Chemical rescue of a site-specific mutant of bacterial copper amine oxidase for generation of the topa quinone cofactor
Chemical rescue of a site-specific mutant of bacterial copper amine oxidase for generation of the topa quinone cofactor
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DOI：
10.1021/bi0361923
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发表时间：
2004-03-02
期刊：
影响因子：
2.9
通讯作者：
Tanizawa, K
中科院分区：
文献类型：
--
作者：
Matsunami, H;Okajima, T;Tanizawa, K
铜胺氧化酶的topa quinone（TPQ）辅因子是通过铜依赖性的自催化过程对特定酪氨酸残基进行翻译后修饰而产生的。我们有位点特异性突变的三个组氨酸残基（His 431，His 433，和His 592）参与结合的铜离子在重组苯乙胺氧化酶从节杆菌球形。突变酶，其中每个组氨酸被丙氨酸取代，在Cu/TPQ-自由的前体形式进行纯化，并通过紫外-可见吸收，共振拉曼，和电子顺磁共振光谱分析其Cu结合和TPQ产生的活动。在三个组氨酸-丙氨酸突变体中，发现只有H592 A在有氧孵育时与Cull离子一起形成TPQ的活性较弱。此外，对于H592 A，外源性咪唑拯救铜的结合，并显着促进TPQ的形成。通过X射线晶体学，建议在H592 A的活性位点中产生的空腔内容纳游离咪唑分子。尽管H592 A突变体中的TPQ辅因子很容易被底物还原，但即使在咪唑存在下，其催化活性也非常低。结合突变酶的晶体结构，这些结果表明了三个铜结合组氨酸残基对TPQ生物合成和催化活性的重要性，微调了必需金属的位置。
The topa quinone (TPQ) cofactor of copper amine oxidase is produced by posttranslational modification of a specific tyrosine residue through the copper-dependent, self-catalytic process. We have site-specifically mutated three histidine residues (His431, His433, and His592) involved in binding of the copper ion in the recombinant phenylethylamine oxidase from Arthrobacter globiformis. The mutant enzymes, in which each histidine was replaced by alanine, were purified in the Cu/TPQ-free precursor form and analyzed for their Cu-binding and TPQ-generating activities by UV-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. Among the three histidine-to-alanine mutants, only H592A was found to show a weak activity to form TPQ upon aerobic incubation with Cull ions. Also for H592A, exogenous imidazole rescued binding of copper and markedly promoted the TPQ formation. Accommodation of a free imidazole molecule within the cavity created in the active site of H592A was suggested by X-ray crystallography. Although the TPQ cofactor in H592A mutant was readily reduced with substrate, its catalytic activity was very low even in the presence of imidazole. Combined with the crystal structures of the mutant enzymes, these results demonstrate the importance of the three copper-binding histidine residues for both TPQ biogenesis and catalytic activity, fine-tuning the position of the essential metal.