Probing the Molecular Mechanisms in Copper Amine Oxidases by Generating Heterodimers
Probing the Molecular Mechanisms in Copper Amine Oxidases by Generating Heterodimers
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DOI：
10.1002/cbic.201402653
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发表时间：
2015-03-02
期刊：
影响因子：
3.2
通讯作者：
McPherson, Michael J.
中科院分区：
文献类型：
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作者：
Gaule, Thembaninkosi G.;Smith, Mark A.;McPherson, Michael J.
对于一些同源二聚体铜胺氧化酶（CuAO），有暗示性的证据表明，在两个活性位点的差异活动，这意味着两个单体之间的潜在的协同性。为了研究球形节杆菌CuAO（AGAO）的这种现象，我们纯化了异二聚体形式的酶与同二聚体进行比较。异二聚体包含活性野生型单体和活性位点酪氨酸突变为苯丙氨酸（Y382 F）的非活性单体。这种突变阻止了三羟基苯丙氨酸醌（TPQ）辅因子的形成。使用pETDuet载体和双重融合标签策略从同源二聚体纯化异源二聚体（WT/Y382 F）。通过蛋白质印迹和非变性PAGE分析确认纯度。光谱和动力学研究支持这样的观点，即无论二聚体中有一个或两个功能单体，每个功能单体的性质是相同的，从而表明在这种细菌酶的活性位点之间没有通信。
For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild-type monomer and an inactive monomer in which an active-site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme.