Lei B, Tu S C
Department of Biology and Biochemistry, University of Houston, Texas 77204-5934, USA.
Biochemistry. 1998 Oct 13;37(41):14623-9. doi: 10.1021/bi981841+.
The mechanisms of reduced flavin transfer in biological systems are poorly understood at the present. The Vibrio harveyi NADPH-FMN oxidoreductase (FRP) and the luciferase pair were chosen as a model for the delineation of the reduced flavin transfer mechanism. FRP, which uses FMN as a cofactor to mediate the reduction of the flavin substrate by NADPH, exhibited a ping-pong kinetic pattern with a Km, FMN of 8 microM and a Km,NADPH of 20 microM in a single-enzyme spectrophotometric assay monitoring the NADPH oxidation. However, the kinetic mechanism of FRP was changed to a sequential pattern with a Km,FMN of 0.3 microM and a Km,NADPH of 0.02 microM in a luciferase-coupled assay measuring light emission. In contrast, the Photobacterium fischeri NAD(P)H-FMN oxidoreductase FRG showed the same ping-pong mechanism in both the single-enzyme spectrophotometric and the luciferase-coupled assays. Moreover, for the FRP, FMN at concentrations over 2 microM significantly inhibited the coupled reaction in both light intensity and quantum yield, and showed apparent noncompetitive and competitive inhibition patterns against NADPH and luciferase, respectively. No inhibition of the NADPH oxidation was detected under identical conditions. These results are consistent with a scheme that the reduced flavin cofactor of FRP is preferentially utilized by luciferase for light emission, the reduced flavin product generated by the reductase is primarily channeled into a dark oxidation, and luciferase competes against flavin substrate in reacting with the FRP reduced flavin cofactor. An FRP derivative containing 2-thioFMN as the cofactor was also used to further examine the mechanism of flavin transfer. Results again indicate a preferential utilization of the reductase reduced flavin cofactor by luciferase for the bioluminescence reaction.
目前，人们对生物系统中黄素还原转移的机制了解甚少。哈氏弧菌NADPH - FMN氧化还原酶（FRP）和荧光素酶对被选作描绘黄素还原转移机制的模型。FRP以FMN作为辅因子，介导NADPH对黄素底物的还原作用。在监测NADPH氧化的单酶分光光度测定中，FRP呈现乒乓动力学模式，其FMN的Km值为8微摩尔，NADPH的Km值为20微摩尔。然而，在测量发光的荧光素酶偶联测定中，FRP的动力学机制转变为有序模式，其FMN的Km值为0.3微摩尔，NADPH的Km值为0.02微摩尔。相比之下，费氏弧菌NAD(P)H - FMN氧化还原酶FRG在单酶分光光度测定和荧光素酶偶联测定中均呈现相同的乒乓机制。此外，对于FRP，浓度超过2微摩尔的FMN在光强度和量子产率方面均显著抑制偶联反应，并且分别对NADPH和荧光素酶表现出明显的非竞争性和竞争性抑制模式。在相同条件下未检测到对NADPH氧化的抑制作用。这些结果与以下方案一致：FRP的还原黄素辅因子优先被荧光素酶用于发光，还原酶产生的还原黄素产物主要进入暗氧化途径，并且荧光素酶在与FRP还原黄素辅因子反应时与黄素底物竞争。还使用了一种以2 - 硫代FMN作为辅因子的FRP衍生物来进一步研究黄素转移机制。结果再次表明，荧光素酶优先利用还原酶的还原黄素辅因子进行生物发光反应。
