Gramicidin S Synthetase. Stability of Reactive Thioester Intermediates and Formation of 3-Amino-2-piperidone

Open Access
Publisher Website
Google Scholar
Abstract
The reactive thioester complees of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recoverd. These processes are strongly accelerated by certain thiol protective agents. In the preesence of 1 mM dithieoerythritol the half-life times of sthese hydrolysis reactions are in the range of 1–90 h at 3 °C. The thieoester comple of gramicidin S synthetase 2 (GS2, the heavy enzyme) with the tripeptide DPhe-Pro-Val is distinguished by the highest stability of all these intermediates. A different decomposition pattern is observed for the thioester comple of GS2 with LOrn. Here 3-amino-2-piperidone (cyclo-LOrn0 is formed in a rapid cyclization reaction. This product specifically blocks the activation center of GS2 for LOrn at the thioester buinding site. All other activation reactions of g ramicidin S synthetase are unaffected. A procedure for a specific labelling of the reaction centers of the multienzyme is outlined.