Reduction kinetics of a flavin oxidoreductase LuxG from Photobacterium leiognathi (TH1): half-sites reactivity.
Reduction kinetics of a flavin oxidoreductase LuxG from Photobacterium leiognathi (TH1): half-sites reactivity.
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DOI：
10.1021/bi1009985
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发表时间：
2010-11-02
期刊：
影响因子：
2.9
通讯作者：
Chaiyen, Pimchai
中科院分区：
文献类型：
--
作者：
Nijvipakul, Sarayut;Ballou, David P.;Chaiyen, Pimchai
细菌生物发光是由双组分FMN依赖性醛单加氧酶系统反应产生的现象，该系统包括细菌荧光素酶和黄素还原酶。细菌荧光素酶（LuxAB）是研究最广泛的双组分单加氧酶之一，而其还原酶伴侣，来自同一操纵子的黄素还原酶（LuxG），最近才以功能形式表达。本工作报告瞬态动力学鉴定的中间体在LuxG反应，使用停流分光光度法。结果表明，整个反应遵循顺序有序的机制，其中NADH首先与酶结合，然后是FMN，导致形成典型的还原吡啶核苷酸和氧化黄素之间的电荷转移中间体1（CT-1）。下一步是FMN的减少，如450 nm处吸光度的大幅降低所示。FMN的减少是双相的。FMN还原的第一阶段与电荷转移中间体2（CT-2）的形成同时发生，而第二阶段与CT-2的衰变同步。当使用同位素标记的底物4（R）[2H] NADH时，第一还原阶段显示出≥ 3.9的主要动力学同位素效应（Dkred），并导致CT-1的更大蓄积。这些结果与CT-1是FMNox：NADH复合物，而CT-2是FMNred：NAD+复合物一致。由于CT-2以2.8 ± 0.2 s-1的速率常数衰减，而从稳态-稳态动力学获得的转换数为1.7 s-1，因此CT-2衰减步骤很可能在很大程度上控制了总反应速率。所有的动力学数据是一致的半网站的反应模型，其中黄素减少发生在一个亚基的时间。第一个减少阶段是由于第一个亚基中FMN的减少，而第二个阶段是由于第二个亚基中FMN的减少。后一阶段受第一亚基中CT-2的衰变速率限制。半位点反应性模型也得到了在预稳态期间爆发动力学检测的支持，其与每摩尔LuxG：NADH复合物还原0.5摩尔FMN相关。这种半位点反应现象的功能重要性尚不清楚。
Bacterial bioluminescence is a phenomenon resulting from the reaction of a two-component FMN-dependent aldehyde monooxygenase system, which comprises a bacterial luciferase and a flavin reductase. Bacterial luciferase (LuxAB) is one of the most extensively investigated two-component monooxygenases, while its reductase partner, the flavin reductase (LuxG) from the same operon, has only been recently expressed in a functional form. This work reports transient kinetics identification of intermediates in the LuxG reaction using stopped-flow spectrophotometry. The results indicate that the overall reaction follows a sequential-ordered mechanism in which NADH binds first to the enzyme, followed by FMN, resulting in the formation of charge-transfer intermediate 1 (CT-1) typical of those between reduced pyridine nucleotides and oxidized flavins. The next step is the reduction of FMN as indicated by a large decrease in absorbance at 450 nm. The reduction of FMN is biphasic. The first phase of FMN reduction occurs concurrently with formation of charge-transfer intermediate 2 (CT-2), while the second phase is synchronous with the decay of CT-2. When the isotope-labeled substrate, 4(R) [2H]NADH, was used, the first reduction phase showed a primary kinetic isotope effect (Dkred) of ≥ 3.9 and resulted in greater accumulation of CT-1. These results are consistent with CT-1 being the FMNox:NADH complex, while CT-2 is the FMNred:NAD+ complex. Because CT-2 decays with a rate constant of 2.8 ± 0.2 s-1, while the turnover number obtained from the steady-steady state kinetics is 1.7 s-1, it is likely that the CT-2 decay step largely controls the overall reaction rate. All kinetic data are consistent with a half-sites reactivity model in which flavin reduction occurs at only at one subunit at a time. The first reduction phase is due to the reduction of FMN in first subunit, while the second phase is due to the reduction of FMN in the second subunit. The latter phase is limited by the rate of decay of CT-2 in the first subunit. The half-sites reactivity model is also supported by detection of burst kinetics during the pre-steady state period that is correlated with 0.5 mol of the FMN being reduced per mole of the LuxG:NADH complex. The functional importance of this half-site reactivity phenomenon is still unclear.
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影响因子：
4.8
作者：
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通讯作者：
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