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

Abstract

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 [Q Q-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.z 1999 Federation of European Biochemical Societies.

Kinetics Profiling of Gramicidin S Synthetase A, a Member of Nonribosomal Peptide Synthetases

“…Such kinetics, manifested by a rapid initial burst followed by a slower increase, has also been observed in homologous enzymes. 14,28,33 Furthermore, our profiling may provide a kinetic basis for previous reports in which a second equivalent of the cognate amino acid and ATP can be consumed before Ppant is discharged again: 33,34 although tight binding between the second equivalent of acyl-adenylate and the NRPS A domain as well as the loaded Ppant may inhibit the next round's onpathway reaction, all of the off-pathway reactions (Scheme 1B(1−3)) could still occur post-thioesterification. These concurrently occurring off-pathway reactions consume the second equivalent acyl-adenylate intermediate at a much slower summed rate (at least 53-fold reduced compared to thioesterification in holo WT), as first reported here, ensuring the observed 2× stoichiometry in the substrate consumption.…”