IDENTIFICATION OF THE ATP BINDING-SITE IN TYROCIDINE SYNTHETASE-1 BY SELECTIVE MODIFICATION WITH FLUORESCEIN 5'-ISOTHIOCYANATE

Identification of the nucleotide binding site in peptide synthetases h as been approached by affinity labeling of tyrocidine synthetase 1 wit h fluorescein 5'-isothiocyanate. Binding was accompanied by irreversib le inhibition of the ATP-dependent phenylalanine activation reaction a nd was prevented in the presence of MgATP(2-). The reaction obeyed pse udo first-order rate kinetics and was accelerated by Mg2+. Complete in hibition corresponded to incorporation of 2.3 mol of fluorescein 5'-is othiocyanate (FITC)/mol of protein. Upon protection by MgATP(2-), abou t 1 mol of FITC is still incorporated; however, this does not affect a ctivity. The modified synthetase was extensively fragmented by tryptic digestion and the labeled fragments isolated by reverse-phase high pe rformance liquid chromatography. Two peptides, DHQVKIR and LDKMPLTPNDK IDR, have been identified by sequencing, and the FITC conjugate of the former peptide has been detected by laser desorption mass spectrometr y. The labeled residues, Lys-422 and Lys-505, are located within highl y conserved segments of this new class of synthetases.