Poulsen L L, Ziegler D M
J Biol Chem. 1979 Jul 25;254(14):6449-55.
A FAD-containing monooxygenase isolated from pig liver microsomes migrates as a single band upon electrophoresis in polyacrylamide gels in the presence of dodecyl sulfate. The minimum molecular weight based on mass of amino acids per mole of flavin is 64,000. However, the catalytically active enzyme exists as aggregating units of the monomer. Neither oxygen nor organic substrates perturbed the spectrum of the oxidized flavoprotein and their binding to this form of the enzyme could not be detected. Anaerobically NADPH bleaches the flavoprotein, and in the presence of both NADPH and oxygen a remarkably stable intermediate form of the enzyme, with an absorption band at 375 nm, is observed. The spectrum of the intermediate resembles that of a peroxyflavin. The monooxygenase catalyzes NADPH- and oxygen-dependent oxygenations of nucleophilic nitrogen- or sulfur-containing compounds. Kinetic studies carried out with a model organic nitrogen substrate (trimethylamine) and a sulfur substrate (methimazole) gave similar patterns. The kinetic data are consistent with an ordered Ter-Bi mechanism with an irreversible step between the second and third substrate where NADPH is added first, followed by oxygen, and the oxidizable organic substrate is added last. If NADPH is the first substrate added, then NADP+ must be the last product released since NADP+ is competitive with NADPH.
从猪肝微粒体中分离出的一种含黄素腺嘌呤二核苷酸（FAD）的单加氧酶，在十二烷基硫酸钠存在下于聚丙烯酰胺凝胶中进行电泳时迁移为单一谱带。基于每摩尔黄素的氨基酸质量计算，其最小分子量为64,000。然而，具有催化活性的酶以单体的聚集单元形式存在。氧气和有机底物均未扰动氧化型黄素蛋白的光谱，且未检测到它们与这种酶形式的结合。在无氧条件下，NADPH会使黄素蛋白褪色，并且在同时存在NADPH和氧气的情况下，会观察到该酶的一种非常稳定的中间形式，其在375 nm处有一条吸收带。该中间产物的光谱类似于过氧黄素的光谱。这种单加氧酶催化亲核含氮或含硫化合物的NADPH和氧气依赖性氧化反应。用模型有机氮底物（三甲胺）和硫底物（甲巯咪唑）进行的动力学研究给出了相似的模式。动力学数据与有序的Ter - Bi机制一致，在第二个和第三个底物之间存在不可逆步骤，其中首先添加NADPH，随后是氧气，最后添加可氧化的有机底物。如果NADPH是首先添加的底物，那么NADP +必定是最后释放的产物，因为NADP +与NADPH具有竞争性。
