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Active site titration of gramicidin S synthetase 2: evidence for misactivation and editing in non‐ribosomal peptide biosynthesis
Author(s) -
Kittelberger R.,
Pavela-Vrancic M.,
von Döhren H.
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01445-3
Subject(s) - active site , titration , amino acid , aminoacylation , gramicidin s , chemistry , biosynthesis , biochemistry , enzyme , peptide , gramicidin , stereochemistry , adenylate kinase , binding site , membrane , transfer rna , inorganic chemistry , rna , gene
The catalytic competence of gramicidin S synthetase 2 (GS2) was determined by following the kinetics of PP i generation using active site titration measurements with [γ‐ 32 P]ATP. The initial ‘burst’ of product formation can be correlated to the generation of the aminoacyl adenylate:enzyme complexes at the four amino acid activation domains and the subsequent aminoacylation of carrier domains, followed by a slow linear turnover of substrate due to breakdown of the intermediate. Simultaneous activation of all four amino acid substrates at a saturating concentration displayed a consumption of 8.3 ATP/GS2. In the presence of single amino acids, a binding stoichiometry higher than the anticipated two ATP per active site was obtained, implying misactivation at non‐cognate domains. Breakdown of acyladenylate intermediates reflects a possible corrective mechanism by which the enzyme controls the fidelity of product formation.