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Vol. 304, Issue 1, 477-487, January 2003
Department of Pharmacology and Toxicology, University of Texas
Medical Branch, Galveston, Texas (M.S., H.L., Q.W., J.R.H.); and
Department of Physiology and Pharmacology, Oregon Health and Science
University, Portland, Oregon (A.D., D.R.K.)
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.
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