Cho U S, Ahn H J, Park E Y, Dong M S, Kim K H
Graduate School of Biotechnology, Korea University, 136-701, Seoul, South Korea.
Biochim Biophys Acta. 2001 Apr 7;1546(2):412-21. doi: 10.1016/s0167-4838(01)00165-0.
Human cytochrome P-450 (P-450) 1A2 expressed in Escherichia coli is readily converted into non-native cytochrome P-420 (P-420) in the presence of detergents. alpha-Naphthoflavone (ANF) has been used to prevent P-450 1A2 inactivation to P-420 during purification. However, the mechanism by which ANF modulates P-450 1A2 is not clearly understood. We observed that recombinant human P-450 1A2 prepared in the absence of ANF has an approx. 5 times higher maximum catalytic activity in the O-deethylation of 7-ethoxycoumarin than that in the presence of ANF, with the same K(m) values. The results revealed that the enzyme purified with ANF is not catalytically fully active, indicating that ANF tightly binds to the enzyme, only to be dissociated by heat denaturation. Furthermore, the inactive P-420 form of the enzyme could be reconverted to P-450 by ANF in high concentrations of detergents. The reconversion was concentration-dependent, confirming ANF-induced regeneration of active P-450 1A2. The reconversion coincided with the conformational change of the enzyme including increased alpha-helix content. The conformation of P-450 1A2 was also stabilized by ANF, resulting in an approx. 5 degrees C increase in thermal stability.
在大肠杆菌中表达的人细胞色素P-450（P-450）1A2在去污剂存在的情况下很容易转化为非天然细胞色素P-420（P-420）。α-萘黄酮（ANF）已被用于防止P-450 1A2在纯化过程中失活为P-420。然而，ANF调节P-450 1A2的机制尚不清楚。我们观察到，在没有ANF的情况下制备的重组人P-450 1A2在7-乙氧基香豆素的O-去乙基化反应中的最大催化活性比在有ANF的情况下高约5倍，而K(m)值相同。结果表明，用ANF纯化的酶没有完全催化活性，这表明ANF与酶紧密结合，只有通过热变性才能解离。此外，在高浓度去污剂中，ANF可以将无活性的P-420形式的酶重新转化为P-450。这种重新转化是浓度依赖性的，证实了ANF诱导活性P-450 1A2的再生。这种重新转化与酶的构象变化一致，包括α-螺旋含量增加。ANF还稳定了P-450 1A2的构象，导致热稳定性提高约5℃。
