Inhibition and activation of porcine squalene epoxidase.
Pig liver squalene epoxidase (SE) has been partially purified from solubilized microsomes by DEAE-Sephacel and Blue Sepharose 4B chromatography. This stable and reproducible preparation was used to investigate the mechanism of several substrate-like inhibitors of SE and to study the effects of pH, metals, detergents, and cofactors on enzyme activity. Most divalent (1 mM) and trivalent (0.1 mM) metal cations had little effect on SE at pH 7.4; only ferrous and cupric ions showed ca. 50% reduction in SE activity. Interestingly, at pH 8.8, EDTA (10 mM) shows 1.8-fold enhancement of enzyme activity. Among the detergents, Triton X-100 was clearly superior for solubilization and purification of porcine SE; Tween 80, Lubrol-PX, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid, octyl beta-glucoside, and three different Zwittergents were much less effective for SE solubilization. Partially purified pig liver SE showed maximal activity at pH 8.8-9.0. Trisnorsqualene alcohol and trisnorsqualene cyclopropylamine were noncompetitive inhibitors at pH 8.8, with Ki values of 4 microM and 180 nM, respectively; these two inhibitors were not substrates for SE. In contrast, 26-hydroxysqualene was both a competitive inhibitor with a Ki value of 4 microM at pH 8.8 and a substrate for SE. An unexpected enhancement (up to 350%) of SE activity was observed at pH 7.4 following preincubation with selected nonpolar derivatives of farnesol and farnesoic acid. At pH 8.8, this effect was less dramatic but still evident.
Bai
M
M
Department of Chemistry, State University of New York, Stony Brook 11794-3400.
Prestwich
G D
GD
D000818:Animals; D001548:Benzalkonium Compounds / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D003902:Detergents; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D005204:Farnesol / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D010105:Oxygenases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D050603:Squalene Monooxygenase; D013379:Substrate Specificity; D013552:Swine
D001548:Benzalkonium Compounds; D003902:Detergents; D008670:Metals; D005182:Flavin-Adenine Dinucleotide; D005204:Farnesol; D003300:Copper; D004492:Edetic Acid; D010105:Oxygenases; D050603:Squalene Monooxygenase; C015163:dimethyldodecylbenzylammonium