Ruggiero C E, Smith J A, Tanizawa K, Dooley D M
Department of Chemistry and Biochemistry, Montana State University, Bozeman 59717, USA.
Biochemistry. 1997 Feb 25;36(8):1953-9. doi: 10.1021/bi9628836.
An alternative purification for apophenylethylamine oxidase from Arthrobacter globiformis has been developed, which avoids the use of possible contaminants that may interfere with the topa quinone (TPQ) self-processing reaction. The binding of Cu(II) and the kinetics of TPQ formation in these enzyme preparations have been reinvestigated. Our results show that Cu(II) is not significantly reduced when added to the apoprotein under anaerobic conditions. The Cu(II) EPR and circular dichroism spectra of the initially formed complex are different from the spectra of the mature Cu(II)/TPQ-containing protein, indicating that the active site structure must be altered during TPQ formation. The kinetics we observe are cleanly first-order in protein [measured subsequent to Cu(II) binding] when dioxygen is present in pseudo-first-order excess (k(obs) = 1.5 min(-1)). We found no rate dependence on copper, so long as one copper per subunit was present. This indicates that tyrosine oxidation to give TPQ depends only on the copper that is bound in the active site. These results differ from those originally reported; an alternative mechanism, which involves attack of an activated copper-oxygen species on a tyrosine radical intermediate, is proposed for TPQ formation.
已开发出一种从球形节杆菌中纯化阿朴苯乙胺氧化酶的替代方法，该方法避免使用可能干扰对苯二酚醌（TPQ）自加工反应的潜在污染物。对这些酶制剂中铜（II）的结合以及TPQ形成的动力学进行了重新研究。我们的结果表明，在厌氧条件下将铜（II）添加到脱辅基蛋白中时，铜（II）不会显著还原。最初形成的复合物的铜（II）电子顺磁共振（EPR）和圆二色光谱与成熟的含铜（II）/TPQ蛋白的光谱不同，这表明在TPQ形成过程中活性位点结构必定发生了改变。当氧气以伪一级过量存在时（k(obs) = 1.5 min(-1)），我们观察到的动力学在蛋白质中是清晰的一级反应（在铜（II）结合后测量）。我们发现只要每个亚基存在一个铜，反应速率就与铜无关。这表明酪氨酸氧化生成TPQ仅取决于结合在活性位点的铜。这些结果与最初报道的结果不同；提出了一种涉及活化的铜 - 氧物种攻击酪氨酸自由基中间体的替代机制来解释TPQ的形成。
