title: Regulation of rat hepatic 3beta-hydroxysterol delta7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation.
abstract: The mechanism for the catalytic reduction of the double bond at C-7, 8 in 7-dehydrocholesterol by 3beta-hydroxysterol Delta7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. The hepatic smooth endoplasmic reticulum was identified as the site of enzyme activity. All putative substrates contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3beta- to 3alpha-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol). Two non-steroidal compounds that inhibit 3beta-hydroxysterol Delta7-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7, 8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 3beta-hydroxysterol Delta7-reductase activity. The double bond at C-7,8 in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 3beta-hydroxysterol Delta7-reductase activity non-competitively. 3beta-Hydroxysterol-Delta7-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was increased by phosphorylation with Mg2+, and ATP. These results demonstrate that the reduction of the double bond at C-7,8 requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5,6. The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3alpha-configuration. 3beta-Hydroxysterol Delta7-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form.
authors.affiliation: Department of Medicine and Liver Center, UMD-NJ Medical School, Newark, NJ 07103, USA.
authors.forename: S
authors.initials: S
authors.lastname: Shefer
authors.forename: G
authors.initials: G
authors.lastname: Salen
authors.forename: A
authors.initials: A
authors.lastname: Honda
authors.forename: A K
authors.initials: AK
authors.lastname: Batta
authors.forename: L B
authors.initials: LB
authors.lastname: Nguyen
authors.forename: G S
authors.initials: GS
authors.lastname: Tint
authors.forename: Y A
authors.initials: YA
authors.lastname: Ioannou
authors.forename: R
authors.initials: R
authors.lastname: Desnick
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D008099:Liver / Q000201:enzymology*; D008297:Male; D015394:Molecular Structure; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
chemical_list: D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019995:7-dehydrocholesterol reductase