On the Mechanism of the Enzymatic Conversion of Cholest-5-ene-3beta,7alpha-diol into 7alpha-Hydroxycholest-4-en-3-one

Abstract

The conversion of cholest-5-ene-3p,7a-diol into 7a-hydroxycholest-4-en-3-one in the presence of rat liver microsomes fortified with NAD was studied with the aid of [4-14C]-, [3a-3H]-, [4p-3H]-, and [4@-2H]cholest-5-ene-3B,7a-diol. The reaction was also carried out in 3H,0 and ,H,O and the incorporation of isotope in the product was determined. It was found that the rate-limiting step in the reaction involved loss of the 3a-hydrogen. This step was coupled with or followed by loss of hydrogen from the 4p-position, of which a small amount was transferred to the 6/?-position of the product.The major pathways for the conversion of cholesterol into chenodeoxycholic acid and cholic acid in rat liver involve cholest-5-ene-3~,7a-diol and 7a-hydroxycholest-4-en-3-one as intermediates [I]. The transformation of cholest-5-ene-3/?,7a-diol into 7a-hydroxycholest-4-en-3-one is catalyzed by the microsoma1 fraction and NAD is the preferred cofactor [2,3]. The detailed enzymatic and stereochemical features of the reaction are not known. I n a study of the transformation of [4/?-SH]cholesterol into bile acids in the bile fistula rat, Green and Samuelsson found that the main part of the 4b-hydrogen was lost and a small part was transferred to the 6@-position [4]. I n view of present knowledge of the pathways of bile acid biosynthesis it is probable that these events occur mainly during the transformation of cholest-5-ene-3@,7a-diol into 7a-hydroxycholest-4-en-3-one. However, it appeared possible that more detailed information concerning this reaction might be provided by studies in vitro. The present communication describes a series of experiments on the formation of 7a-hydroxycholest-4-en-3-one from cholest-5-ene-3B,7a-diol labeled with 3H and 2H in specific positions.

Mechanism of Microbial Transformation of Cholesterol into Coprostanol

“…Coprostanol was oxidized with chromic-acid [8] to yield coprostanone. The product was purified by means of thin-layer chromatography with benzeneethyl acetate (9 : 1, vlv), as solvent.…”

“…The isotope effect found is smaller than would be expected if breaking of the C-H bond was the only rate-determining factor in the conversion of cholesterol into coprostanol. The conversion of 5-[3a-3H]cholestene-3/?,7a-diol into 7a-hydroxy-4-cholesten-3-one by rat liver microsomes has been shown to involve a kinetic isotope effect of about 5 [8]. The presence of an isotope effect of about 2 and the retention of about 50°/, of the tritium in the conversion of [3a-3H]cholesterol into coprostanol would a t first sight indicate that mechanism I and mechanism I1 are responsible each for about 50°/, of the reaction.…”

Hydroxylations in Biosynthesis of Bile Acids. Cytochrome P-450 LM4 and 12alpha-Hydroxylation of 5beta-Cholestane-3alpha,7alpha-diol

“…5~-[7~-3H]Cholestane-3a,7a-diol (500 Ci/mol) and 5b-[7~-3H]cholestane-3a,7a,12a-triol (500 Ci/mol) were prepared as described previously [12,13]. p-Naphthoflavone was obtained from Serva.…”

On the Conversion of Cholestanol into Allocholic Acid in Rat Liver

“…Homogenates of liver, 20°/, (w/v), were prepared in a modified Bucher medium [13] and fractionated as described in previous communications [4,10]. I n studies of 7a-hydroxylation, livers from rats with a biliary fistula were used since 7a-hydroxylation of cholestanol as well as of cholesterol is stimulated by biliary drainage [9,14].…”

“…Cholestanol, 5a-cholestan-3-one, 501-cholestan-3a-01 and the corresponding 7a-hydroxylated compounds were used as internal standards. The different chromatographic zones were detected and assayed for radioactivity as described in previous communications [4,10,14]. I n experiments concerning oxidation of 5~~-[ 6 p -~H ] -cholestane-3/3,7a-diol and reduction of 7a-hydroxy5a-[6p-3H]cholestan-3-one, 50 pg of the steroid dissolved in 50 pl of acetone were incubated for 20 min a t 37" with 3 ml of microsomal fraction or 100000 xg supernatant fluid or 3 ml of a solution containing 200-fold purified 3a-hydroxysteroid dehydrogenase [15].…”