor site is a thiol site.