Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase.
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.
Lee
Hee-Kyoung
HK
Department of Biochemistry and Cell Biology, The University at Stony Brook, NY 11794-5215, USA.
Zheng
Yi Feng
YF
Xiao
Xiao-Yi
XY
Bai
Mei
M
Sakakibara
Jun
J
Ono
Teruo
T
Prestwich
Glenn D
GD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001391:Azo Compounds / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D003488:Cyanogen Bromide / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012862:Skatole / Q000031:analogs & derivatives* / Q000737:chemistry; D013185:Squalene / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D050603:Squalene Monooxygenase; D013379:Substrate Specificity; D014316:Tritium
D000596:Amino Acids; D001391:Azo Compounds; D010455:Peptides; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D014316:Tritium; C005904:BNPS-skatole; D013185:Squalene; D012862:Skatole; D010105:Oxygenases; D050603:Squalene Monooxygenase; D003488:Cyanogen Bromide