ATP BINDING IN PEPTIDE SYNTHETASES - DETERMINATION OF CONTACT SITES OF THE ADENINE MOIETY BY PHOTOAFFINITY-LABELING OF TYROCIDINE SYNTHETASE-1 WITH 2-AZIDOADENOSINE TRIPHOSPHATE

Characterization of the nucleotide binding domain in peptide synthetas es was approached by photoaffinity labeling of tyrocidine synthetase 1 (TY1) with 2-azidoadenosine triphosphate (2-azido-ATP). Exposure of T Y1 in the presence of photolabel to irradiation with ultraviolet light resulted in a time-dependent covalent modification of the enzyme with a concomitant loss of catalytic activity. Inactivation was not observ ed if incubation was performed in the absence of either light or the n ucleotide analogue. Specificity of labeling was indicated by the abili ty of 2-azido-ATP to serve as a substrate in the amino acid activation reaction. The modified protein was subjected to tryptic digestion, an d the fragments labeled by the nucleotide analogue were purified by re verse-phase high-performance liquid chromatography. Sequence analysis identified three tryptic peptides corresponding to residues G373-K384, W405-R416, and L483-K494, derived from the N-terminal half of the TY1 sequence. As this region shows similarity to strongly conserved regio ns in other peptide synthetases and acyl-CoA synthetases, it is consid ered to be the region catalyzing aminoacyl adenylate formation. The id entified sequences appear to define components of the nucleotide bindi ng domain found in close proximity to the adenine ring in ATP. Conserv ation of primary structure and homology to other carboxyl-activating e nzymes of this superfamily, including peptide synthetases, insect luci ferases, and acyl-CoA synthetases, is discussed.