Cloning, heterologous expression, and enzymological characterization of human squalene monooxygenase.
The cDNA for human squalene monooxygenase, a key enzyme in the committed pathway for cholesterol biosynthesis, was amplified from a human liver cDNA library and cloned, and the protein was expressed in Escherichia coli and purified. Kinetic analysis of the purified enzyme revealed an apparent K(m) for squalene of 7.7 microM and an apparent k(cat) of 1.1 min(-1). For FAD the apparent K(m) is 0.3 microM, consistent with a loosely bound flavin. The apparent K(m) for NADPH-cytochrome P450 reductase, the requisite electron transfer partner, is 14 nM. The amount of reductase needed for maximal activity is about threefold less than the amount of squalene monooxygenase present in the assay; thus, electron transfer to the monooxygenase is not likely to be rate limiting. Previous reports have implicated inhibition of this enzyme as the cause of a peripheral demyelination seen in weanling rats fed a diet containing tellurium. As no data were available for humans, the ability of a number of tellurium and related elemental compounds to inhibit the recombinant human enzyme was examined. Tellurite, tellurium dioxide, selenite, and selenium dioxide were inhibitory; the tellurium compounds were more potent than the selenium compounds, as indicated by their IC(50) values (17 and 37 microM, respectively). Kinetic analysis of the inhibition by tellurite suggests multiple sites of interaction with the enzyme in a noncompetitive manner with respect to squalene.
Laden
B P
BP
Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536-0082, USA.
Tang
Y
Y
Porter
T D
TD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D016366:Open Reading Frames; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018036:Selenium Compounds / Q000494:pharmacology; D064588:Selenium Oxides; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018038:Sodium Selenite / Q000494:pharmacology; D013185:Squalene / Q000378:metabolism; D050603:Squalene Monooxygenase; D013691:Tellurium / Q000494:pharmacology
D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D018036:Selenium Compounds; D064588:Selenium Oxides; D005182:Flavin-Adenine Dinucleotide; D013185:Squalene; D010105:Oxygenases; D050603:Squalene Monooxygenase; D009251:NADPH-Ferrihemoprotein Reductase; D018038:Sodium Selenite; C026660:tellurous acid; D013691:Tellurium