Reactive Sulfhydryl Groups Involved in the Aminoacyl Adenylate Activation Reactions of the Gramicidin S Synthetase 2

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Abstract
Six accessible sulfhydryl groups of the light component of gramicidin S synthetase (GS 1) were titrated with N-ethylmaleimide (MalNEt) as well as 3,3''-dithiobis(6-nitrobenzoic acid) (Nbs2). Twenty-four thiols were detected in the heavy enzyme (GS 2) using MalNEt as the modifier. Substrate amino acid-induced protection of GS 2 against deactivation by MalNEt indicates that in addition to the specific thiols at the thiotemplates of gramicidin S synthetase reactive SH groups are also involved in the primary aminoacyl adenylate activation reactions. Obviously 2 sulfhydryl groups are essential for the adenylation of each L-Pro, L-Val and L-Leu, while 3 thiols were detected for the ornithine activation. Agents like MalNEt or Nbs2 inhibit the thiolation of the substrate amino acids of gramicidin S synthetase and gramicidin S formation more severely than the aminoacyl adenylate activation reactions which are affected at 10-100-fold higher inhibitor concentrations. These processes can be studied separately if the thioester formation sites are inhibited at low concentrations of sulfhydryl inhibitors.

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