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The multienzyme gramicidin S synthetase 2, composed of one polypeptide chain, was treated with trypsin and chymotrypsin to give fragments retaining partial enzyme activities. Previously, a tryptic fragment of this multienzyme has been identified as a structural and functional domain. In this study two more fragments, activating Leu and Val, respectively, are shown to represent domains. Careful inspection of the data on limited proteolysis, from this study as well as from previous work, suggests that domains are not simply connected like pearls on a string, and a model for the structure of gramicidin S synthetase, with implications for other peptide synthetase multienzymes, is presented. It is suggested that gramicidin S synthetase 2 is constructed from core catalytic domains and intervening framework. Such an interpretation is in accordance with all published data on limited proteolysis of peptide synthetases, but needs an interplay with gene structural studies in order to be validated and refined. [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]

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]