Investigation of the Component Reactions of Oxidative Sterol Demethylation OXIDATION OF A 4,4-DIMETHYL STEROL TO A 4β-METHYL-4α-CARBOXYLIC ACID DURING CHOLESTEROL BIOSYNTHESIS

Abstract Microsomal enzymes of rat liver catalyze the oxidative demethylation of 4,4-dimethyl-5α-cholest-7-en-3β-ol. Demethylation requires oxygen and both reduced and oxidized pyridine nucleotides. Aerobic incubation of 30,31-14C-4,4-dimethyl-5α-cholest-7-en-3β-ol with a microsomal preparation depleted of NAD+ and containing an NADPH-generating system yields oxidative attack of substrate but no release of 14CO2. Following incubation, sterols are extracted from the microsomal protein with acetone. Only one oxygenated sterol is formed in high yield. Homogeneity and purity of the new sterol have been established by thin layer chromatography on plates of silica gel. Nuclear magnetic resonance and mass spectrometry have been used to identify the new sterol as 3β-hydroxy-4β-methyl-5α-cholest-7-ene-4α-carboxylic acid. Isolation and identification of the 4α-carboxylic acid demonstrates that the 4α-methyl group of the 4,4-dimethyl sterol may be attacked initially. Furthermore, the rates of 4α-carboxylic acid formation and decarboxylation (NAD+-dependent) are sufficient to account for the over-all rate of demethylation. Demethylation of 4,4-dimethyl-5α-cholest-7-en-3β-ol and 4α-methyl-5α-cholest-7-en-3β-ol is inhibited similarly by additions of cyanide, cytochrome c, glycolithocholate, and dithiothreitol; prior treatment of the enzyme system with trypsin and heat affected the rates similarly: dietary cholestyramine enhanced the amount of demethylation of each sterol to the same extent. It is clear now that the 4α-methyl group of both sterol substrates is oxidatively attacked; oxidation of each substrate requires the same cofactors; thus, the similar behavior of the enzyme system under the above treatments is consistent with the conclusion that the same enzymic system may catalyze the removal of the 4α-methyl group from both the 4,4-dimethyl and 4α-monomethyl sterol substrates.