Active site titration of gramicidin S synthetase 2: evidence for misactivation and editing in non-ribosomal peptide biosynthesis

The catalytic competence of gramicidin S synthetase 2 (GS2) was determined by following the kinetics of PPi generation using active site titration mea surements with [gamma-P-32]ATP. The initial 'burst' of product formation ca n be correlated to the generation of the aminoacyl adenylate:enzyme complex es at the four amino acid activation domains and the subsequent aminoacylat ion of carrier domains, followed by a slow linear turnover of substrate due to breakdown of the intermediate. Simultaneous activation of all four amin o 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 intermedi ates reflects a possible corrective mechanism by which the enzyme controls the fidelity of product formation, (C) 1999 Federation of European Biochemi cal Societies.