Mechanistic Aspects of Horseradish Peroxidase Elucidated through Single-Molecule Studies
Mechanistic Aspects of Horseradish Peroxidase Elucidated through Single-Molecule Studies
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DOI：
10.1021/ja9008858
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发表时间：
2009-05-06
影响因子：
15
通讯作者：
Walt, David R.
中科院分区：
文献类型：
--
作者：
Gorris, Hans H.;Walt, David R.
在化学蚀刻到玻璃光纤束表面的50000飞升室阵列中，通过荧光显微镜分离和观察了许多单独的辣根过氧化物酶（HRP）分子。我们很容易地分析了数百个HRP分子的底物周转情况，观察到的大量分子提供了很好的统计数据。与用于单分子研究的其他酶相比，在飞升阵列中产物形成的速率平均比在散装溶液中低10倍。我们将这种现象归因于HRP特殊的氧化还原反应机制，该机制涉及两个独立的产物形成步骤。HRP首先将荧光底物分子如Amplex Red氧化为自由基中间体。两个自由基分子随后经历不依赖于酶的突变反应，当限制在飞升室时，其速率降低，导致产物减少。广泛使用的Amplex Red以及其他荧光底物的这两步反应机制经常被忽视。该机制不仅影响HRP单分子研究，也影响低底物周转率下的体反应。
Many individual horseradish peroxidase (HRP) molecules were isolated and observed simultaneously by fluorescence microscopy in an array of 50 000 femtoliter chambers chemically etched into the surface of a glass optical fiber bundle. The substrate turnovers of hundreds of individual HRP molecules were readily analyzed, and the large number of molecules observed provided excellent statistics. In contrast to other enzymes used for single-molecule studies, the rates of product formation in the femtoliter array were, on average, 10 times lower than in bulk solution. We attribute this phenomenon to the particular redox-reaction mechanism of HRP that involves two separate steps of product formation. HRP first oxidizes fluorogenic substrate molecules like Amplex Red to radical intermediates. Two radical molecules subsequently undergo an enzyme-independent dismutation reaction, the rate of which is decreased when confined to a femtoliter chamber resulting in less product. This two-step reaction mechanism of the widely used Amplex Red, as well as other fluorogenic substrates, is often overlooked. The mechanism not only affects single-molecule studies with HRP but also bulk reactions at low substrate turnover rates.