Regulation of rat hepatic 3 beta-hydroxysterol Delta(7)-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation

The mechanism for the catalytic reduction of the double bond at C-7,8 in 7- dehydrocholesterol by 3 beta-hydroxysterol Delta(7)-reductase was investiga ted by testing structurally related sterols as substrates and potential inh ibitors. The hepatic smooth endoplasmic reticulum was identified as the sit e of enzyme activity. All putative substrates contained 27 carbons, but dif fered from 7-dehydrocholesterol by the addition of either an ethyl substitu ent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl subs tituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3 beta - to 3 alpha-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol), Two non-steroidal compounds that inhibit 3 be ta-hydroxysterol Delta(7)-reductase in vivo (AY 9944 and BM 15.766) were al so tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol wer e reduced at C-7,8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol, Increasin g concentrations of these sterols competitively inhibited 3 beta-hydroxyste rol Delta(7)-reductase activity, The double bond at C-7,8 in lathosterol wa s not reduced, AY 9944 and BM 15.766 inhibited 3 beta-hydroxysterol Delta(7 )-reductase activity non-competitively. 3 beta-Hydroxysterol-Delta(7)-reduc tase activity declined after microsomes were exposed to alkaline phosphatas e, 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 r equires binding of the enzyme protein with the B-ring of the sterol substra te that contains a double bond at C-5,6, The reaction is hindered by substi tuents located on the apolar side-chain and epimerization of the hydroxyl g roup in ring A to a 3 alpha-configuration, 3 beta-Hydroxysterol Delta(7)-re ductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form.