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. α-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 Km 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 α-helix content. The conformation of P-450 1A2 was also stabilized by ANF, resulting in an approx. 5°C increase in thermal stability.
|Number of pages
|Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
|Published - 2001 Apr 7
Bibliographical noteFunding Information:
This study was supported by a grant (No. HMP-98-D-5-0046) of R&D Project, the Ministry of Health and Welfare, Korea. We also wish to acknowledge the financial support of the Korea Research Foundation made in the program year of 1998. U.S.C., H.J.A. and E.Y.P. were supported by the Brain Korea 21 program of the Ministry of Education.
- Conformational activation
- Human cytochrome P-450 1A2
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology