An electrochemical method for investigation of conformational flexibility of active sites of Trametes versicolor laccase based on sensitive determination of copper ion with cysteine-modified electrodes.
An electrochemical method for investigation of conformational flexibility of active sites of Trametes versicolor laccase based on sensitive determination of copper ion with cysteine-modified electrodes.
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DOI：
10.1021/ac302241a
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发表时间：
2012-10
影响因子：
7.4
通讯作者：
Xianchan Li;Ping Yu;Lifen Yang;Fuyi Wang;L. Mao
中科院分区：
文献类型：
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作者：
Xianchan Li;Ping Yu;Lifen Yang;Fuyi Wang;L. Mao
利用半胱氨酸修饰的Au电极对变色栓菌（Trametes versicolor，Tv）漆酶中游离的铜离子（Cu（2+））进行灵敏测定，为研究Tv漆酶活性中心的构象柔性提供了一种简便有效的电化学方法.在天然状态下，多铜活性位点深埋在Tv漆酶的多肽中，因此即使在半胱氨酸修饰的Au电极上也不能电化学检测。盐酸胍（GdnHCl）诱导的Tv漆酶解折叠后，铜离子从肽链上解离下来，从而被电化学还原，并在半胱氨酸修饰的Au电极上被检测到。这一性质可用于研究Tv漆酶多铜活性位点的构象柔性。我们发现，在Tv漆酶的多铜活性位点的构象和酶活性的变化与存在下的低浓度的盐酸钆变性剂（中点，其中50%的酶是未折叠的，在0.7 M）。该浓度低于诱导Tv漆酶分子作为一个整体的构象变化所需的浓度（中点在3.4 M），如通过Tv漆酶的固有荧光所研究的。这一观察结果表明，多铜活性位点形成的相对较弱的相互作用，因此可能是构象更灵活的比完整的酶。电化学方法简单有效，可用于研究变性剂诱导多铜氧化酶构象变化的热力学和动力学。
This study demonstrates a facile yet effective electrochemical method to investigate the conformational flexibility of the active sites of Trametes versicolor (Tv) laccase based on sensitive determination of copper ions (Cu(2+)) dissociated from the enzyme with the cysteine-modified Au electrodes. In the native state, the multicopper active sites are deeply buried in the polypeptide of Tv laccase and are thus not electrochemically detectable even at the cysteine-modified Au electrodes. Upon the unfolding of Tv laccase induced by guanidine hydrochloride (GdnHCl), copper ions dissociate from the peptide chain and, as a consequence, are electrochemically reduced and thus detected at the cysteine-modified Au electrodes. Such a property could be used to investigate the conformational flexibility of multicopper active sites of Tv laccase in a simple way. We find that both the conformation of the multicopper active sites in Tv laccase and the enzyme activity change with the presence of a low concentration of GdnHCl denaturant (midpoint, where 50% of the enzyme is unfolded, at 0.7 M). This concentration is lower than that required to induce the conformational changes of Tv laccase molecule as a whole (midpoint at 3.4 M), as investigated by the intrinsic fluorescence of Tv laccase. This observation suggests that the multicopper active sites are formed by relatively weak interactions and hence may be conformationally more flexible than the intact enzyme. The electrochemical method demonstrated in this study is technically simple yet effective and could be potentially useful for investigation on the thermodynamics and kinetics of the conformational changes of multicopper oxidases induced by different denaturants.