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 has been approached by affinity labeling of tyrocidine synthetase 1 with fluorescein 5'-isothiocyanate. Binding was accompanied by irreversible inhibition of the ATP-dependent phenylalanine activation reaction and was prevented in the presence of MgATP2-. The reaction obeyed pseudo first-order rate kinetics and was accelerated by Mg2+. Complete inhibition corresponded to incorporation of 2.3 mol of fluorescein 5'-isothiocyanate (FITC)/mol of protein. Upon protection by MgATP2-, about 1 mol of FITC is still incorporated; however, this does not affect activity. The modified synthetase was extensively fragmented by tryptic digestion and the labeled fragments isolated by reverse-phase high performance liquid chromatography. Two peptides, DHQVKIR and LDKMPLTPNDKIDR, have been identified by sequencing, and the FITC conjugate of the former peptide has been detected by laser desorption mass spectrometry. The labeled residues, Lys-422 and Lys-505, are located within highly conserved segments of this new class of synthetases.
Pavela-Vrancic
M
M
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Federal Republic of Germany.
Pfeifer
E
E
Schröder
W
W
von Döhren
H
H
Kleinkauf
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D016650:Fluorescein-5-isothiocyanate; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010453:Peptide Synthases / Q000378:metabolism*; D016415:Sequence Alignment
D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C035180:tyrocidine synthetase; D016650:Fluorescein-5-isothiocyanate