Pember S O, Villafranca J J, Benkovic S J
Biochemistry. 1986 Oct 21;25(21):6611-9. doi: 10.1021/bi00369a042.
Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.
来自紫色杆菌的蝶呤依赖性苯丙氨酸羟化酶含有化学计量的铜（Cu2+，每摩尔酶1摩尔）。该酶的电子顺磁共振光谱表明它是一种II型含铜蛋白。氧化态的酶必须通过单电子还原才能具有催化活性。发现二硫苏糖醇是该酶的有效还原剂。电子顺磁共振数据和动力学结果表明，在二硫苏糖醇对酶进行需氧还原过程中形成了酶-硫醇复合物。6,7-二甲基四氢蝶呤也能还原激活该酶，但仅在有底物存在时，且在动力学上比二硫苏糖醇效果差。金属中心不会因正常周转而重新氧化。然而，数据表明存在一条导致酶缓慢重新氧化的替代途径。在酶的周转过程中观察到了6-甲基四氢蝶呤的4a-水合物（4a-甲醇胺）。在含铁的哺乳动物酶催化的反应中也观察到了这种中间体，这表明两种酶的氧激活机制相似。
