Reactive sulfhydryl groups involved in the aminoacyl adenylate activation reactions of the gramicidin S synthetase 2.
Six accessible sulfhydryl groups of the light component of gramicidin S synthetase (GS 1) were titrated with N-ethylmaleimide (Ma1NEt) as well as 3,3'-dithiobis(6-nitrobenzoic acid) (Nbs2). Twenty-four thiols were detected in the heavy enzyme (GS 2) using Ma1NEt as the modifier. Substrate amino acid-induced protection of GS 2 against deactivation by Ma1NEt 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 Ma1NEt 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.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000591:Amino Acid Isomerases / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009579:Nitrobenzoates / Q000494:pharmacology*; D010453:Peptide Synthases / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; D009097:Multienzyme Complexes; D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2; D005033:Ethylmaleimide