Abstract
Human CYP2B6 and CYP2E1 were used to investigate the extent to which differential substrate selectivities between cytochrome P450 subfamilies reflect differences in active-site residues as opposed to distinct arrangement of the backbone of the enzymes. Reciprocal CYP2B6 and CYP2E1 mutants at active-site positions 103, 209, 294, 363, 367, and 477 (numbering according to CYP2B6) were characterized using the CYP2B6-selective substrate 7-ethoxy-4-trifluoromethylcoumarin, the CYP2E1-selective substrate p-nitrophenol, and the common substrates 7-ethoxycoumarin, 7-butoxycoumarin, and arachidonic acid. This report is the first to study the active site of CYP2E1 by systematic site-directed mutagenesis. One of the most intriguing findings was that substitution of CYP2E1 Phe-477 with valine from CYP2B6 resulted in significant 7-ethoxy-4-trifluoromethylcoumarin deethylation. Use of three-dimensional models of CYP2B6 and CYP2E1 based on the crystal structure of CYP2C5 suggested that deethylation of 7-ethoxy-4-trifluoromethylcoumarin by CYP2E1 is impeded by van der Waals overlaps with the side chain of Phe-477. Interestingly, none of the CYP2B6 mutants acquired enhanced ability to hydroxylatep-nitrophenol. Substitution of residue 363 in CYP2E1 and CYP2B6 resulted in significant alterations of the metabolite profile for the side chain hydroxylation of 7-butoxycoumarin. Probing of CYP2E1 mutants with arachidonic acid indicated that residues Leu-209 and Phe-477 are critical for substrate orientation in the active site. Overall, the study revealed that differences in the side chains of active-site residues are partially responsible for differential substrate selectivities across cytochrome P450 subfamilies. However, the relative importance of active-site residues appears to be dependent on the structural similarity of the compound to other substrates of the enzyme.
Footnotes
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↵1 Permanent address: Center for Drug Discovery and Design, The State Key Laboratory of New Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P. R. China.
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↵2 To simplify the comparison of CYP2B6 and CYP2E1, the CYP2B6 numbering will be used throughout the paper.
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↵3 The assays of the CYP2E1dH mutants were performed with a different lot of 7-EFC, which resulted in lower specific activity than that used for CYP2B6dH mutants.
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↵4 The double mutant CYP2E1dH L367V-F477V did not produce sufficient stable hemeprotein for further studies.
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This work was supported by National Institutes of Health Grants ES03619 (J.R.H.) and AA08608 (D.R.K.) and Center Grant ES06676. This work was presented at the 2nd Southwest P450 Meeting, Camp Allen, TX, May 2002.
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DOI: 10.1124/jpet.102.043323
- Abbreviations:
- P450
- cytochrome P450
- pNP
- p-nitrophenol
- 7-EFC
- 7-ethoxy-4-trifluoromethylcoumarin
- 7-HFC
- 7-hydroxy-4-trifluoromethylcoumarin
- 7-EC
- 7-ethoxycoumarin
- 7-HC
- 7-hydroxycoumarin
- AA
- arachidonic acid
- PCR
- polymerase chain reaction
- βME
- β-methylmercaptoethanol
- Ni2+-NTA
- nickel-nitrilotriacetic acid
- EET
- epoxyeicosatrienoic acid
- Received August 15, 2002.
- Accepted September 30, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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