Replacement of phenylalanine in gramicidin S by other amino acids

Abstract

This decapeptide is synthesized by gramicidin S synthetase which consists of two enzymes, the light and the heavy [I]. The light enzyme which is also a racemase, activates and thioesterbinds phenylalanine. The growth of the peptide chain is initiated by transfer of the thioester-bound D-phenylalanyl group to the heavy enzyme. This enzyme activates and thioesterbinds L-proline, Gvaline, L-ornithine and L-leucine and catalyzes the formation of the peptide bonds in gramicidin S. In the past, substitution of phenylalanine in gramicidin S by other amino acids using gramicidin S synthetase has been claimed to take place. For instance it has been reported that p-fluorophenylalanine and fl-thienylalanine will substitute [2]. However, in neither case have the cyclic decapeptides been identified chromatographically and separated from gramicidin S. Furthermore, there is conflicting evidence on whether or not tyrosine can replace phenylalanine [2,3]. It has also been claimed that tryptophan could not replace this amino acid [3]. We have reexamined these reports and present evidence that p-fluoro-, p-chloro-, p-bromophenylalanine, /3-thienylalanine, tyrosine and tryptophan can replace phenylalanine. Evidence for the formation of a hybrid decapeptide containing one residue of tyrosine and one of phenylalanine is also presented. In addition, cyclohexylalanine, phenylglycine and a-methyl phenylalanine have been examined for their ability to substitute for phenylalanine. None of these could substitute in the synthesis. However, it was found that in the presence of cyclohexylalanine and proline only, synthesis is initiated since the dipeptide cyclohexylalanylproline is produced. If valine and leucine were present, the formation of the dipeptide was inhibited. This probably explains why no synthesis of the cyclic decapeptide takes place in a complete incubation mixture.