Linked Life Data

20812728

Source:http://linkedlifedata.com/resource/pubmed/id/20812728

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
41
pubmed:dateCreated
2010-10-12
pubmed:databankReference
pubmed:abstractText
The use of molecular modeling in conjunction with site-directed mutagenesis has been extensively used to study substrate orientation within cytochrome P450 active sites and to identify potential residues involved in the positioning and catalytic mechanisms of these substrates. However, because docking studies utilize static models to simulate dynamic P450 enzymes, the effectiveness of these studies is strongly dependent on accurate enzyme models. This study employed a cytochrome P450 3A4 (CYP3A4) crystal structure (Protein Data Bank entry 1W0E) to predict the sites of metabolism of the known CYP3A4 substrate raloxifene. In addition, partial charges were incorporated into the P450 heme moiety to investigate the effect of the modified CYP3A4 model on metabolite prediction with the ligand docking program Autodock. Dehydrogenation of raloxifene to an electrophilic diquinone methide intermediate has been linked to the potent inactivation of CYP3A4. Active site residues involved in the positioning and/or catalysis of raloxifene supporting dehydrogenation were identified with the two models, and site-directed mutagenesis studies were conducted to validate the models. The addition of partial charges to the heme moiety improved the accuracy of the docking studies, increasing the number of conformations predicting dehydrogenation and facilitating the identification of substrate-active site residue interactions. On the basis of the improved model, the Phe215 residue was hypothesized to play an important role in orienting raloxifene for dehydrogenation through a combination of electrostatic and steric interactions. Substitution of this residue with glycine or glutamine significantly decreased dehydrogenation rates without concurrent changes in the rates of raloxifene oxygenation. Thus, the improved structural model predicted novel enzyme-substrate interactions that control the selective dehydrogenation of raloxifene to its protein-binding intermediate.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1520-4995
pubmed:author
pubmed:issnType
Electronic
pubmed:day
19
pubmed:volume
49
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
9011-9
pubmed:dateRevised
2011-10-19
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Improved cytochrome P450 3A4 molecular models accurately predict the Phe215 requirement for raloxifene dehydrogenation selectivity.
pubmed:affiliation
Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City, UT 84112, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, N.I.H., Extramural