Analysis of differential substrate selectivities of CYP2B6 and CYP2E1 by site-directed mutagenesis and molecular modeling

Margit Spatzenegger, Hong Liu, Qinmi Wang, Andrea De Barber, Dennis Koop, James R. Halpert

Research output: Contribution to journalArticle

57 Citations (Scopus)

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 hydroxylate p-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.

Original languageEnglish (US)
Pages (from-to)477-487
Number of pages11
JournalJournal of Pharmacology and Experimental Therapeutics
Volume304
Issue number1
DOIs
StatePublished - Jan 1 2003

Fingerprint

Cytochrome P-450 CYP2E1
Site-Directed Mutagenesis
Catalytic Domain
Arachidonic Acid
Cytochrome P-450 Enzyme System
Cytochrome P-450 CYP2B6
Valine
Enzymes
Hydroxylation

ASJC Scopus subject areas

  • Pharmacology

Cite this

Analysis of differential substrate selectivities of CYP2B6 and CYP2E1 by site-directed mutagenesis and molecular modeling. / Spatzenegger, Margit; Liu, Hong; Wang, Qinmi; De Barber, Andrea; Koop, Dennis; Halpert, James R.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 304, No. 1, 01.01.2003, p. 477-487.

Research output: Contribution to journalArticle

Spatzenegger, Margit ; Liu, Hong ; Wang, Qinmi ; De Barber, Andrea ; Koop, Dennis ; Halpert, James R. / Analysis of differential substrate selectivities of CYP2B6 and CYP2E1 by site-directed mutagenesis and molecular modeling. In: Journal of Pharmacology and Experimental Therapeutics. 2003 ; Vol. 304, No. 1. pp. 477-487.
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