Gannett Peter M, Kabulski Jarod, Perez Felio A, Liu Zhongyuan, Lederman David, Locuson Charles W, Ayscue Robyn R, Thomsen Nissa M, Tracy Timothy S
Basic Pharmaceutical Sciences, West Virginia University, P.O. Box 9530, Morgantown, WV 26506, USA.
J Am Chem Soc. 2006 Jul 5;128(26):8374-5. doi: 10.1021/ja0608693.
The cytochrome P450 enzymes represent an important class of heme-containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological-based chips for standardizing cytochrome P450 metabolism or for high-throughput screening of pharmaceutical agents. Previous studies have generally immobilized P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely, superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed.
细胞色素P450酶是一类重要的含血红素酶。人们对将这些酶固定在表面上有着浓厚兴趣，这样就可以研究单个酶与其他物质之间关于电子转移、同二聚体或异二聚体相互作用的情况，或者用于构建基于生物的芯片，以标准化细胞色素P450代谢或用于药物高通量筛选。以往的研究通常是将P450酶固定在基质或表面上。在这里，我们已将CYP2C9附着于金基底上，使得所得构建体在存在NADPH和细胞色素P450还原酶的情况下保持结合和代谢底物的能力。这些芯片的活性直接取决于用于附着CYP2C9的连接子以及酶附着过程中活性位点关键分子的存在。一种检测底物 - 酶结合的新方法，即超导量子干涉装置（SQUID）磁力测定法，被用于监测底物的结合。最重要的是，已经开发出了能够使可测量的CYP2C9代谢发生的条件。
