Thermodynamic analysis of the binding of oxidized and reduced FMN cofactor to Vibrio harveyi NADPH-FMN oxidoreductase FRP apoenzyme
Thermodynamic analysis of the binding of oxidized and reduced FMN cofactor to Vibrio harveyi NADPH-FMN oxidoreductase FRP apoenzyme
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DOI：
10.1021/bi0610956
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发表时间：
2006-12-12
期刊：
影响因子：
2.9
通讯作者：
Tu, Shiao-Chun
中科院分区：
文献类型：
--
作者：
Li, Xi;Chow, Dar-Chone;Tu, Shiao-Chun
哈维氏弧菌NADPH特异性黄素还原酶FRP遵循乒乓球机制，但在荧光素酶偶联反应中切换为顺序机制。结合的FMN与FRP共分离，在单酶反应中作为真正的辅助因子，在荧光素酶偶联反应中作为预结合底物发挥作用，一旦被还原就直接转移到荧光素酶[Lei，B.，and Tu，S.-C(1998)BioChemical 37,14623-14629]。为了更好地了解FMN在FRP全酶中的作用，本研究对FRP脱辅酶与氧化和还原FMN结合的热力学性质进行了定量和比较。通过在pH 7.0和15-30℃的不同缓冲液中的恒温滴定量热(ITC)测量，发现apo-FRP与FMN的结合是非合作的、放热的、主要由热能驱动的。在此温度范围内，结合自由能变化(即缔合常数)几乎不变。当FMN结合时，FRP的构象发生了显著的变化。黄素依赖的蛋白质平衡结合还原黄素的研究很少。在这项工作中，通过ITC测量发现，在三组实验条件下，apo-FRP与还原FMN结合的热力学性质与FMN结合的热力学性质非常相似，在一种情况下，荧光猝灭。现在，通过直接测量氧化和还原的FMN辅因子的结合亲和力，支持了FRP的动力学推导的乒乓球机制。这些发现也与FRP在FRP-荧光素酶对中作为还原黄素供体的功能有关。
The Vibrio harveyi NADPH-specific flavin reductase FRP follows a ping-pong mechanism but switches to a sequential mechanism in the luciferase-coupled reaction. The bound FMN co-isolated with FRP, while acting as a genuine cofactor in the single-enzyme reaction, functions in the luciferase-coupled reaction as a prebound substrate and is directly transferred to luciferase once it is reduced [Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623-14629]. With the aim of better understanding the functions of FMN in the FRP holoenzyme, this study was undertaken to quantify and compare the thermodynamic properties of the binding of oxidized and reduced FMN by the FRP apoenzyme. By isothermal titration calorimetry (ITC) measurements in various buffers at pH 7.0 and 15-30 degrees C, the binding of FMN by apo-FRP was found to be noncooperative, exothermic, and primarily enthalpy driven. The binding free energy change (hence, the association constant) was nearly invariant over this temperature range. Significant conformational changes in FRP upon binding of FMN were indicated. Equilibrium bindings of reduced flavins by flavin-dependent proteins have rarely been studied. In this work, the thermodynamic properties of binding of reduced FMN by apo-FRP were found to closely resemble those of FMN binding under three sets of experimental conditions via ITC measurements and, in one case, fluorescence quenching. The kinetically deduced ping-pong mechanism of FRP is now supported by direct measurements of binding affinities of the oxidized and reduced FMN cofactors. These findings are also discussed in relation to the function of FRP as a reduced flavin donor in the FRP-luciferase couple.