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The tripeptide intermediate D-Phe-Pro-Val in the biosynthesis of gramicidin S was labeled by incorporation of either L-[14C]phenylalanine or L-[14C]valine in an in vitro biosynthetic assay. The gramicidin S synthetase 2-tripeptide complex was first digested with CNBr and subsequently by Staphylococcus aureus V8 protease. The active site peptide carrying the radioactively labeled tripeptide was isolated in pure form by reversed phase high performance liquid chromatography technology and analyzed by liquid phase sequencing, mass spectrometry, and amino acid analysis. It was demonstrated that D-Phe-Pro-Val is attached to the 4'-phosphopantetheine cofactor at the thiolation center for valine of gramicidin S synthetase 2. In this way the attachment site of a peptide intermediate in nonribosomal peptide biosynthesis was identified for the first time. Our results are in full agreement with the multiple carrier model of nonribosomal peptide biosynthesis (Stein, T., Vater, J., Kruft, V., Otto, A., Wittmann-Liebold, B., Franke, P., Panico, M., McDowell, R., and Morris, H. R. (1996) J. Biol. Chem. 271, 15426-15435), which predicts that the growing peptide chain in the elongation process should always be bound to the thiotemplate site specific for its C-terminal amino acid component.

Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.

Aminoacyl-tRNA synthetases (amino acid-tRNA ligases, EC 6.1.1.-) catalyze the aminoacylation of specific amino acids onto their cognate tRNAs with extraordinary accuracy. Recent reports, however, indicate that this class of enzymes may play other roles in cellular metabolism. Several aminoacyl-tRNA synthetases are herein shown to catalyze the AMP----ADP and ADP----ATP exchange reactions (in the absence of tRNAs) by utilizing a transfer of the gamma-phosphate of ATP to reactive AMP and ADP intermediates that are probably the mixed anhydrides of the nucleotide and the corresponding amino acid. AMP and ADP produce active intermediates with amino acids by entering the back-reaction of amino acid activation, reacting with labile covalent amino acid-enzyme intermediates. Gramicidin synthetases 1 and 2, which are known to activate certain amino acids through the formation of intermediate thiol-esters of the amino acids and the enzymes, catalyze the same set of reactions with similar characteristics. Several lines of evidence suggest that these activities are an inherent part of the enzymatic reactions catalyzed by the aminoacyl-tRNA synthetases and gramicidin synthetases and are not due to impurities of adenylate kinase, NDP kinase, or low levels of tRNAs bound to the enzymes. The covalent amino acid-enzyme adducts are likely intermediates in the aminoacylation of their cognate tRNAs. The use of gramicidin synthetases has thus helped to illuminate mechanistic details of amino acid activation catalyzed by the aminoacyl-tRNA synthetases.