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 PP(i) generation using active site titration measurements with [gamma-(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.
Kittelberger
R
R
Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Franklinstrasse 29, 10587, Berlin, Germany.
Pavela-Vrancic
M
M
von Döhren
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000591:Amino Acid Isomerases / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D011756:Diphosphates / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D010452:Peptide Biosynthesis; D010453:Peptide Synthases / Q000378:metabolism*
D011756:Diphosphates; D009097:Multienzyme Complexes; D000255:Adenosine Triphosphate; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2