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ACTIVATION, RACEMIZATION AND ELONGATION OF SUBSTRATES BY GRAMICIDIN S SYNTHETASE
Abstract
The use of dipeptides as substrates for gramicidin S synthetase (GSS) was investigated. The dipeptides studied were Leu-Phe, Val-Phe, Val-Leu, Leu-Val, Gly-Val, Val-Gly, Gly-Leu, Leu-Gly and Phe-Phe. The dipeptides should contain at least one amino acid found in gramicidin (GS) to be a substrate. Dipeptides containing glycine and leucine or valine gave fairly good synthesis of a cyclopeptide when the remaining four amino acids necessary for GS synthesis were added to the reaction mixture. The glycine was unable to replace any of the amino acids and its position in the dipeptide had no effect on the ability of the dipeptide to synthesize a cyclopeptide. The nature of the incorporation of the amino acids present in the dipeptide was examined. The dipeptides are not cleaved during the synthesis since free amino acids were undetected in the reaction media. There is a possibility that the whole dipeptide is incorporated into the cyclopeptide. When Leu-Gly was utilized as the dipeptide in the synthesis reaction, the hydrolysate of the purified product indicated the presence of glycine. The methyl thioesters of D and L phenylalanine were prepared. The reaction of these thioesters, in the absence of ATP, indicated that only the D-isomer is capable of synthesizing GS. The thioesters failed to synthesize GS in the absence of the light enzyme or in the presence of NEM-treated light enzyme, demonstrating the importance of the light enzyme. The light enzyme was shown to be able to transfer the enzyme-bound thioester of phenylalanine to methyl mercaptan in solution. The transfer resulted in the synthesis of ('14)C-phenylalanine methyl thioester. The thioester was analysed for the amount of D-phenylalanine present using D-amino acid oxidase. The results indicated that only 34% of the phenylalanine in the thioester was D-phenylalanine. Several inhibitors were investigated. The alcohol analogues usually inhibited their respective amino acid ATP-PPi exchange. Leucinol was found to be a competive inhibitor of the leucine exchange (K(,I) = 0.68mM). The N-methylated derivates also demonstrated some inhibition.
Subject Area
Biochemistry
Recommended Citation
FELDHAUS, RODGER WARREN, "ACTIVATION, RACEMIZATION AND ELONGATION OF SUBSTRATES BY GRAMICIDIN S SYNTHETASE" (1981). ETD collection for University of Nebraska-Lincoln. AAI8208349.
https://digitalcommons.unl.edu/dissertations/AAI8208349