Kittelberger R, Pavela-Vrancic M, von Döhren H
Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Franklinstrasse 29, 10587, Berlin, Germany.
FEBS Lett. 1999 Nov 19;461(3):145-8. doi: 10.1016/s0014-5793(99)01445-3.
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.
通过使用[γ-(32)P]ATP进行活性位点滴定测量来跟踪PP(i)生成的动力学，从而确定短杆菌肽S合成酶2（GS2）的催化活性。产物形成的初始“爆发”可与四个氨基酸活化结构域处氨基酰腺苷酸：酶复合物的生成以及载体结构域随后的氨基酰化相关，随后由于中间体的分解，底物出现缓慢的线性周转。在饱和浓度下同时激活所有四种氨基酸底物时，显示消耗8.3个ATP/GS2。在存在单一氨基酸的情况下，获得的结合化学计量高于每个活性位点预期的两个ATP，这意味着在非同源结构域存在错误激活。酰腺苷酸中间体的分解反映了一种可能的校正机制，通过该机制酶控制产物形成的保真度。
