The proline‐activating activity of the multienzyme gramicidin S synthetase 2 can be recovered on a 115‐kDa tryptic fragment

Abstract

The multienzyme gramicidin S synthetase 2 was treated with trypsin to obtain fragments capable of activating proline. Three different active fragments were detected. The course of proteolysis was simulated by using a concentration range of trypsin; the cleavage pattern indicated that one of the fragments was particularly stable. This fragment was purified and shown to have a molecular mass of 115 kDa. It was compared chromatographically, by SDS/PAGE, and enzymatically to a Pro-activating fragment produced by a gramicidin-S-negative mutant. It can be concluded that the proteolytic fragment represents a structure which is contained on a continuous part of the polypeptide chain of gramicidin S synthetase 2 and has a relatively compact structure. This provides evidence that the multienzyme gramicidin S synthetase 2 is, at least in part, constructed from functional domains. An approach towards extending these studies to other parts of the gramicidin S synthetase 2 molecule has also been devised. This work complements recombinant DNA studies in the area, providing stable functional fragments.

On the domain construction of the multienzyme gramicidin S synthetase 2

“…4, one single, symmetrical peak was detected. When compared to gramicidin S synthetase 2 and fragments thereof [6], the peak corresponded to about 120 kDa.…”

“…Within 5-7 min of termination of trypsination, the gelfiltered sample was applied to a Mono Q column and eluted as described [6] with a gradient of 0 -500 mM NaC1, gradient rate 16.7 mM/ml and eluent flow rate 1.0 ml/min. The eluate was monitored by Azso.…”

“…The determination of amino acid activation was performed as described [6]. Assays contained 2 mM amino acid, 2.5 mM ATP and ImM PPi.…”

“…They may or may not also have significant homology. We recently demonstrated that the region of gramicidin S synthetase 2 which activates Pro can be removed from the multienzyme by trypsin, and collected on a proteaseresistant structural characteristic of structural domains [6]. A similar Pro-activating fragment is produced by a gramicidin S negative mutant (n7) presumably by early termination [6 -101.…”

“…This provides evidence that to some degree the gramicidin S synthetase 2 multienzyme is divided into structural and functional domains. In order to characterize the Pro-activating domain [6], we used a range of trypsin concentrations for one short, fixed period of time to investigate the course of proteolysis. This approach, in combination with FPLC, made it possible to analyze the release of active fragments.…”