Xun L, Orser C S
Department of Bacteriology and Biochemistry, University of Idaho, Moscow 83843.
J Bacteriol. 1991 Jul;173(14):4447-53. doi: 10.1128/jb.173.14.4447-4453.1991.
A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.
一种五氯苯酚（PCP）羟化酶被鉴定出来，它在NADPH和氧气存在的情况下，催化PCP转化为2,3,5,6 - 四氯对苯二酚，并从三碘苯酚中释放出碘化物。该酶通过硫酸鱼精蛋白沉淀、硫酸铵沉淀、疏水色谱、阴离子交换色谱、凝胶过滤色谱和结晶进行纯化。该酶是一种分子量为63,000的单体。在某些条件下，也观察到二聚体和多聚体构象。该酶的pI为pH 4.3。活性的最佳条件是pH值为7.5至8.5，温度为40摄氏度。每个酶分子含有一个黄素腺嘌呤二核苷酸分子。PCP的Km为30微摩尔，Vmax为16微摩尔/分钟/毫克蛋白质。每摩尔卤化底物的酶促反应需要2摩尔NADPH。根据我们提供的数据，PCP羟化酶很可能是一种黄素蛋白单加氧酶。向反应混合物中添加黄素并不能刺激酶促反应；然而，我们发现黄素单核苷酸或核黄素与光可使三碘苯酚和三溴苯酚发生光降解，但PCP不会。
