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Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase
- Source :
-
Biochemical & Biophysical Research Communications . Feb2004, Vol. 315 Issue 1, p1. 9p. - Publication Year :
- 2004
-
Abstract
- 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 (<f>Ki=18.4</f> μM), [3H]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 [3H]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 [3H]TNSA-Dza. This constitutes the first evidence as to the identity of the substrate-binding site of SE. [Copyright &y& Elsevier]
- Subjects :
- *MUTAGENESIS
*PHOTOAFFINITY labeling
*PLANT growing media
*SQUALENE
Subjects
Details
- Language :
- English
- ISSN :
- 0006291X
- Volume :
- 315
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Biochemical & Biophysical Research Communications
- Publication Type :
- Academic Journal
- Accession number :
- 12100045
- Full Text :
- https://doi.org/10.1016/j.bbrc.2004.01.012