Photoaffinity labeling and site-directed mutagenesis of rat squalene epoxidase

Squalene epoxidase (SE) (EC 1.14.99.7) is a flavin-requiring, non-cytochrom e P-450 oxidase that catalyzes the conversion of squalene to (3S)-2,3-oxido squalene, Photolabeling and site-directed mutagenesis were performed on rec ombinant rat SE (rrSE) to elucidate the location and roles of active-site r esidues important for catalysis, Two new benzophenone-containing analogs of NB-598, a nanomolar inhibitor of vertebrate SE, were synthesized in tritiu m-labeled form. These photoaffinity analogs (PDA-I and PDA-II) became coval ently attached to SE when irradiated at 360 nm, Lys-C digestion and HPLC pu rification of [H-3]PDA-I-labeled rrSE resulted in isolation of a single maj or peptide. MALDI-TOF mass spectrometry of this peptide indicated a covalen t adduct between PDA-I and a tripeptide, Asp-Ile-Lys, beginning at Asp-426 of rat SE. Based on the labeling results, three mutant constructs were made . First, the D426A and K428A constructs showed a 5- to 8-fold reduction in SE activity compared with wild-type enzyme, while little change was observe d in the I427A mutant. Second, a set of five mutant constructs was prepared for the conserved region based on the structure of the flavoprotein p-hydr oxybenzoate hydroxylase (PHBH), Compared with wild-type, D284A and D407A sh owed less than 25% SE activity. This reduction also appeared to correlate w ith reduced affinity of the mutant proteins for FAD, Finally, each of the s even Cys residues of rrSE were individually mutated to Ala, Three Cys subst itutions had no effect on SE activity, and substitutions at Cys-500 and Cys -533 showed a 50% lower SE activity. Mutations at Cys-490 and Cys-557 produ ced proteins with negligible SE activity, implicating these residues as bei ng either structurally or catalytically essential. Chemical modification of wild-type and Cys mutants with a thiol-modifying reagent support the exist ence of a disulfide bond between Cys-490 and Cys-557. (C) 2000 Academic Pre ss.