CHEMICAL AND ENZYMATIC INVESTIGATIONS OF THE MECHANISM OF THE REMOVAL OF THE 14ALPHA-METHYL GROUP OF LANOSTEROL

[摘要] A variety of 14(alpha)-methyl, 14(alpha)-hydroxymethyl, and 14(alpha)-hydroxy sterols were prepared by chemical synthesis for use in investigations of the mechanism of the enzymatic removal of the 14(alpha)-methyl group (carbon atom 32) of lanosterol during the biosynthesis of cholesterol. A comparison of the in vitro metabolism of {2,4-(;;3)H}14(alpha)-hydroxymethyl-5(alpha)-cholest-7-en-3(beta)-ol (I) and {2,4-(;;3)H}14(alpha)-hydroxymethyl-5(alpha)-cholest-7-ene-3(beta),15(alpha)-diol (II) was made using 10,000 x g supernatant fractions of liver homogenates from male rats. Compound I was found to be efficiently converted to tritium-labeled cholesterol upon aerobic incubation with these enzyme preparations supplemented with NAD and NADP; in the absence of a cofactor supplement, a variety of other labeled C(,27) monohydroxy sterols, in addition to cholesterol, were isolated and characterized, including 5(alpha)-cholest-8(14)-en-3(beta)-ol, 5(alpha)-cholest-7-en-3-ol, 5(alpha)-cholesta-8,14-dien-3(beta)-ol, and cholesta-5,7-dien-3(beta)-ol. Compound II, which differed from I only by the addition of a 15(alpha)-hydroxyl group, was not significantly metabolized to cholesterol in either the presence or the absence of a cofactor supplement. These results do not favor the intermediary role of 15(alpha)-hydroxy sterols during the enzymatic removal of carbon atom 32 of 14(alpha)-methyl sterols. Incubation of compound I with a washed microsomal preparation (derived from the livers of male rats) supplemented with NAD and an NADPH generator under aerobic conditions yielded cholesterol and 5(alpha)-cholest-7-en-3(beta)-ol as the principal labeled C(,27) monohydroxy sterol products. However, incubation of I with washed microsomes under aerobic conditions in the absence of added cofactors or in the presence or absence of supplemental NAD and NADP under anaerobic conditions yielded 5(alpha)-cholest-8(14)-en-3(beta)-ol as the major ((GREATERTHEQ) 90%) labeled C(,27) monohydroxy sterol product. The extent of conversion of I to C(,27) monohydroxy sterols was roughly the same in all of the microsomal incubations, regardless of the presence or absence of oxygen and/or a cofactor supplement. These results indicate that oxygen is not required for the enzymatic removal of carbon atom 32 of 14(alpha)-hydroxymethyl-5(alpha)-cholest-7-en-3(beta)-ol. It is further suggested by these results that oxygen is required only for the initial hydroxylation of the 14(alpha)-methyl group during the removal of carbon atom 32 of lanosterol, and that the immediate product of this demethylation process is a sterol containing a (DELTA)(;;8(14)) double bond. Washed microsomal preparations were also found to catalyze, under anaerobic conditions, the isomerization of 14(alpha)-methyl-5(alpha)-cholest-7-en-3(beta)-ol and 14(alpha)-hydroxymethyl-5(alpha)-cholest-7-en-3(beta)-ol to 14(alpha)-methyl-5(alpha)-cholest-8-en-3(beta)-ol and 14(alpha)-hydroxymethyl-5(alpha)-cholest-8-en-3(beta)-ol, respectively. Two sterols with the ;;unnatural;; cis-C-D ring junction were prepared in labeled form: {2,4-(;;3)H}5(alpha),14(beta)-cholest-7-en-3(beta),15(beta)-diol (III) and {2,4-(;;3)H}5(alpha),14(beta)-cholest-7-en-3(beta),15(beta)-diol (IV). Compound III, but not its 15(beta)-hydroxy epimer (IV), was found to be efficiently converted to cholesterol in 10,000 x g supernatant fractions of liver homogenates from male and female rats. After the incubation of III, a number of other labeled products, in addition to cholesterol, were isolated and characterized, including 5(alpha)-cholest-8-en-3(beta),ol, 5(alpha)-cholest-7-en-3(beta)-ol, 5(alpha)-cholesta-8,14-dien-3(beta)-ol, and cholesta-5,7-dien-3(beta)-ol.

[发布机构] Rice University