Hastings J W, Balny C, Peuch C L, Douzou P
Ecole des Hautes Etudes, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, Paris 5 France.
Proc Natl Acad Sci U S A. 1973 Dec;70(12 Pt 1-2):3468-72. doi: 10.1073/pnas.70.12.3468.
Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide by molecular oxygen; long-chain aldehyde is required for light emission. At 20 degrees the bioluminescence has a lifetime of tens of seconds, while excess reduced flavin is removed by way of nonenzymatic autoxidation in less than a second. This observation indicates the existence of a long-lived enzyme intermediate, which has been postulated to be a peroxide of the enzyme-bound reduced flavin. This intermediate was isolated and studied at low temperature (-20 degrees ), where it has a lifetime measured in days. It has an absorption with a single band peaking at 372 nm, and fluorescence emission centered at about 485 nm, which might be expected for the postulated flavin peroxide. Upon conversion to product, flavin mononucleotide-like absorption and fluorescence appear, supporting the postulate that flavin turns over in the reaction. Upon injection into buffer at 20 degrees with added aldehyde, bioluminescence occurs. Based on a stoichiometry of one flavin per luciferase molecule, the specific activity of the intermediate is equal to that of pure luciferase.
细菌荧光素酶催化还原型黄素单核苷酸被分子氧氧化；发光需要长链醛。在20摄氏度时，生物发光的寿命为数十秒，而过量的还原型黄素通过非酶自氧化在不到一秒的时间内被去除。这一观察结果表明存在一种长寿命的酶中间体，据推测它是与酶结合的还原型黄素的过氧化物。这种中间体在低温（-20摄氏度）下被分离并进行了研究，在该温度下其寿命以天来衡量。它有一个吸收峰，单一谱带在372纳米处达到峰值，荧光发射集中在约485纳米处，这对于推测的黄素过氧化物来说是可以预期的。在转化为产物时，出现了类似黄素单核苷酸的吸收和荧光，支持了黄素在反应中周转的推测。当在20摄氏度下注入添加了醛的缓冲液中时，会发生生物发光。基于每个荧光素酶分子一个黄素的化学计量，中间体的比活性与纯荧光素酶的比活性相等。
