Linked Life Data

12490624

Source:http://linkedlifedata.com/resource/pubmed/id/12490624

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2002-12-19
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0022-3565
pubmed:author
pubmed:issnType
Print
pubmed:volume
304
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
477-87
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:12490624-Amino Acid Sequence, pubmed-meshheading:12490624-Arachidonic Acid, pubmed-meshheading:12490624-Aryl Hydrocarbon Hydroxylases, pubmed-meshheading:12490624-Binding Sites, pubmed-meshheading:12490624-Computer Simulation, pubmed-meshheading:12490624-Cytochrome P-450 CYP2E1, pubmed-meshheading:12490624-Dealkylation, pubmed-meshheading:12490624-Humans, pubmed-meshheading:12490624-Hydroxylation, pubmed-meshheading:12490624-Kinetics, pubmed-meshheading:12490624-Models, Molecular, pubmed-meshheading:12490624-Mutagenesis, Site-Directed, pubmed-meshheading:12490624-Mutation, pubmed-meshheading:12490624-Oligonucleotide Probes, pubmed-meshheading:12490624-Oxidation-Reduction, pubmed-meshheading:12490624-Oxidoreductases, N-Demethylating, pubmed-meshheading:12490624-Protein Conformation, pubmed-meshheading:12490624-Substrate Specificity
pubmed:year
2003
pubmed:articleTitle
Analysis of differential substrate selectivities of CYP2B6 and CYP2E1 by site-directed mutagenesis and molecular modeling.
pubmed:affiliation
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031, USA. jhalpert@utmb.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.