Macheroux P, Ghisla S, Hastings J W
Faculty of Biology, University of Konstanz, Germany.
Biochemistry. 1993 Dec 28;32(51):14183-6. doi: 10.1021/bi00214a017.
The bioluminescent reaction of luciferase isolated from Vibrio harveyi, strain M17, was initiated by mixing the luciferase-bound flavin 4a-hydroperoxide intermediate, purified in advance, with a long-chain aldehyde (dodecanal or octanal) at -4 degree C. Measurements of absorbance changes from 300 to 600 nm during the course of the reaction revealed the existence of three sets of isosbestic points and three kinetic phases, the second of which parallels kinetically the decay of bioluminescence, measured concurrently. The absorbance changes in this second step and the decay of light emission exhibited similar deuterium isotope effects; this is postulated to be the step giving rise to the excited state and the enzyme-bound flavin 4a-hydroxide. The first step of the reaction, however, did not show an isotope effect; the intermediate thereby formed, observed here for the first time, is postulated to correspond to the luciferase-bound flavin 4a-peroxyhemiacetal.
从哈维弧菌M17菌株中分离出的荧光素酶的生物发光反应，是通过将预先纯化的与荧光素酶结合的黄素4a - 氢过氧化物中间体与长链醛（十二醛或辛醛）在 -4℃混合来启动的。在反应过程中对300至600nm吸光度变化的测量揭示了存在三组等吸收点和三个动力学阶段，其中第二个阶段在动力学上与同时测量的生物发光衰减平行。第二步中的吸光度变化和发光衰减表现出相似的氘同位素效应；据推测这是产生激发态和与酶结合的黄素4a - 氢氧化物的步骤。然而，反应的第一步没有显示出同位素效应；在此首次观察到由此形成的中间体，据推测它对应于与荧光素酶结合的黄素4a - 过氧半缩醛。
