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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. [ABSTRACT FROM AUTHOR]

The amino acid analog L-cyclohexylalanine, which may be considered an analog of the hydrophobic amino acids leucine and valine, was found to thioester-bind to the heavy enzyme of gramicidin S synthetase. The results indicate that it preferably binds to the thiol site of leucine, although binding to the valine site also occurs. In the synthesis of the cyclic decapeptide by gramicidin S synthetase, the results suggest that L-cyclohexylalanine can replace L-leucine and L-valine. We have also found that in the synthesis of the cyclic decapeptidase L-leucine can replace L-valine and vice versa. The results further indicate that the previously reported thioester binding of D and L-phenylalanine to the heavy enzyme takes place at the thiol site of leucine. [ABSTRACT FROM AUTHOR]

Columns containing one of the amino acids in gramicidin S bound through its carboxyl group to the amino group of amino-alkyl-Sepharose (CNBr-activated Sepharose + 3,3′-diaminodipropylamine) have been prepared and were found to exhibit affinity for the corresponding enzymes (light or heavy) of gramicidin S synthetase. The D-phenylalanyl ligand will bind the light enzyme and the L-propyl, L-valyl, L-ornithyl or L-leucyl ligand, the heavy enzyme. These columns permitted a study of the interaction of the light and heavy enzyme. The results show that the area of the light enzyme which has affinity for the heavy enzyme is that part which activates phenylalanine. The area of the heavy enzyme which interacts with the light enzyme is not that part of the enzyme which activates proline. However, it is probably located close to the activation sites for proline and leucine. The columns, in particular with proline as ligand, provide a quick and convenient method for the separation of the light and heavy enzyme of gramicidin S synthetase. [ABSTRACT FROM AUTHOR]

The results show that enzyme I of gramicidin S synthetase activates D-leucine in a manner analogous to that of L-leucine (ATP + D-leucine ↔ D-leucyl-adenylate +. PPi) and binds two moles of D-leucine in thioester linkage. It has therefore two thiol sites for D-leucine, compared to one for the L-isomer. One of the sites seems to be the thiol site for L-leucine which is therefore stereo-unspecific. The results also indicate that the site for the formation of the aminoacyl adenylates is the same for the two isomers. In contrast to thioester-bound L-leucine, enzyme-bound D-leucine does not catalyze the ATP-[14C]AMP exchange reaction. At a molar ratio of L-leucine: D-leucine equal to 4, gramicidin S synthesis is almost completely d and the growth of the peptide chain stops at the tetrapeptide stage (D-Phe-L-ProL-Val-L-Orn). Enzyme-bound D-leucine is thus not able to replace L-leucine in the formation of the pentapeptide. [ABSTRACT FROM AUTHOR]