Kondo S, Sakaki T, Ohkawa H, Inouye K
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan.
Biochem Biophys Res Commun. 1999 Apr 13;257(2):273-8. doi: 10.1006/bbrc.1999.0455.
The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of Vmax were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains.
通过使用含有天然酶或其融合酶的重组酵母微粒体，分析了大鼠细胞色素P450 1A1与酵母NADPH - P450还原酶之间的静电相互作用。当反应混合物的离子强度为0.1 - 0.15时，含有大鼠细胞色素P4501A1和酵母NADPH - P450还原酶的重组微粒体（混合体系）中7 - 乙氧基香豆素O - 脱乙基反应的Vmax最大。然而，对于大鼠细胞色素P450 1A1与酵母NADPH - P450还原酶之间的融合酶（融合体系），随着离子强度增加，活性均匀降低。混合体系和融合体系之间Vmax的pH曲线也不同。基于这些结果，我们提出一个假说，即细胞色素P450和NADPH - P450还原酶具有不止一种结合模式。混合体系在离子强度为0.1 - 0.15时的最大活性可以通过结合模式的变化来解释。另一方面，由于细胞色素P450和NADPH - P450还原酶结构域的拓扑结构有限，融合酶似乎只有一种结合模式。
