Versatile peroxidase (VP) from Bjerkandera adusta is an enzyme able to oxidize bulky and high-redox substrates trough a Long-Range Electron Transfer (LRET) pathway. In this study, the introduction of radical-forming aromatic amino acids by chemical modification of the protein surface was performed, and the catalytic implications of these additional surface active-sites on the oxidation of 2,6-dimethylphenol, Mn2+ and Remazol Brilliant Blue R (RBBR) were determined. These three different substrates are oxidized in different active-sites of enzyme molecule, of which the high redox RBBR the only one that is transformed by an external radical formed on the protein surface. Both catalytic constants kcat and KM were significantly affected by the chemical modifications. Tryptophan- and tyrosine-modified VP showed higher catalytic transformation than the unmodified enzyme for RBBR, while the Mn2+ oxidation was significantly reduced by all chemical modifications. Electron Paramagnetic Resonance studies demonstrated the formation of additional protein-based radicals after the chemical modification with radical-forming amino acids. In addition, the catalytic rate of the LRET-mediated transformation showed a good correlation with the ionization energy of the additional amino acid on the protein surface.

中文翻译：

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在通用过氧化物酶表面添加新的催化位点以增强 LRET 催化

来自 Bjerkandera adusta 的多功能过氧化物酶 (VP) 是一种能够通过长程电子转移 (LRET) 途径氧化大体积和高氧化还原底物的酶。在这项研究中，通过蛋白质表面的化学修饰引入了形成自由基的芳香族氨基酸，以及这些额外的表面活性位点对 2,6-二甲基苯酚、Mn2+ 和 Remazol Brilliant Blue R 氧化的催化影响(RBBR) 被确定。这三种不同的底物在酶分子的不同活性位点被氧化，其中高氧化还原RBBR是唯一一种被蛋白质表面形成的外部自由基转化的底物。催化常数 kcat 和 KM 都受到化学修饰的显着影响。色氨酸和酪氨酸修饰的 VP 对 RBBR 显示出比未修饰的酶更高的催化转化，而所有化学修饰都显着降低了 Mn2+ 氧化。电子顺磁共振研究表明，在用形成自由基的氨基酸进行化学修饰后，会形成额外的基于蛋白质的自由基。此外，LRET 介导的转化的催化速率与蛋白质表面额外氨基酸的电离能显示出良好的相关性。