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

FEBS Letters

von Döhren, H.¹  Pavela-Vrancic, M.¹  Kittelberger, R.¹ 

[1] Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Franklinstrasse 29, 10587 Berlin, Germany

关键词: Multienzyme;    Peptide synthetase;    Gramicidin S synthetase 2;    Aminoacyl adenylation;    Misactivation;    Editing;   

DOI  :  10.1016/S0014-5793(99)01445-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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-³²P]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.

【 授权许可】

Unknown