Vol. 41, No. 5, 1970 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ENZYME-BOUND PHOSPHOPANTFTHEINE IN TYROCIDINE BIOSYNTHESIS Hors-t Kleinkauf, Wieland Gevers, Robert Roskoski, Jr., and Fritz Lipmann The Rockefeller University New York, N.Y. 10021 Received October 23. 1970 SUMMARY: Phosphopantetheine has been demonstrated to be an enzyme-bound cofactor in one of the three enzyme fractions (light, intermediate, and heavy) required for tyrocidine biosynthesis. The enzyme-bound pantothenate had to be liberated by heating with alkali and treating the product with alkaline phosphatase. One mole of cofactor was estimated to be present per mole of heavy enzyme. It was absent from the light and intermediate enzyme fractions. We have reported that in the biosynthesis of the two cyclic decapeptides gramicidin S (GS) and tyrocidine (Ty), a polyenzyme system performs amino acid polymerization from protein-bound amino acids linked as thioesters (l-5). GS has the following sequence: D-y+ Pro+ Valj Or--+ Leu ! L Leu +-Orn +Val +-Pro +D-Phe (1) The amino acid sequence of Ty is as follows: D-Phej Pro-J Phe+ D-Phej Asn m Ji 2x1 wrn +--Val <-Tyr t-Gin (Phe) (2) The solid arrows show the direction of one-by-one addition; the stippled arrows indicate ring closure (ls5). The similarity between antibiotic polypeptide syntheses and fatty acid elongation in the polyenzymes of Iynen (6) and Vagelos (7) made us propose a possible participation of pantetheine (1). Our first attempts to show i4C-pantothenate incorporation into enzyme in vivo by the procedure used by Vagelos (7) to demonstrate its incorporation into E_. - coli acyl carrier protein were unsuccesful. Gilhuus-Moe et al. (8) used the more suitable microbiological assay for pantothenic acid. Pantothenic acid was liberated from the enzyme fraction by the method of 1218