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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
1
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pubmed:dateCreated |
2004-3-11
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pubmed:abstractText |
Squalene epoxidase (SE) catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) in order to identify the location of the substrate-binding site and the roles of key residues in catalysis. Truncated 50-kDa rrSE was purified and photoaffinity labeled by competitive SE inhibitor (Ki=18.4 microM), [(3)H]TNSA-Dza. An 8-kDa CNBr/BNPS-skatole peptide was purified and the first 24 amino acids were sequenced by Edman degradation. The sequence PASFLPPSSVNKRGVLLLGDAYNL corresponded to residues 388-411 of the full-length rat SE. Three nucleophilic residues (Lys-399, Arg-400, and Asp-407) were labeled by [(3)H]TNSA-Dza. Triple mutants were prepared in which bulky groups were used to replace the labeled charged residues. Purified mutant enzymes showed lower enzymatic activity and reduced photoaffinity labeling by [(3)H]TNSA-Dza. This constitutes the first evidence as to the identity of the substrate-binding site of SE.
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pubmed:grant |
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Amino Acids,
http://linkedlifedata.com/resource/pubmed/chemical/Azo Compounds,
http://linkedlifedata.com/resource/pubmed/chemical/BNPS-skatole,
http://linkedlifedata.com/resource/pubmed/chemical/Cyanogen Bromide,
http://linkedlifedata.com/resource/pubmed/chemical/Oxygenases,
http://linkedlifedata.com/resource/pubmed/chemical/Peptides,
http://linkedlifedata.com/resource/pubmed/chemical/Photoaffinity Labels,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Skatole,
http://linkedlifedata.com/resource/pubmed/chemical/Squalene,
http://linkedlifedata.com/resource/pubmed/chemical/Squalene Monooxygenase,
http://linkedlifedata.com/resource/pubmed/chemical/Tritium
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pubmed:status |
MEDLINE
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pubmed:month |
Feb
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pubmed:issn |
0006-291X
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:day |
27
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pubmed:volume |
315
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
1-9
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pubmed:dateRevised |
2007-11-14
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pubmed:meshHeading |
pubmed-meshheading:15013417-Amino Acid Sequence,
pubmed-meshheading:15013417-Amino Acid Substitution,
pubmed-meshheading:15013417-Amino Acids,
pubmed-meshheading:15013417-Animals,
pubmed-meshheading:15013417-Azo Compounds,
pubmed-meshheading:15013417-Binding, Competitive,
pubmed-meshheading:15013417-Binding Sites,
pubmed-meshheading:15013417-Cyanogen Bromide,
pubmed-meshheading:15013417-Molecular Sequence Data,
pubmed-meshheading:15013417-Mutagenesis, Site-Directed,
pubmed-meshheading:15013417-Oxygenases,
pubmed-meshheading:15013417-Peptides,
pubmed-meshheading:15013417-Photoaffinity Labels,
pubmed-meshheading:15013417-Rats,
pubmed-meshheading:15013417-Recombinant Proteins,
pubmed-meshheading:15013417-Skatole,
pubmed-meshheading:15013417-Squalene,
pubmed-meshheading:15013417-Squalene Monooxygenase,
pubmed-meshheading:15013417-Substrate Specificity,
pubmed-meshheading:15013417-Tritium
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pubmed:year |
2004
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pubmed:articleTitle |
Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase.
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pubmed:affiliation |
Department of Biochemistry and Cell Biology, The University at Stony Brook, NY 11794-5215, USA.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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